Synthesis and anti-liver fibrotic activity study of a chalcone derivative through anti-inflammatory effects and inhibition of JNK/NF-κB signaling pathways

ChunweiLv1

LeiZhang2

ChenxuWang1

TingtingJin1

ZhishunZhang1

YixiLi1

DianHe1✉Emailhed@lzu.edu.cn

QuanyiZhao1

LifangZheng1

1Institute of Medicinal ChemistrySchool of Pharmacy of Lanzhou University730000LanzhouChina

2Gansu Medical Device Inspection And Testing Institute730000LanzhouChina

Chunwei Lv^1,#, Lei Zhang^2,#, Chenxu Wang¹,Tingting Jin¹,Zhishun Zhang¹,Yixi Li¹, Dian He¹*, Quanyi Zhao¹, Lifang Zheng¹

(¹ Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University, Lanzhou 730000, China.² Gansu Medical Device Inspection And Testing Institute,Lanzhou 730000, China.)

^*Corresponding author: hed@lzu.edu.cn (Dian He).

Chunwei Lv and Lei Zhang contributed equally to this work.

Abstract

Liver fibrosis is a progressive disease caused by chronic inflammation and the activation of hepatic stellate cells (HSCs). This disease manifests as the abnormal proliferation and migration of HSCs, as well as the excessive deposition of the extracellular matrix. Chalcone analogues exhibit various biological activities, including anti-inflammatory, anti-proliferative, and apoptotic modulation properties, making them promising candidates for anti-fibrotic drug development. To enhance anti-fibrotic activities and decrease their side-effects, 31 novel chalcone derivatives were synthesized and evaluated. Among all the compounds, c31 exhibited the strongest anti-inflammatory activity, and its IC₅₀ is 3.05 ± 0.12 µM; and it effectively inhibited the activation and proliferation of HSC-T6 cells. Mechanistic studies revealed that c31 inhibits HSC activation by downregulating the expression levels of inflammatory factors, such as TNF-α, IL-6, and IL-1β, and by interfering with NF-κB and JNK signaling pathways. Additionally, c31 inhibited HSC-T6 proliferation and promoted apoptosis by blocking a G2/M phase cell cycle; and it also significantly inhibited HSC-T6 cell migration. In a rat model of CCl₄-induced liver fibrosis, c31 improved pathological symptoms, decreasing collagen deposition, fibrotic protein expression, and ALT and AST levels. Meanwhile, it also reduced the secretion of inflammatory factors, thereby alleviating CCl₄-induced liver fibrosis. In summary, c31 had significant anti-inflammatory and anti-fibrotic effects in both in vivo and in vitro; this indicates c31 has the potential to be used as a therapeutic candidate for hepatic inflammation and fibrosis.

Keywords:

Chalcone derivatives

Inflammation

Liver fibrosis

HST-6

JNK /NF-κB signaling pathway

1. Introduction

Liver fibrosis is a sustained healing response of the liver to damage and inflammation, and it is caused by cholestasis, hepatitis virus infection, medication toxicity, and nonalcoholic steatohepatitis (NASH) [1–3]. Persistent liver fibrosis can progress to cirrhosis, liver cancer, liver failure and even death [4]. The primary characteristic of liver fibrosis is excessive extracellular matrix (ECM) deposition [5]. During liver damage, the normally quiet hepatic stellate cells (HSCs) become continually activated and develop into myofibroblasts (MFs), which are the primary source of ECM [6, 7]. It has been shown that liver damage induces an inflammatory response that activates and recruits hepatic macrophages. Activated hepatic macrophages can promote the activation of HSCs by inducing the release of inflammatory mediators, pro-fibrotic cytokines, and reactive oxygen species [8, 9]. Therefore, the compounds which effective suppression of liver inflammatory responses and hepatic stellate cell (HSC) activation will possibly be drugs to delay or even prevent the progression of liver fibrosis.

Natural products have long been a vital source for drug development, and they are valuable for designing and optimizing novel drugs because of their structural diversity and natural selectivity [10]. Among numerous natural products, chalcones are a class of natural compounds and their derivatives featuring a 1,3- diphenyl − 2-propenone skeleton, widely distributed across various plants [11, 12]. Owing to their simple structure and excellent modifiability, chalcones are not only amenable to optimization in chemical synthesis but also exhibit diverse biological activities, including anti-inflammatory, antioxidant, antiproliferative, and apoptosis-regulating effects [13,–16].

In recent years, numerous in vitro and in vivo studies have demonstrated that chalcones and their derivatives exhibit significant antifibrotic effects in multiple liver injury models [17, 18]. As shown in Fig. 1, in a carbon tetrachloride-induced liver injury model, chalcone compounds DMC and 4-Hydroxychalcone effectively reduced serum liver marker levels (such as AST, ALT, and ALP), inhibited the release of pro-inflammatory factors (such as TNF-α, IL-6, and IL-8), alleviated oxidative stress responses, and reduced excessive extracellular matrix deposition. These effects slowed the pathological progression of liver fibrosis [19, 20].

Fig. 1

Structures of chalcone derivatives for the treatment of liver fibrosis through anti-inflammatory effects or inhibition of hepatic stellate cell (HSC) activation

Chalcone derivatives have anti-fibrotic effects through multiple pathways, primarily via two mechanisms [21]. According to reports, chalcone compounds Lophirone B, Lophirone C, and Xanthohumol (Fig. 1) suppressed inflammatory responses and oxidative stress by blocking inflammatory signaling pathways, such as NF-κB and JNK; and they reduced the release of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6. Meanwhile, the phenylpropyl ketone confers potent free radical scavenging capacity, thereby reducing reactive oxygen species (ROS) production and mitigating the promotion of liver fibrosis [22, 23]. It was also reported that Hydroxy crocin A, TMMC, BUTEIN, and Isoliquiritigenin (Fig. 1) acted directly on hepatic stellate cells (HSCs) by inhibiting the TGF-β1 and JNK/NF-κB signaling pathways and downregulating the expression of α-SMA, collagen I, and other collagens. This action prevented HSC activation and proliferation. These compounds also promoted apoptosis in activated HSCs by activating caspase-3, which ultimately reduced excessive extracellular matrix deposition and delayed or even reversed the fibrotic process [24–26]. In summary, chalcones are effective scaffolds for treating liver fibrosis. However, chalcone derivatives in previous studies primarily targeted single functions, either anti-inflammatory activities or the inhibition of hepatic stellate cell (HSC) activation. The low activity of these mono-functional compounds renders them ineffective against the complex, multifactorial pathological milieu of liver fibrosis, resulting in limited efficacy in vivo.

Fig. 2

Strategies for Structural Modification of Chalcone Derivatives

To address the limitations, this study utilizes the natural chalcone skeleton as a lead structure. Through structural modifications, it aim is to synthesize compounds which having both significant anti-inflammatory activity and inhibition of hepatic stellate cell (HSC) activation, achieving synergistic intervention in liver fibrosis through a dual mechanism of action. Hydroxyl and amide groups were introduced into the A ring to enhance hydrogen-bond donor/acceptor capabilities and electronic effects, thereby boosting antioxidant and anti-inflammatory activity. The introduction of electron-withdrawing or electron-donating groups into the B-ring modulates the overall electron distribution of the molecule, thereby optimizing interactions with the target protein (Fig. 2).

Based on this strategy, we designed and synthesized 31 chalcone derivatives, systematically evaluating the most potent compounds for their anti-inflammatory and anti-liver fibrosis activities in vitro and in vivo. We further explored their mechanisms of action, providing potential candidates for treating inflammation and fibrosis associated with chronic liver disease.

2. Results and discussion

2.1 Chemistry

In this study, 5-acetyl-2-hydroxybenzamide was used as the starting material and condensed with various substituted benzaldehydes via a Claisen–Schmidt condensation reaction to obtain 31 chalcone derivatives. The synthetic route of compounds c1- c31 is shown in Scheme 1 and their specific structures are listed in Table 1. In the Claisen-Schmidt condensation reaction, strong alkaline conditions are commonly employed to enhance the reactivity of aldehydes and ketones. However, these conditions are unsuitable for compounds such as 5-acetyl-2-hydroxybenzamide, which contains sensitive functional groups including an amide and an ortho-hydroxyl group. Under high pH, the amide bond is prone to hydrolysis, and the phenolic hydroxyl group deprotonate, leading to side reactions. On the other hand, acid catalysis is typically achieved by directly adding hydrochloric acid. However, this method subjects the reaction system to a transient strong acidic environment, which can easily trigger hydrolysis or side reactions between the amide and hydroxyl groups in 5-acetyl-2-hydroxybenzamide. Additionally, hydrochloric acid introduces excessive moisture into the system, disrupting the anhydrous reaction conditions. This may not only lead to the hydration of the aldehyde substrate, but also reduce the purity of the target chalcone product. In contrast, this study uses HCl generated by the in-situ reaction of SOCl₂ and anhydrous ethanol as the acid source. This can slowly release acid in an anhydrous environment, avoiding the side reactions caused by instantaneous strong acid, while ensuring the uniformity and mildness of the reaction, which is more conducive to the transformation of sensitive substrates and improves the yield and purity of products. All the synthesized compounds were confirmed by ¹H NMR, ¹³C NMR and ESI-MS, and their purity was determined by HPLC.

Scheme 1

Reagents and conditions: ⅰ) SOCl₂, EtOH, rt, 2 h.

