Alginate oligosaccharides promote wound healing and induce macrophage M2 polarisation by activating the PI3K/AKT1 signalling pathway
A
LeiLiu
MD
1BohanPan
MD
2ZihanTao
MD
2ShihuiZhu
MD
1✉Emaildoctorzhushihui@163.comï¿¿QingsongQingsongLiu
MD
1JianJin
MD
1,3,4Emailjinjiannavy@163.comLiu
MD
1Emailchemliuqinsong@126.com1Department of Burns and Plastic SurgeryShanghai Children’s Medical Center, Shanghai Jiaotong University School of MedicineShanghaiChina
2Department of Burn SurgeryFirst Affiliated Hospital of Naval Military Medical UniversityShanghaiChina
3Shanghai Depeac Biotechnology Co., LtdShanghaiChina
4Department of Polymer ScienceFudan University, Shanghai Depeac Biotechnology Co., LtdShanghai, ShanghaiChina, China
Lei Liu, MD1,†, Bohan Pan, MD2,†, Zihan Tao, MD2,†, Shihui Zhu, MD1, Qingsong Liu, MD1,Jian Jin, MD3,4
1 Department of Burns and Plastic Surgery, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, China
2 Department of Burn Surgery, First Affiliated Hospital of Naval Military Medical University, Shanghai, China
3 Department of Burns and Plastic Surgery, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, China
4 Shanghai Depeac Biotechnology Co., Ltd, Shanghai, China
Corresponding author: Shihui Zhu, MD, Department of Burns and Plastic Surgery, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, China, doctorzhushihui@163.com;Qingsong Liu, MD, Department of Burns and Plastic Surgery, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, China,chemliuqinsong@126.com; and Jian Jin, MD, Department of Polymer Science, Fudan University, Shanghai, China and Shanghai Depeac Biotechnology Co., Ltd, Shanghai, China, jinjiannavy@163.com.
Lei Liu MD, Bohan Pan MD, Zihan Tao MD and Liu MD contributed equally to this work.
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Funding:
This research was funded by the National Natural Science Foundation of China, grant number 81871578; Naval Military Medical University Basic Research Project, grant number 2022MS010; Shanghai Municipal Commission of Health and Family Planning Clinical Research Program, grant number 20184Y0113; Medical and Health Science and Technology Project of Hangzhou, grant number B20200432; Hangzhou Science and Technology Development Plan Project, grant number 20210133X01; Zhejiang Medical and Health Science and Technology Plan Project, grant numbers 2018KY631 and 2022KY282; Baoshan Special Project of Shanghai Science and Technology Action, grant number 21SQBS00400; Shanghai Special Fund for Promoting High Quality Industrial Development – Biomedicine, Medical and Health Science and Technology Project of Zhejiang Province, grant number 2022RC237; and Special policy for science and technology park around Shanghai University, grant number 2021-HSD-8-1-004.
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Data Availability
All data and materials can be obtained from the corresponding author at jinjiannavy@163.com.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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Author Contribution
Conceptualization: L.L., B-h.P., and Z-h.T.; Investigation: S-h.Z.; Methodology: S-h.Z., Q-s.L., and J.J.; Formal analysis: Q-s.L.; Writing – original draft: L.L., B-h.P., and Z-h.T.; Writing – review & editing: S-h.Z., Q-s.L., and J.J.
Products, devices, drugs, etc., used in the manuscript:
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AOs were purchased from Shaanxi Longzhou Biotechnology Co., Ltd. (China). Mouse mononuclear macrophages and human keratinocytes (HaCat) were purchased from Procell (Wuhan, China). Standard Escherichia coli CMCC (B) 64941 and Staphylococcus aureus CMCC (B) 26001 strains were obtained from the National Center for Medical Culture Collections (Beijing, China). Experimental Sprague Dawley rats were provided by the Experimental Centre of the Second Military Medical University (license number, SCXK [Shanghai] 2012-0003). Anti-CD68 antibody (ab283654), anti-inducible nitric oxide synthase (iNOS) antibody (ab178945), anti-IL-1 beta antibody (ab254360), anti-caspase-3 antibody (ab184787), anti-PI3 kinase p85 alpha antibody (ab191606), anti-AKT1 (phospho S473) antibody (ab81283), the rat IL-1 beta ELISA Kit (ab255730), the rat IL-10 ELISA Kit (ab214566), the rat TNF alpha ELISA Kit (ab236712), the mouse iNOS ELISA Kit (ab253219), the mouse Arginase 1 ELISA Kit (ab269