Table 1
Summary of specific compound structures
Compd.	R¹	R²	R³	R⁴	R⁵	Compd.	R¹	R²	R³	R⁴	R⁵
c1	H	H	H	H	H	c17	OCH₃	H	H	H	H
c2	F	H	H	H	H	c18	H	OCH₃	H	H	H
c3	H	F	H	H	H	c19	H	H	OCH₃	H	H
c4	H	H	F	H	H	c20	CH₃	CH₃	H	H	H
c5	Cl	H	H	H	H	c21	CH₃	H	CH₃	H	H
c6	H	Cl	H	H	H	c22	CH₃	H	H	CH₃	H
c7	H	H	Cl	H	H	c23	OCH₃	OCH₃	H	H	H
c8	Br	H	H	H	H	c24	OCH₃	H	OCH₃	H	H
c9	H	Br	H	H	H	c25	OCH₃	H	H	OCH₃	H
c10	H	H	Br	H	H	c26	OCH₃	H	H	H	OCH₃
c11	CH₃	H	H	H	H	c27	H	OCH₃	OCH₃	H	H
c12	H	CH₃	H	H	H	c28	OCH₃	OCH₃	OCH₃	H	H
c13	H	H	CH₃	H	H	c29	OCH₃	H	OCH₃	OCH₃	H
c14	C₂H₅	H	H	H	H	c30	OCH₃	H	OCH₃	H	OCH₃
c15	H	C₂H₅	H	H	H	c31	H	OCH₃	OCH₃	OCH₃	H
c16	H	H	C₂H₅	H	H

2.2 Screening of compounds

2.2.1 Inhibitory effects of compounds on LPS-induced NO production in RAW 264.7 macrophages

The pathogenesis of hepatic fibrosis is intimately linked to chronic inflammation, characterized by excessive secretion of inflammatory mediators from hepatic macrophages [27–29]. In this study, we employed an LPS - induced RAW 264.7 macrophage inflammation model to evaluate the anti-inflammatory activity of compounds by measuring the production of nitric oxide (NO).

In order to ensure that the effects observed in the subsequent inflammatory model were due to the pharmacological action of the compound rather than cell death, We evaluated the cytotoxicity of the compounds to RAW 264.7 cells by MTT method and determined the safe concentration for subsequent anti-inflammatory studies. The results showed that almost all compounds did not exhibit significant cytotoxicity at 50 µM for 24 h treatment, and the cell viabilities were more than 80% compared with control. (Fig.S1 in supporting information). The results of MTT experiment showed that the safe concentration of all compounds was higher than 50 µM. Therefore, the anti-inflammatory activity was evaluated in the range of 0–50 µM.

Subsequently, we evaluated the inhibitory effects of the compounds at different concentrations (2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM) on LPS-induced NO release from RAW 264.7 cells using the Griess method [30]. The classical anti-inflammatory drug dexamethasone and the anti-inflammatory compound curcumin with a bichalcone structure were used as positive control drugs. The experimental results showed that compounds c5, c6, c9, c11, c12, c14, c15, c17-20, c22, c23, c25, c27-29, and c31 inhibited the NO expression level in RAW 264.7 cells more than the positive control drug, among which, compound c31 showed the most prominent performance, with the IC₅₀ = 3.05 ± 0.12 µM for NO inhibition (Table 2).

Table 2
The IC₅₀ values of compounds for NO inhibition in LPS-stimulated RAW 264.7 cells
Compd.	IC₅₀ (µM) ^a	Compd.	IC₅₀ (µM) ^a
c1	7.94 ± 0.61	c17	6.54 ± 1.06
c2	8.01 ± 0.22	c18	3.63 ± 0.21
c3	7.36 ± 0.15	c19	7.28 ± 0.98
c4	11.76 ± 0.86	c20	9.28 ± 1.15
c5	5.78 ± 0.49	c21	17.86 ± 1.42
c6	3.86 ± 0.66	c22	8.27 ± 0.52
c7	7.48 ± 0.39	c23	4.02 ± 0.25
c8	> 40	c24	19.94 ± 2.03
c9	4.08 ± 0.60	c25	4.57 ± 0.51
c10	22.91 ± 0.72	c26	> 40
c11	4.16 ± 0.34	c27	3.28 ± 0.45
c12	3.75 ± 0.52	c28	3.40 ± 0.36
c13	16.70 ± 1.20	c29	4.33 ± 0.22
c14	8.08 ± 0.95	c30	13.08 ± 1.14
c15	6.37 ± 0.58	c31	3.05 ± 0.12
c16	14.77 ± 0.67	CUR	12.58 ± 0.76
DXM	6.04 ± 0.98

^aThe IC₅₀ (µM) were the concentrations of compounds that inhibited 50% of NO expression compared to the LPS stimulated group. Values represent mean ± SD from at least three independent experiments.

2.2.2 Inhibitory effects of compounds on TGF-β1-activated HSC-T6 cell proliferation

The activation, proliferation and migration of HSCs are important links in the development of liver fibrosis [31, 32]. Therefore, evaluating the inhibitory effects of a compound on activated hepatic stellate cells (HSCs) is a reliable method for assessing its anti-fibrotic activity.

Here we selected TGF-β-induced HSC-T6 as a hepatic fibrosis cell model and LO2 as a normal hepatocyte model with curcumin as a positive control. The IC₅₀ values of all compounds on the proliferation of activated HSC-T6 were determined by MTT assay. The results are shown in Table 3, all compounds inhibited the proliferation of HSC-T6 activated by TGF-β1 (5 ng/ml) to some extent, with IC₅₀ values ranging from 42.97 µM to 118.87 µM. Among them, compound c31 showed the best inhibitory effect on activated HSC-T6, even slightly better than the activity of curcumin. Although curcumin had better activity against activated HSC-T6, its toxicity to normal hepatocytes LO2 was also higher, with a selectivity factor of only 1.41. In contrast, compound c31 achieved a markedly higher selectivity factor of 3.65, significantly outperforming curcumin. Moreover, c31 exhibits excellent cellular safety without compromising its anti-fibrotic efficacy.

Table 3
The IC₅₀ values of compounds on HSC-T6 cells and LO2 cells.
Compd	IC₅₀ (µM)^a		Selectivity Index^c	Compd	IC₅₀ (µM)^a		Selectivity Index^c
Compd	HSC-T6^b	LO2	Selectivity Index^c	Compd	HSC-T6^b	LO2	Selectivity Index^c
c1	87.54 ± 1.78	168.73 ± 17.10	1.92	c17	78.72 ± 2.75	371.97 ± 12.14	4.73
c2	68.82 ± 1.51	242.63 ± 13.96	3.53	c18	61.56 ± 1.51	202.17 ± 9.26	3.28
c3	66.09 ± 1.41	138.27 ± 4.18	2.09	c19	89.59 ± 3.79	195.67 ± 10.47	2.18
c4	92.83 ± 9.46	129.33 ± 5.09	1.39	c20	93.24 ± 2.17	208.24 ± 4.17	2.33
c5	61.23 ± 4.51	125.28 ± 2.76	2.05	c21	107.61 ± 5.29	314.62 ± 6.40	2.92
c6	57.64 ± 1.74	102.19 ± 7.50	1.77	c22	66.90 ± 1.04	269.18 ± 2.65	4.02
c7	63.57 ± 2.05	132.08 ± 4.57	2.08	c23	59.77 ± 3.96	173.41 ± 4.33	2.90
c8	107.23 ± 2.11	359.19 ± 14.41	3.35	c24	67.23 ± 5.58	133.17 ± 2.68	1.98
c9	70.82 ± 1.61	157.53 ± 12.63	2.22	c25	60.88 ± 3.60	242.11 ± 7.83	3.98
c10	67.54 ± 1.42	129.33 ± 5.09	1.92	c26	140.93 ± 1.05	> 400	> 2.84
c11	104.44 ± 2.52	216.99 ± 16.36	2.08	c27	57.61 ± 0 .47	239.40 ± 7.13	4.15
c12	48.14 ± 1.15	168.73 ± 17.10	3.50	c28	49.14 ± 1.83	210.29 ± 2.39	4.28
c13	110.21 ± 3.48	346.72 ± 12.21	3.15	c29	58.92 ± 4.08	192.94 ± 3.28	3.27
c14	64.62 ± 2.03	159.17 ± 6.52	2.46	c30	113.37 ± 4.59	323.15 ± 6.56	2.85
c15	58.71 ± 1.27	114.83 ± 3.26	1.96	c31	42.97 ± 0.93	157.23 ± 3.79	3.66
c16	103.38 ± 5.91	241.18 ± 2.77	2.33	CUR	45.79 ± 0.78	64.74 ± 7.87	1.41

^a The IC₅₀ values represent mean ± SD from at least three independent experiments.

^b HSC-T6 cells were activated by TGF-β1 (5 ng/mL).

^c Selectivity index, representing the ratio of the IC50 value of LO2 cells to the IC₅₀ value of activated HSC-T6 cells.

Based on the chalcone backbone, the relationship between compound structure and anti-inflammatory and inhibition of activated HSC-T6 activity was proposed (Fig. 3).The in vitro anti-inflammatory activity and anti-liver fibrosis activity of compounds exhibit similar structure-activity relationships. Specifically, compounds with strong anti-inflammatory activity in vitro (such as c6, c12, c27, c28, and c31) also demonstrate pronounced inhibitory effects on the proliferation of activated HSC-T6 cells.When the B-ring in the chalcone structure is monosubstituted (except for bromo-substituted compounds c8-c10), the anti-inflammatory and anti-liver fibrosis activities of the compounds follow the order:meta-position > ortho-position > para-position. When the B-ring is di-substituted with electron-donating groups (such as methyl or methoxy), the compounds with meta-substitution also exhibit significantly activities, and are higher than those without meta-substitution. Compound c31 exhibited the strongest in vitro anti-inflammatory and anti-liver fibrosis activities. Therefore, we selected compound c31 for further investigation of its anti-inflammatory and anti-liver fibrosis activities.

Fig. 3

Conformational relationships for in vitro anti-inflammatory and anti-liver fibrosis activities of compounds

2.3 Evaluation of in vitro anti-fibrotic activity of compound c31

2.3.1 Anti-proliferative activity of compound c31 against activated HSC-T6 cells

The regulation of the essential functions of HSCs has been the most critical event in the process of liver fibrosis. HSCs activated by TGF-β1 and LPS induce fibrosis through increased proliferation, excessive deposition of ECM and expression of pro-fibrotic cytokines. Inhibiting the activation and proliferation of HSCs and promoting the apoptosis of activated HSCs can inhibit or even reverse liver fibrosis [33].

To further study the anti-proliferative activity of c31 against TGF-β1-induced HSC-T6, we first evaluated its time- and dose-dependent effects following treatment. As shown in Fig. 4A,the inhibitory effect of c31 on cell viability had a significant dose-dependent and time-dependent trend. The cell viability curve at 24 h showed that compound c31 did not exhibit significant cytotoxicity at concentrations below 30 µM, and the cell survival rate was above 80%. Consequently, to avoid significant cytotoxicity, subsequent scratch wound, migration, and colony formation assays were performed after treating the cells with compound c31 (10, 20, or 30 µM) for 24 hour

Then, the proliferation of TGF-β1-induced HSC-T6 cells treated with compound c31 was observed more accurately by the colony formation assay. As illustrated in Fig. 4B, TGF-β1 treatment was able to promote the proliferation of HSC-T6 with a significant increase in the number of colonies. Compound c31 inhibited colony formation in a concentration-dependent manner, indicating that the compound effectively inhibited TGF-β1-induced proliferation of HSC-T6 cells.

To further investigate the mechanism of the anti-cell proliferation activity of compound c31, the effect of c31 on the cycle progression of TGF-β1-induced HSC-T6 cells was examined by flow cytometry. As depicted in Fig. 4C,compound c31 blocked the cell cycle of TGF-β1-induced HSC-T6 cells in a dose-dependent manner in the G2/M phase, and the proportion of activated HSC-T6 cells in the G2/M phase increased from 15.50% to 41.29% as the c31 concentration increased from 0 µM to 60 µM. In sum, compound c31 may inhibit the proliferation of activated HSC-T6 cells by inducing their cycle arrest in the G2/M phase.

Fig. 4

The effects of compound c31 on the proliferation ability of TGF-β1-induced HSC-T6 cells. The cells were incubated with TGF-β1 (5 ng/mL) for 12 h, then compound c31 was added and continued to incubate for 24 h. (A) The cell viability curves of activated HSC-T6 cells. (B) The formation of colonies of activated HSC-T6 cells treated with compound c31. (C) The cell cycle distribution of HSC-T6 was recorded with flow cytometry and quantitatively analyzed.

2.3.2 Pro-apoptotic effects of compound c31 on activated HSC-T6 cells

Apoptosis-induced programmed cell death can reduce the number of activated HSC-T6 cells and serves as a valuable target for the treatment of liver fibrosis [34]. Through Gimsa staining, it was found that the cells in the c31 treatment group showed deepening of nuclear staining, nuclear condensation and fragmentation, accompanied by the formation of apoptotic bodies, while the nuclei of the control group were intact and arranged regularly (Fig. 5A). These results suggested that c31 could induce apoptosis. To further verify whether compound c31 induced apoptosis in TGF-β1-actived HSC-T6 cells, the apoptosis induced effect was determined with annexin V/PI apoptotic staining. After treatment with different concentrations of compound c31, the percentage of activated HSC-T6 cells with early apoptosis increased from 1.24% to 13.12% and the percentage of cells with late apoptosis increased from 4.71% to 27.34% (Fig. 5B), indicating that compound c31 effectively induced the apoptosis of activated HSC-T6.

Fig. 5

The compound c31 induced apoptosis in TGF-β1-induced HSC-T6 cells. The HSC-T6 cells were pre-incubated with TGF-β1 (5 ng/mL) for 12 h before the treatment with compound c31 for 24 h. (A) Geimsa staining assay manifested the morphological changes in HSC-T6 cells (magnification 20×, scale bar = 100 µm). (B) The types and proportions of apoptosis were measured in activated HSC-T6 cells by flow cytometry. Data represent the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Apoptosis is a programmed process, and its regulatory mechanisms mainly involve mitochondria-dependent endogenous pathways and extracellular signal-induced signaling pathways [35]. To determine whether compound c31-induced apoptosis is associated with the mitochondrial pathway, TMRE was used as a probe to detect mitochondrial membrane potential (MMP). As illustrated in Fig. 6A, c31 caused a significant change in the MMP of TGF-β1-induced HSC-T6 cells, with the proportion of TMRE-positive cells decreasing from 96.39% to 48.00% in a concentration -dependent manner. The above phenomenon demonstrated that c31-induced apoptosis in activated HSC-T6 cells may be completed through a mitochondria-mediated pathway.

Mitochondria-associated apoptotic signaling is regulated by a balance between pro-apoptotic proteins, such as Bax, and anti-apoptotic proteins, such as Bcl-2[36]. Based on the MMP-related pro-apoptotic profile of compound c31, the effect of c31 on the mitochondria-mediated apoptotic pathway was further confirmed by western blot assay. Compound c31 at concentrations of 20 µM, 40 µM, and 60 µM elicited a dose-dependent upregulation of the pro-apoptotic proteins Bax, cytochrome C, and apoptotic effector cleaved caspase-3, and a downregulation of the anti-apoptotic protein Bcl-2. (Fig. 6B). The evident results above proved that compound c31 mediated the apoptosis of activated HSC-T6 cells through the mitochondrial pathway.

Fig. 6

Effects of compound c31 on mitochondrial pathway-mediated apoptosis in activated HSC-T6 cells. (A) Changes in mitochondrial membrane potential (ΔΨm) in activated HSC-T6 cells after treatment with compound c31; (B) Expression levels of apoptosis-related proteins (Bcl-2, Bax, cytochrome C, cleaved-caspase-3) in activated HSC-T6 cells following compound c31 treatment. All data represent mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 indicate statistically significant differences compared with TGF-β1 group; ns denotes no significant difference versus blank control group.

2.3.3 Anti-migratory effects of compound c31 on activated HSC-T6 cells

In liver injury, activated hepatic stellate cells (HSCs) migrate toward the site of damage in response to chemokine signaling, thereby exacerbating the progression of liver fibrosis [37]. Studies have indicated that the migration of HSCs serves an important role in liver fibrosis, including ECM and growth factor production [38].

The inhibitory effect of compound c31 on the migration of activated HSC-T6 cells was evaluated using scratch wound and transwell assays. The results of the scratch assay (Fig. 7A) showed that the wound of the TGF-β1 induced-HSC-T6 cell group was almost healed, while migration of c31-treated cells was significantly inhibited in a dose-dependent manner. Meanwhile, in the transwell migration assay (Fig. 7B), the number of cells migrating into the lower compartment in the c31-treated group was markedly less than that in the TGF-β1- induced group. Based on the aforementioned results, treatment with compound c31 at varying concentrations significantly inhibited the migration capacity of TGF-β1-induced HSC-T6 cells.

Fig. 7

c31 induced migration in TGF-β1-actived HSC-T6 cells. (A) Scratch test results of activated HSC-T6 cells before and after treatment with c31 (magnification 10×, scale bar = 100 µm). (B) Transwell migration assay results of activated HSC-T6 cells before and after treatment with c31 (magnification 10×, scale bar = 100 µm). (C) Statistical analysis of the scratch results was performed by Image J. (D) Statistical analysis of the number of cells passing through the Transwell chamber. Data represent the mean ± SD. Curcumin is represented by CUR ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Fig. 7

Compound c31 induced migration in TGF-β1-actived HSC-T6 cells. (A) Scratch test results of activated HSC-T6 cells before and after treatment with c31 (magnification 10×, scale bar = 100 µm). (B) Transwell migration assay results of activated HSC-T6 cells before and after treatment with c31 (magnification 10×, scale bar = 100 µm). (C) Statistical analysis of the scratch results was performed by Image J. (D) Statistical analysis of the number of cells passing through the Transwell chamber. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

2.3.4 Compound c31 suppresses HSC-T6 activation via α-SMA /Col1α1 /TGF-β1 downregulation

The main role of normal HSCs is to store fat and vitamin A. However, their activation leads to excessive deposition of ECM proteins, which is a feature of liver fibrosis [39]. Production of α-smooth muscle actin (α-SMA) and type I collagen α-1 (Col1α1) are the most common hallmarks for identifying activated HSCs [40]. In addition, activated HSCs release an excess of the pro-fibrotic cytokine TGF-β1, which promotes the proliferation, migration and ECM production of activated HSCs [41].

To evaluate the effect of c31 on HSC-T6 cell activation, the action of c31 on α-SMA, Col1α1 and TGF-β1 expression in LPS-induced HSC-T6 cells was detected with ELISA kits. As shown in Fig. 8A-C, the levels of α-SMA, Col1α1 and TGF-β1 were considerably increased in HSC-T6 cells after LPS (10 µg/mL) stimulation. But after c31 treatment at concentration gradients (10 µM, 20 µM, and 30 µM), the expression levels of the above three proteins were all remarkably reduced.These findings demonstrate that c31 attenuates liver fibrogenesis through suppression of HSC-T6 activation and extracellular matrix deposition.

Fig. 8

The effect of c31 on activation of TGF-β1-induced HSC-T6 cells. The HSC-T6 cells were pre-incubated with TGF-β1 (5 ng/mL) for 12 h before the treatment with compound c31 for 24 h. (A - C) The expression levels of fibrosis-related proteins α-SMA, Col1α1 and TGF-β1 in activated HSC-T6 cells treated with c31. Data represent the mean ± SD. Curcumin is represented by CUR ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Fig. 8

Compound c31 effect on activation of TGF-β1-induced HSC-T6 cells. The HSC-T6 cells were pre-incubated with TGF-β1 (5 ng/mL) for 12 h before the treatment with compound c31 for 24 h. (A - C) The expression levels of fibrosis-related proteins α-SMA, Col1α1 and TGF-β1 in activated HSC-T6 cells treated with c31. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

2.4 Mechanistic study on the anti-fibrotic effects of c31

2.4.1 Regulatory effects of c31 on inflammatory factor expression in RAW 264.7 macrophages

During the inflammatory response that accompanies the development of liver fibrosis, hepatic macrophages are recruited to the site of liver injury, producing large amounts of inflammatory mediators such as COX-2, TNF-α, and IL-1β, leading to overactive liver inflammation and intensifying the severity of liver fibrosis [42, 43]. Thus, To further investigate whether c31 exerts its anti-inflammatory effects by modulating macrophage function,the effect of c31 on the secretion of three cytokines, TNF-α, IL-6 and IL-1β, and the expression of two inflammatory proteins, iNOS and COX-2, was determined in LPS-induced RAW 264.7 cells.

First, the levels of TNF-α, IL-6 and IL-1β in the cell supernatant were measured using the ELISA kit.The levels of TNF-α, IL-6 and IL-1β decreased in a dose-dependent manner after 24 h of c31 treatment, with IC₅₀ values of around 21.35 ± 0.67 µM for TNF-α, 13.12 ± 1.21 µM for IL-6 and 19.43 ± 1.21 µM for IL-1β.(Fig. 9A-C) Next, the western blot experiment was performed to examine the expression of intracellular inflammatory proteins iNOS and COX-2. c31 had a significant inhibitory effect on the expression of iNOS and COX-2 proteins as well (Fig. 9D-E). Therefore, c31 inhibits inflammation by reducing the levels of critical inflammatory factors, namely TNF-α, IL-6, IL-1β, iNOS, and COX-2.

Fig. 9

The effects of c31 on production of inflammatory mediators in LPS-induced RAW 264.7 cells. The cells were incubated with LPS (1 µg/mL) and c31 for 24 hours. (A-C) The concentrations of TNF-α, IL-6 and IL-1β were measured by the ELISA kit. (D&E) The proteins expressions of iNOS and COX-2 were detected by Western Blot. All data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs the LPS group; ^###p < 0.001 vs the Control group.

2.4.2 Inhibition of c31 on the JNK/NF-κB pathway

The NF-κB signaling pathway plays a pivotal role in regulating cell death, inflammation, and tissue repair, serving as a central mechanism in maintaining liver homeostasis and managing the inflammatory-fibrotic-cancer progression [44]. By activating macrophages and hepatic stellate cells while promoting the production of pro-inflammatory factors and extracellular matrix (ECM), it perpetuates a vicious cycle of inflammation and fibrosis[3].

To verify whether c31 ameliorates liver inflammation and liver fibrosis by inhibiting the JNK/NF-κB signaling pathway, the effect of c31 on the JNK/NF-κB pathway in RAW 264.7 cells and HSC-T6 cells was examined by western blot assay. As shown in Fig. 10A-D, IKBα and NF-κB p65 subunit phosphorylation levels were significantly increased and IKBα was degraded in RAW 264.7 cells after stimulation by LPS (10 µg/mL). In RAW264.7 cells, c31 demonstrated dose-dependent reversal of LPS-induced IκBα degradation and IκBα/NF-κB p65 phosphorylation, while significantly inhibiting JNK phosphorylation (Fig. 10E-F). In HSC-T6 cells, c31 also exhibited dose-dependent inhibition of LPS-induced IκBα degradation and phosphorylation of IκBα, NF-κB p65, and JNK (Fig. 11A-F). Additionally, c31 markedly reduced LPS-induced COX-2 expression in both cell lines (Fig. 9E,11G-H). These results suggest that c31 can reduce liver inflammation and fibrosis by inhibiting the activation of hepatic macrophages and hepatic stellate cells through inhibition of JNK/NF-κB signaling pathway.

Fig. 10

Effect of c31 on JNK/NF-κB pathway in RAW264.7 cells induced by LPS. The RAW264.7 cells were pre-incubated with LPS (10 µg/mL) for 12 h before the treatment with c31 for 24 h. (A - F) Expression of NF-κB p65, phosphorylated NF-κB p65, IκBα, phosphorylated IκBα, JNK and phosphorylated JNK protein in RAW264.7 cells treated with c31. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Fig. 11

Effect of c31 on LPS-induced JNK/NF-κB pathway in HSC-T6 cells. (A-H) Expression of NF-κB p65, phosphorylated NF-κB p65, IκBα, phosphorylated IκBα, JNK, phosphorylated JNK protein and COX-2 protein after treatment of activated HSC-T6 cells with c31. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

2.4.4 Molecular docking of c31 with target proteins

In order to further explore the interactions between c31 and key proteins, a computer simulation model of protein-ligand complexes was constructed using Discovery Studio software. The binding modes of c31 with NF-κB (PDB: 8TQD) [45], JNK (PDB: 3ELJ) [46], COX-2 (PDB: 5IKT) [47] and iNOS (PDB: 3E7G) [48] protein binding modes were then analyzed.

Molecular docking results demonstrate that c31 forms stable interactions with multiple targets. Within the NF-κB protein, c31 establishes hydrogen bond networks with key residues including Gln98, Leu99, Glu119, Asp120, Gly121, Thr124, Arg156, and Arg163, indicating strong binding affinity (Fig. 12A). In the JNK kinase pocket, c31 forms hydrogen bonds with residues such as Ala113, Met111, and Asn156, while also creating hydrophobic interactions with Ile32 and Val158, thereby enhancing binding stability (Fig. 12B). Regarding COX-2, c31 forms hydrogen bonds with residues including Ser78, Gln96, and Asn81, deeply embedding into the active site to exhibit higher binding stability (Fig. 12C). Additionally, the methoxy and amino groups of c31 can establish stable hydrogen bonds with Gln371 and Glu377 in iNOS, demonstrating high affinity for the iNOS active site (Fig. 12D).

In summary, c31 can stably bind to the active sites of several key target proteins. During the binding process, it forms hydrogen bonds with various amino acid residues and has hydrophobic interactions with hydrophobic residues. This significantly enhances the binding stability with target proteins and reveals its potential molecular mechanism of action.

Fig. 12

The schematic diagram of the binding between c31 and different target proteins shows that green dashed lines represent hydrogen bonds, while pink dashed lines indicate hydrophobic interactions. (A) c31 in complex with NF-κB (PDB:8TQD), (B) c31 in complex with JNK (PDB:3ELJ) (C) c31 in complex with COX-2(PDB:5IKT) (D) c31 in complex with iNOS (PDB:3E7G)

2.5 Compound c31 against CCl₄ -induced hepatic fibrosis in vivo

2.5.1 Inhibition of c31 on the CCl₄-induced liver fibrosis in rats

CCl₄ can induce various hepatic histopathological changes in vivo [49], and its induced liver injury model has been widely used in the research of hepatoprotective drugs and anti-fibrosis candidate molecules [50]. This study selected this model to preliminarily evaluate the hepatoprotective and anti-fibrotic potential of c31. Acute toxicity test results showed that mice administered a single oral dose of 2000 mg/kg of c31 exhibited no mortality and significant behavioral abnormalities. Histopathological examination revealed no notable pathological changes in major organs (Fig. 13). These findings indicate that the oral LD₅₀ at this dose exceeds 2000 mg/kg. Based on these results, the experimental dose for in vivo administration was determined to be 100 mg/kg.

Fig. 13

Effect of c31 on major organs after 14 days of single-dose gavage (magnification: 20 ×, scale bar: 100 µm).

The H&E staining results (Fig. 14A) showed that the hepatic lobules of vehicle treated control group were structurally intact and the hepatocytes were neatly arranged in a cord-like pattern. After CCl₄ treatment, liver tissues showed severe damage, including disordered hepatocyte architecture, ballooning degeneration and hepatocellular necrosis, along with infiltration of fibroblasts and inflammatory cells around the hepatic lobules. These pathological changes were markedly ameliorated in the livers of rats treated with c31. Masson trichrome staining results (Fig. 14B) indicated that high-density collagen staining (blue portion) and fibrotic intervals with significant proliferation of collagen fibers were seen around the central vein of the liver in CCl₄-treated rats. However, c31 treatment significantly reduced collagen deposition and attenuated liver fibrosis, with pathological changes almost restored to the control level.

Fig. 14

Effect of c31 on CCl₄-induced liver fibrosis in rats. (A) H&E staining and Masson trichrome staining results of liver sections, magnification: 20 ×, scale bar: 100 µm, red arrows indicate cell swelling, necrosis, and inflammatory cell infiltration, black arrows indicate fiber and collagen deposition; (B-C) levels of α-SMA and Col1α1 protein in liver; (D-E) ALT and AST levels in serum. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

The expression of α-SMA and COL1α1 was significantly increased in rat liver after CCl₄ treatment, while c31 at a dose of 100 mg/kg significantly down-regulated the levels of these two collagens (Fig. 14C). Moreover, serum ALT and AST are important biochemical indicators of liver injury accompanied with fibrosis. The data in Fig. 14D - E showed serum ALT and AST levels were remarkably rise induced by CCl₄, but this was reversed after c31 treatment. The results showed that c31 could effectively reduce CCl₄ induced liver injury and fibrosis, showing significant anti-fibrotic potential in vivo.

Fig. 14

Effect of c31 on CCl₄-induced liver fibrosis in rats. (A) H&E staining and Masson trichrome staining results of liver sections, magnification: 20 ×, scale bar: 100 µm, red arrows indicate cell swelling, necrosis, and inflammatory cell infiltration, black arrows indicate fiber and collagen deposition; (B-C) levels of α-SMA and Col1α1 protein in liver; (D-E) ALT and AST levels in serum. Data represent the mean ± SD. Curcumin is represented by CUR ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

2.5.2 Inhibition of c31 on inflammation in CCl₄-induced liver fibrosis model in rats

Liver inflammation is known to be closely associated with the progression of chronic liver disease and liver fibrosis [51]. To further investigate whether c31 can ameliorate hepatic inflammation associated with liver fibrosis in vivo, we measured the levels of inflammatory factors in the serum of rats from each group using ELISA kits. As shown in Fig. 15A-D, in the CCl₄ model group, serum levels of inflammatory mediators such as TNF-α, IL-6, and IL-1β were significantly elevated compared to the blank control group. These findings indicate that CCl₄-induced rat liver fibrosis is accompanied by pronounced inflammatory responses. c31 exhibited a superior inhibitory potency over curcumin in reducing the levels of these inflammatory factors, particularly IL-6, compared to the CCl₄ model group, a finding that aligns with the results from our in vitro studies.These results indicate that c31 effectively alleviates liver inflammation associated with fibrosis.

Fig. 15

Effect of c31 on serum cytokines in CCl₄-induced liver fibrosis model in rats. (A-D) Serum levels of cytokines such as TNF-α, IL-6, IL-1β and TGF-β1. Data represent the mean ± SD.Curcumin is represented by CUR ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Fig. 15

Effect of c31 on serum cytokines in CCl₄-induced liver fibrosis model in rats. (A-D) Serum levels of cytokines such as TNF-α, IL-6, IL-1β and TGF-β1. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

3. Conclusion

In conclusion, we synthesized 31 chalcone derivatives and evaluated their anti-inflammatory and anti-fibrotic activities. The mechanism of the anti- inflammatory and anti-liver fibrosis activities of c31 is shown in Fig. 16. In cellular model assays, c31 exhibited stronger anti-inflammatory activity, inhibitory activity of HSCs and lower toxicity than others. Firstly, c31 inhibited the inflammatory response in vitro by downregulating the levels of TNF-α, IL-6, IL-1β, iNOS and COX-2 in macrophage RAW 264.7 cells. Secondly, c31 inhibited the proliferation of activated HSC-T6 cells through cycle arrest and induced apoptosis of activated HSC-T6 cells via mitochondrial pathway. Thirdly, c31 significantly inhibited the activation of HSC-T6 cells by reducing the expression of hepatic fibrosis markers α-SMA and COL1α1 protein as well as the pro-fibrotic cytokine TGF-β1. Afterwards, Western blot analysis revealed that c31 inhibited the phosphorylation of JNK, IKBα and NF-κB p65 subunits and the degradation of IKBα protein in RAW 264.7 cells and HSC-T6 cells, indicating that compound-induced activation and in vitro inflammation in HSC-T6 cells are likely related to the JNK/NF-κB signaling pathway. Furthermore, in a CCl₄-induced liver fibrosis mouse model, oral administration of c31 for two weeks significantly alleviated liver fibrosis and the associated inflammatory response, indicating that c31 possesses promising anti -fibrotic potential and may serve as a candidate compound for further investigation and development.

Fig. 16

Schematic diagram of the anti-inflammatory and anti-liver fibrotic mechanism of c31.

Fig. 16

Schematic diagram of the anti-inflammatory and anti-liver fibrotic mechanism of c31.

Synthesis and anti-liver fibrotic activity study of a chalcone derivative through anti-inflammatory effects and inhibition of JNK/NF-κB signaling pathways

Chunwei Lv^1,#, Lei Zhang^2,#, Chenxu Wang¹,Tingting Jin¹,Zhishun Zhang¹,Yixi Li¹, Dian He¹*, Quanyi Zhao¹, Lifang Zheng¹

(¹ Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University, Lanzhou 730000, China.² Gansu Medical Device Inspection And Testing Institute,Lanzhou 730000, China.)

^*Corresponding author: hed@lzu.edu.cn (Dian He).

^# These authors contributed equally to the work

4. Experimental section

4.1 Chemistry

All chemicals used are of analytical or chemical grade. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were recorded using a Bruker AVANCE III 400 spectrometer (Bruker Company, Germany). Chemical shifts (δ) are quoted in ppm relative to tetramethylsilane (TMS) as an internal standard, where (δ) TMS = 0.00 ppm. The mass spectra were recorded on a Bruker Daltonics APEXII49e spectrometer (Bruker Company, Germany) with an ESI source as the ionization source. The purity of all compounds was characterized by HPLC analysis (Hitachi Chromaster system) on a Hitachi Lachrom C18 (4.6 × 250 mm, 5 µm) column with an injection volume of 10 µL and detection at 254 nm. The target compounds were eluted with methanol: water (80:20, v/v) as the mobile phase at a flow rate of 1 mL/min. The purity of the measured compounds was ≥ 95%.

4.1.1 General procedure for the synthesis of compounds

The compounds c1 - c31 were prepared as follows: 5mmol of 5-acetyl − 2-hydroxybenzamide and 5mmol of different substituted benzaldehyde were dissolved in 10 ml of anhydrous ethanol, and the reaction solution was stirred while slowly adding 1.0 ml of thionyl chloride (SOCl₂) dropwise at room temperature (25 ℃) for 2 h. The stirring was stopped, and the reaction solution was left to stand for 12 h. The reaction solution was then filtered and the filter cake was dried after being rinsed twice with ice-cold ethanol. The mixture was cooled and allowed to cool down to room temperature naturally. After adding acetone to dissolve the filter cake and remove the insoluble material, the excess solvent was removed by spin evaporation, the crude product was purifed by silica gel column chromatography.

4.1.2 Characterization and Purity Analysis of Synthesized Compounds

This article only lists the NMR and HRMS data for some of the compounds. The complete spectra and related characterization data of all compounds are provided in the Supplementary Information.

(E)-2-hydroxy-5-(3-(o-tolyl)acryloyl)benzamide ( c11 ).

Pale yellow solid, yield: 68.2%.¹H NMR (400 MHz, DMSO-d₆) δ 13.99 (s, 1H), 8.80 (s, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.26–8.15 (m, 2H), 8.03–7.93 (m, 2H), 7.87 (d, J = 15.4 Hz, 1H), 7.39–7.25 (m, 3H), 7.05 (d, J = 8.7 Hz, 1H), 2.44 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.22, 172.30, 166.07, 141.00, 138.40, 134.82, 133.97, 131.28, 130.75, 130.09, 129.00, 127.21, 126.81, 123.12, 118.76, 114.21, 19.78. ESI-MS: calcd. for C₁₇H₁₅NO₃ [M + H]⁺ 282.1124, found 282.1193.

(E)-2-hydroxy-5-(3-(m-tolyl)acryloyl)benzamide ( c12 ).

White solid,yield:37.8%.¹H NMR (400 MHz, DMSO-d₆) δ 13.98 (s, 1H), 8.81 (s, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.29–8.15 (m, 2H), 7.95 (d, J = 15.5 Hz, 1H), 7.76–7.67 (m, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 2.37 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.19, 172.31, 166.02, 144.06, 138.65, 135.21, 134.83, 131.73, 130.06, 129.54, 129.27, 129.04, 126.64, 121.95, 118.67, 114.27, 21.38. ESI-MS: calcd. for C₁₇H₁₅NO₃ [M + H]⁺ 282.1124, found 282.1191.

(E)-2-hydroxy-5-(3-(3-methoxyphenyl)acryloyl)benzamide ( c18 ).

Pale pink solid, yield: 77.3%.¹H NMR (400 MHz, DMSO-d₆) δ 13.98 (s, 1H), 8.83 (s, 1H), 8.70 (d, J = 2.2 Hz, 1H), 8.22 (dd, J = 8.8, 2.1 Hz, 2H), 7.97 (d, J = 15.5 Hz, 1H), 7.73 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 6.8 Hz, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 3.84 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.20, 172.23, 166.15, 160.12, 143.84, 136.68, 134.88, 130.43, 130.18, 128.88, 122.49, 121.75, 118.69, 116.71, 114.40, 114.37, 55.77. ESI-MS: calcd. for C₁₇H₁₅NO₄ [M + H]⁺ 298.1073, found 298.1179.

(E)-5-(3-(2,5-dimethoxyphenyl)acryloyl)-2-hydroxybenzamide ( c25 ).

Pale yellow solid, yield: 78.7%.¹H NMR (400 MHz, DMSO-d₆) δ 13.92 (s, 1H), 8.87–8.63 (m, 2H), 8.18 (t, J = 9.2 Hz, 2H), 8.11–7.98 (m, 1H), 7.97–7.83 (m, 1H), 7.53 (m, 1H), 7.05 (m, 3H), 3.82 (d, J = 16.9, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.44, 173.17, 165.94, 154.71, 153.62, 138.54, 135.40, 131.51, 129.68, 125.78, 123.13, 119.74, 117.79, 116.20, 113.97, 113.05, 58.47, 56.18. ESI-MS: calcd. for C₁₈H₁₇NO₅ [M-H]⁻ 326.1035, found 326.1036.

(E)-5-(3-(3,4-dimethoxyphenyl)acryloyl)-2-hydroxybenzamide ( c27 ).

Pale yellow solid, yield: 78.7%.¹H NMR (400 MHz, DMSO-d₆) δ 13.90 (s, 1H), 8.79 (s, 1H), 8.69 (d, J = 2.1 Hz, 1H), 8.26–8.15 (m, 2H), 7.84 (d, J = 15.4 Hz, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.51 (s, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 3.85 (d, J = 15.6 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.22, 172.26, 165.72, 151.67, 149.45, 144.38, 134.83, 130.01, 129.30, 128.09, 123.86, 119.87, 118.43, 114.47, 112.12, 111.78, 56.23, 56.09. ESI-MS: calcd. for C₁₈H₁₇NO₅ [M + Na]⁺ 350.0999, found 350.1010.

(E)-2-hydroxy-5-(3-(2,3,4-trimethoxyphenyl)acryloyl)benzamide ( c28 ).

Pale yellow solid, yield: 70.5%.¹H NMR (400 MHz, DMSO-d₆) δ 13.91 (s, 1H), 8.78 (s, 1H), 8.67 (s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 15.6 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.88 (d, J = 3.3 Hz, 6H), 3.79 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.25, 172.26, 165.77, 156.17, 153.49, 142.27, 138.16, 134.72, 129.92, 129.28, 123.37, 121.65, 120.61, 118.57, 114.35, 108.90, 62.04, 60.93, 56.54. ESI-MS: calcd. for C₁₉H₁₉NO₆ [M-H]⁻ 356.1140, found 356.1147.

(E)-2-hydroxy-5-(3-(2,4,5-trimethoxyphenyl)acryloyl)benzamide ( c29 )

Yellow solid, yield: 67.3%.¹H NMR (400 MHz, DMSO-d₆) δ 13.83 (s, 1H), 8.82–8.61 (m, 2H), 8.26–8.17 (m, 1H), 8.13 (s, 1H), 8.05 (d, J = 15.5 Hz, 1H), 7.74 (d, J = 15.7 Hz, 1H), 7.50 (d, J = 4.4 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.75 (d, J = 4.3 Hz, 1H), 3.99–3.80 (m, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 187.87, 172.80, 166.08, 155.29, 154.00, 144.09, 138.83, 135.32, 130.46, 130.09, 119.54, 118.86, 115.44, 115.08, 112.28, 98.63, 57.60, 57.43, 56.86. ESI-MS: calcd. for C₁₉H₁₉NO₆ [M-H]⁻ 356.1140, found 356.1133.

(E)-2-hydroxy-5-(3-(3,4,5-trimethoxyphenyl)acryloyl)benzamide (c31).

Pale yellow solid, yield: 78.7%.¹H NMR (400 MHz, DMSO-d₆) δ 13.91 (s, 1H), 8.76 (s, 1H), 8.69 (d, J = 2.2 Hz, 1H), 8.26 (dd, J = 8.7, 2.1 Hz, 1H), 8.19 (s, 1H), 7.89 (d, J = 15.5 Hz, 1H), 7.72 (d, J = 15.4 Hz, 1H), 7.23 (s, 2H), 7.06 (d, J = 8.7 Hz, 1H), 3.88 (s, 6H), 3.73 (s, 3H). ¹³C NMR (100 MHz, DMSO) δ 187.37, 172.20, 165.75, 153.60, 144.35, 140.25, 134.93, 130.82, 130.17, 121.60, 118.36, 114.63, 107.04, 65.49, 60.63, 56.63, 30.47, 19.12, 14.00. ESI-MS: calcd. for C₁₉H₁₉NO₆ [M + H]⁺ 358.1291, found 358.1356.

4.2 Biology assays

4.2.1 Cell lines and cell cultures

RAW 264.7, HSC-T6, LO2 cell lines were purchased from the China Center for Type Collection (CCTCC, China) with STR identification. All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mLstreptomycin at 37°C in a humidified atmosphere containing 5% CO₂. To ensure stable cell status and good viability, subsequent experiments were conducted using cells digested and passaged with trypsin at passages 5 to 10.The concentration of dimethyl sulfoxide (DMSO) was below 0.5% in all treated cells (control group and administration group).

4.2.2 Cell viability assay

Cell viability was measured by MTT assay. Cells were inoculated into 96-well plates at 6×10³ cells/well, and after 12 h incubation, the compounds were added at final concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 µM for treatment. Of which, HSC-T6 cells were pretreated with 5 ng/mL TGF-β1 for 12 h to activate the cells before adding the compounds. After 24 h of compound treatment, 10 µL MTT reagent (5 mg/mL) was added and further incubated for 4 h. Then, the supernatant was discarded and 100 µL DMSO was added to dissolve formazone crystals. The absorbance was measured at 570 nm using a microplate reader (TECAN spark, Switzerland).

4.2.3 NO level assay in RAW 264.7 cells

RAW 264.7 cells were inoculated in 24-well plates (7×10⁴ cells/well). After 12 h of incubation, cells were treated with 1 µg/mL of LPS and final concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 µM compounds for 24 h. Cell culture supernatants were collected and NO levels were assayed with Griess reagent (1% p-anilinesulfonic acid and 0.5% naphthyl ethylenediamine dihydrochloride in 5% phosphoric acid). The optical density was measured at 540 nm using a microplate reader (TECAN Spark, Switzerland).

4.2.4 Colony formation assay

HSC-T6 cells were plated into six-well plates with 1×10³ cells per well. After the cells were cultured overnight, TGF-β1 was added to continue the incubation for 12 h. The cells were treated with different concentrations (10, 20, 30 µM) of c31 and 20 µM curcumin for 1–2 weeks until the colony formation. After that, the medium was discarded and the cells were fixed with 4% paraformaldehyde for 15 min before stained with 1% crystalline violet dye. Finally, observe and photograph under an optical microscope (Moticam Pro 282B, China).

4.2.5 Cell cycle analysis

HSC-T6 cells (7×10⁴ cells/well) were seeded into six-well plates and maintained overnight and then co-cultured with c31 at the indicated concentrations (20, 40, 60 µM) and curcumin (40 µM) for 24 h before pretreatment with TGF-β1 (5 ng/mL) for 12 h. Thereafter, the cells were collected and washed twice with pre-chilled PBS before adding 500 µL of DNA staining solution and 5 µL permeabilization solution, and next incubated for 30 min in the dark at 37°C. The cell cycle was monitored using a cell death detection kit (Caisun Innovation Bio- Technology Co., Ltd., Beijing, China) and the samples were processed accordingly. The cell cycle analysis was detected by flow cytometry (Beckman, Germany). ModFit LT 4.0 software was employed for processing statistical analysis.

4.2.6 Giemsa staining observation

HSC-T6 in the quantity of 7×10⁴ cells/well was incubated overnight in six-well plates and then pretreated with TGF-β1 (5 ng/mL) for 12 h. c31 (20, 40, 60 µM) and curcumin (40 µM) were added for 24 h of treatment. And then the cells were rinsed twice with PBS and fixed with 4% paraformaldehyde for 15 min. After dyeing with diluted Giemsa solution for 15 minutes at room temperature, cells were flushed with water every two minutes and cell morphology was closely observed under a light microscope (Moticam Pro 282B, China).

4.2.7 Cell apoptosis detection

In six-well plates, HSC-T6 cells (7 × 10⁴ per well) were inoculated and cultured until the cells were attached to the wall. Following pretreatment with 5 ng/mL TGF-β1 for 12 h, cells were incubated with different concentrations (20, 40, 60 µM) of c31 and 40 µM curcumin for 24 h. Subsequently, the cells were collected, washed with cold PBS three times, and resuspended in 100 µL 1 × Binding Buffer. And then, 5 µL Annexin V and 10 µL PI (Yeasen, Cat. #: 40302ES60, China) were added sequentially for incubation in the dark for 15 min. After dilution with 400 µL 1 × Binding Buffer, the cells were detected using a flow cytometer (Beckman, Germany).

4.2.8 Mitochondrial membrane potential measurement

HSC-T6 cells were seeded into six-well plates and cultured overnight, then preincubated with TGF-β1 (5 ng/mL) for 12 h. After that, c31 (20, 40, 60 µM) and curcumin (40 µM) were added to treat the cells for 24 h. Cells were harvested and incubated with TMRE solution (Biyotime, diluted in serum-free medium) for 30 min at 37°C. Finally, TMRE-labeled cells were detected in flow cytometers (Beckman, Germany).

4.2.9 Wound-healing assay

HSC-T6 cells were inoculated in six-well plates and maintained overnight, followed by the treatment with 5 ng/mL TGF-β1. When the cell density reached 90%, cell monolayers were scratched with a 10 µL sterile pipette tip. Dropped debris and cells were gently washed with PBS and removed. Subsequently, serum-free medium containing different concentrations (10, 20, 30 µM) of c31 and curcumin (20 µM) was added to each well. The migration of cells at 0 h and 24 h was observed using a light microscope (Moticam Pro 282B, China), and the results were processed using Image J software .

4.2.10 Transwell migration assay

In transwell migration assay, HSC-T6 cells (2 × 10⁵ cells/well) containing serum-free medium were inoculated in the upper chamber of the transwell after 2 h of starvation culture in serum-free medium. A volume of 600 µl of DMEM medium containing 20% FBS was added to the bottom chamber of the transwell. TGF-β1 (5 ng/mL), c31 (10, 20, 30 µM) and curcumin (20 µM) were added to the upper chamber and co-incubated with the cells for 24 hours. Unmigrated cells on the surface of the upper chamber were gently wiped off with a cotton swab, and cells passing through the upper chamber were fixed with 4% (w/v) paraformaldehyde for 10 min and then stained with 0.1% crystal violet for 15 min. Finally, the excess crystalline violet dye was washed away and images were captured with a microscope (Moticam Pro 282B, China).

4.2.11 ELISA assay

The levels of TNF-α, IL-6 and IL-1β in the supernatant of RAW 264.7 cells and rat serum, as well as the expression of α-SMA, COL1α and TGF-β1 in HSC-T6 cells and rat liver homogenates, were measured using commercial ELISA kits (Elabscience Biotechnology, China) according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (TECAN Spark, Switzerland).

4.2.12 Western blot

Cell samples were lysed on ice using RIPA lysis solution (Solarbio, item number: R0010, China) containing protease and phosphatase inhibitors (Biyotime, item number: P-1045, China) after drug treatment. After protein concentration was determined using the BCA method (Solarbio, item number: PC0020), samples were diluted by adding the corresponding volume of 4× loading buffer (Solarbio, item number: P1016). An equal amount of protein (60 µg) was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane, which was closed in TBST buffer containing 5% skimmed milk powder for 2 h and incubated with primary antibody (dilution of 1:500-1:1000) at 4 ℃ overnight. After washing the membrane, secondary antibody (goat anti-rabbit IgG H + L, 1:3000, Affinity Bioscience) was added and incubated at room temperature for 2 h. Protein bands were visualized by chemiluminescence imaging system (Tanon 5500, China) and quantitatively analyzed by ImageJ software. The antibodies used in the experiments were purchased from Affinity Biosciences (China).

4.2.13 Acute toxicity study

All animals were purchased from Lanzhou University.

The animals were housed under a 12-hour light/dark cycle and were fed ad libitum according to the Institutional Animal Care and Use Committee (IACUC) guidelines. In addition, the animals were acclimatized to the environment for 7 days before the experiments.

All animal experiments were conducted following the Animal Research: Reporting of In Vivo Experiment (ARIVAL) guidelines, and were approved by the Ethics Committee of Lanzhou University.

Overall, 12 Kunming mice of 19–24 g body weight were randomized into control group and c31 group (n = 6). Next, 2000 mg/kg c31 was dissolved in saline containing 5% polyoxyethylene castor oil and 4% DMSO and then administered orally at a volume of 200 µL for each mouse. The control group was orally given the same amount of saline. The incidence of abnormal behavior or death was monitored daily for 14 consecutive days. After 14 days, histological examinations of the heart, liver, spleen, lungs, and kidneys were performed to identify signs of structural alterations.

4.2.14 Establishment of CCl₄-induced liver fibrosis model in rats

Twenty-four Wistar male rats weighing 200 ± 20 g were randomly divided into four groups: control group, CCl₄ group, CCl₄ + curcumin group, and CCl₄ + c31 group (n = 6). 40% CCl₄ (v/v, diluted in olive oil) was administered intraperitoneally at a dose of 2 mL/kg twice a week for 6 weeks to establish a rat liver fibrosis model. In the fifth week of CCl₄ administration, according to the group, curcumin (150 mg/kg) and c31 (150 mg/kg) were given orally once daily for 2 weeks. The control group was given olive oil intraperitoneally, and saline was given orally at the 5th week as in the CCl₄ group. Twenty-four hours after the last administration, the rats were euthanized with ether, and blood and liver tissues were collected for subsequent analyses.

4.2.15 Histopathological analysis

Liver tissues from Wistar rats were fixed in 4% paraformaldehyde for 48 hours and then embedded in paraffin. Next, 5 µm-thick fixed sections were stained using hematoxylin and eosin (H&E) and Masson trichrome dyes. The sections were sealed with gelatin and visualized with an optical microscope (Moticam Pro 282B, China).

4.2.16 Biochemical assay

The collected blood was coagulated at room temperature for 2 hours. After that, the serum was obtained by centrifugation at 2000 rpm for 12 minutes. Then, ALT and AST were measured using a fully automated biochemical analyzer (Beckman Coulter Chemistry Analyzer AU5800, Germany).

4.2.17 Molecular docking

Molecular docking experiments were performed in Discovery Studio software. The molecular structure of c31 was directly constructed in DS software, and the structure optimization, charge adjustment, addition of hydrogen atoms and position optimization were carried out to reduce the energy state of c31 by Prepare ligand module. The 3D structure of the target protein was downloaded from the RCSB Protein Data Bank(https://www.rcsb.org/), and the Prepare Protein module was used to remove water molecules, add hydrogen atoms and complete missing amino acid residues. The binding site was defined according to the original ligand binding pocket. Docking molecules were performed using the CDOCKER algorithm, and the docking results were visualized and processed using PyMOL.

4.2.18 Statistical Analysis.

All experiments were repeated at least three times and experimental data were processed using GraphPad Prism 8.0 software, and one-way ANOVA (one-way ANOVA) was used to assess the differences between groups, with a p-value of < 0.05 considered statistically significant.

CRediT authorship contribution statement

Chunwei Lv: Writing - review & editing, Writing - original draft, Data curation. Lei Zhang: Investigation, Formal analysis, Data curation. Chenxu Wang: Formal analysis. Zhishun Zhang: Software. Tingting Jin:Data curation. Dian He: Writing - & editing, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.Quanyi Zhao:Writing - review & editing Lifang Zheng:Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data that has been used is confidential.

Funding sources

This work was supported by the National Science and Technology Ministry (2017ZX09101001),Gansu Science and Technology Bureau Program Funds (2021-1-141, 2021-RC-86), and the fund grant of NMPA Key Laboratory for Quality Control of Traditional Chinese Medicine(2021GSMPA-KL11 and 2021GSMPA -AJ01).

Electronic Supplementary Material

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Supplementary Material 1

Author Contribution

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Synthesis and anti-liver fibrotic activity study of a chalcone derivative through anti-inflammatory effects and inhibition of JNK/NF-κB signaling pathways

Chunwei Lv^1,#, Lei Zhang^2,#, Chenxu Wang¹,Tingting Jin¹,Zhishun Zhang¹,Yixi Li¹ ,Dian He¹*, Quanyi Zhao¹, Lifang Zheng¹

(¹ Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University, Lanzhou 730000, China.² Gansu Medical Device Inspection And Testing Institute,Lanzhou 730000, China.)

^*Corresponding author: hed@lzu.edu.cn (Dian He).

^# These authors contributed equally to the work

Figure 1 Structures of chalcone derivatives for the treatment of liver fibrosis through anti-infla-mmatory effects or inhibition of hepatic stellate cell (HSC) activation

Figure 2 Strategies for Structural Modification of Chalcone Derivatives

Scheme 1 Reagents and conditions: ⅰ) SOCl₂, EtOH, rt, 2 h.

Figure 3. Conformational relationships for in vitro anti-inflammatory and anti-liver fibrosis activities of compounds

Figure 4 The effects of compound c31 on the proliferation ability of TGF-β1-induced HSC-T6 cells. The cells were incubated with TGF-β1 (5 ng/mL) for 12 h, then compound c31 was added and continued to incubate for 24 h. (A) The cell viability curves of activated HSC-T6 cells. (B) The formation of colonies of activated HSC-T6 cells treated with compound c31. (C) The cell cycle distribution of HSC-T6 was recorded with flow cytometry and quantitatively analyzed.

Figure 5 The compound c31 induced apoptosis in TGF-β1-induced HSC-T6 cells. The HSC-T6 cells were pre-incubated with TGF-β1 (5 ng/mL) for 12 h before the treatment with compound c31 for 24 h. (A) Geimsa staining assay manifested the morphological changes in HSC-T6 cells (magnification 20×, scale bar = 100 µm). (B) The types and proportions of apoptosis were measured in activated HSC-T6 cells by flow cytometry. Data represent the mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Figure 6 Effects of compound c31 on mitochondrial pathway-mediated apoptosis in activated HSC-T6 cells. (A) Changes in mitochondrial membrane potential (ΔΨm) in activated HSC-T6 cells after treatment with compound c31; (B) Expression levels of apoptosis-related proteins (Bcl-2, Bax, cytochrome C, cleaved-caspase-3) in activated HSC-T6 cells following compound c31 treatment. All data represent mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 indicate statistically significant differences compared with TGF-β1 group; ns denotes no significant difference versus blank control group.

Figure 9 The effects of c31 on production of inflammatory mediators in LPS-induced RAW 264.7 cells. The cells were incubated with LPS (1 µg/mL) and c31 for 24 hours. (A-C) The concentrations of TNF-α, IL-6 and IL-1β were measured by the ELISA kit. (D&E) The proteins expressions of iNOS and COX-2 were detected by Western Blot. All data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs the LPS group; ^###p < 0.001 vs the Control group.

Figure 10 Effect of c31 on JNK/NF-κB pathway in RAW264.7 cells induced by LPS. The RAW264.7 cells were pre-incubated with LPS (10 µg/mL) for 12 h before the treatment with c31 for 24 h. (A - F) Expression of NF-κB p65, phosphorylated NF-κB p65, IκBα, phosphorylated IκBα, JNK and phosphorylated JNK protein in RAW264.7 cells treated with c31. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Figure 11 Effect of c31 on LPS-induced JNK/NF-κB pathway in HSC-T6 cells. (A-H) Expression of NF-κB p65, phosphorylated NF-κB p65, IκBα, phosphorylated IκBα, JNK, phosphorylated JNK protein and COX-2 protein after treatment of activated HSC-T6 cells with c31. Data represent the mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs the TGF-β1 group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs the Control group.

Figure 12 The schematic diagram of the binding between c31 and different target proteins shows that green dashed lines represent hydrogen bonds, while pink dashed lines indicate hydrophobic interactions. (A) c31 in complex with NF-κB (PDB:8TQD), (B) c31 in complex with JNK (PDB:3ELJ) (C) c31 in complex with COX-2(PDB:5IKT) (D) c31 in complex with iNOS (PDB:3E7G)

Figure 13 Effect of c31 on major organs after 14 days of single-dose gavage (magnification: 20 ×, scale bar: 100 µm).

Table 1. Summary of specific compound structures

Compd.	R¹	R²	R³	R⁴	R⁵	Compd.	R¹	R²	R³	R⁴	R⁵
c1	H	H	H	H	H	c17	OCH₃	H	H	H	H
c2	F	H	H	H	H	c18	H	OCH₃	H	H	H
c3	H	F	H	H	H	c19	H	H	OCH₃	H	H
c4	H	H	F	H	H	c20	CH₃	CH₃	H	H	H
c5	Cl	H	H	H	H	c21	CH₃	H	CH₃	H	H
c6	H	Cl	H	H	H	c22	CH₃	H	H	CH₃	H
c7	H	H	Cl	H	H	c23	OCH₃	OCH₃	H	H	H
c8	Br	H	H	H	H	c24	OCH₃	H	OCH₃	H	H
c9	H	Br	H	H	H	c25	OCH₃	H	H	OCH₃	H
c10	H	H	Br	H	H	c26	OCH₃	H	H	H	OCH₃
c11	CH₃	H	H	H	H	c27	H	OCH₃	OCH₃	H	H
c12	H	CH₃	H	H	H	c28	OCH₃	OCH₃	OCH₃	H	H
c13	H	H	CH₃	H	H	c29	OCH₃	H	OCH₃	OCH₃	H
c14	C₂H₅	H	H	H	H	c30	OCH₃	H	OCH₃	H	OCH₃
c15	H	C₂H₅	H	H	H	c31	H	OCH₃	OCH₃	OCH₃	H
c16	H	H	C₂H₅	H	H

Table 2 The IC₅₀ values of compounds for NO inhibition in LPS-stimulated RAW 264.7 cells

Compd.	IC₅₀ (µM) ^a	Compd.	IC₅₀ (µM) ^a
c1	7.94 ± 0.61	c17	6.54 ± 1.06
c2	8.01 ± 0.22	c18	3.63 ± 0.21
c3	7.36 ± 0.15	c19	7.28 ± 0.98
c4	11.76 ± 0.86	c20	9.28 ± 1.15
c5	5.78 ± 0.49	c21	17.86 ± 1.42
c6	3.86 ± 0.66	c22	8.27 ± 0.52
c7	7.48 ± 0.39	c23	4.02 ± 0.25
c8	> 40	c24	19.94 ± 2.03
c9	4.08 ± 0.60	c25	4.57 ± 0.51
c10	22.91 ± 0.72	c26	> 40
c11	4.16 ± 0.34	c27	3.28 ± 0.45
c12	3.75 ± 0.52	c28	3.40 ± 0.36
c13	16.70 ± 1.20	c29	4.33 ± 0.22
c14	8.08 ± 0.95	c30	13.08 ± 1.14
c15	6.37 ± 0.58	c31	3.05 ± 0.12
c16	14.77 ± 0.67	CUR	12.58 ± 0.76
DXM	6.04 ± 0.98

Table 3 The IC₅₀ values of compounds on HSC-T6 cells and LO2 cells.

Compd	IC₅₀ (µM)^a		Selectivity Index^c	Compd	IC₅₀ (µM)^a		Selectivity Index^c
Compd	HSC-T6^b	LO2	Selectivity Index^c	Compd	HSC-T6^b	LO2	Selectivity Index^c
c1	87.54 ± 1.78	168.73 ± 17.10	1.92	c17	78.72 ± 2.75	371.97 ± 12.14	4.73
c2	68.82 ± 1.51	242.63 ± 13.96	3.53	c18	61.56 ± 1.51	202.17 ± 9.26	3.28
c3	66.09 ± 1.41	138.27 ± 4.18	2.09	c19	89.59 ± 3.79	195.67 ± 10.47	2.18
c4	92.83 ± 9.46	129.33 ± 5.09	1.39	c20	93.24 ± 2.17	208.24 ± 4.17	2.33
c5	61.23 ± 4.51	125.28 ± 2.76	2.05	c21	107.61 ± 5.29	314.62 ± 6.40	2.92
c6	57.64 ± 1.74	102.19 ± 7.50	1.77	c22	66.90 ± 1.04	269.18 ± 2.65	4.02
c7	63.57 ± 2.05	132.08 ± 4.57	2.08	c23	59.77 ± 3.96	173.41 ± 4.33	2.90
c8	107.23 ± 2.11	359.19 ± 14.41	3.35	c24	67.23 ± 5.58	133.17 ± 2.68	1.98
c9	70.82 ± 1.61	157.53 ± 12.63	2.22	c25	60.88 ± 3.60	242.11 ± 7.83	3.98
c10	67.54 ± 1.42	129.33 ± 5.09	1.92	c26	140.93 ± 1.05	> 400	> 2.84
c11	104.44 ± 2.52	216.99 ± 16.36	2.08	c27	57.61 ± 0 .47	239.40 ± 7.13	4.15
c12	48.14 ± 1.15	168.73 ± 17.10	3.50	c28	49.14 ± 1.83	210.29 ± 2.39	4.28
c13	110.21 ± 3.48	346.72 ± 12.21	3.15	c29	58.92 ± 4.08	192.94 ± 3.28	3.27
c14	64.62 ± 2.03	159.17 ± 6.52	2.46	c30	113.37 ± 4.59	323.15 ± 6.56	2.85
c15	58.71 ± 1.27	114.83 ± 3.26	1.96	c31	42.97 ± 0.93	157.23 ± 3.79	3.66
c16	103.38 ± 5.91	241.18 ± 2.77	2.33	CUR	45.79 ± 0.78	64.74 ± 7.87	1.41

^a The IC₅₀ values represent mean ± SD from at least three independent experiments.

^b HSC-T6 cells were activated by TGF-β1 (5 ng/mL).

^c Selectivity index, representing the ratio of the IC50 value of LO2 cells to the IC₅₀ value of activated HSC-T6 cells.

Yes

Abstract

Liver fibrosis is a progressive disease caused by chronic inflammation and the activation of hepatic stellate cells (HSCs). This disease manifests as the abnormal proliferation and migration of HSCs, as well as the excessive deposition of the extracellular matrix. Chalcone analogues exhibit various biological activities, including anti-inflammatory, anti-proliferative, and apoptotic modulation properties, making them promising candidates for anti-fibrotic drug development. To enhance anti-fibrotic activities and decrease their side-effects, 31 novel chalcone derivatives were synthesized and evaluated. Among all the compounds, c31 exhibited the strongest anti-inflammatory activity, and its IC50 is 3.05 ± 0.12 μM; and it effectively inhibited the activation and proliferation of HSC-T6 cells. Mechanistic studies revealed that c31 inhibits HSC activation by downregulating the expression levels of inflammatory factors, such as TNF-α, IL-6, and IL-1β, and by interfering with NF-κB and JNK signaling pathways. Additionally, c31 inhibited HSC-T6 proliferation and promoted apoptosis by blocking a G2/M phase cell cycle; and it also significantly inhibited HSC-T6 cell migration. In a rat model of CCl₄-induced liver fibrosis, c31 improved pathological symptoms, decreasing collagen deposition, fibrotic protein expression, and ALT and AST levels. Meanwhile, it also reduced the secretion of inflammatory factors, thereby alleviating CCl₄-induced liver fibrosis. In summary, c31 had significant anti-inflammatory and anti-fibrotic effects in both in vivo and in vitro; this indicates c31 has the potential to be used as a therapeutic candidate for hepatic inflammation and fibrosis.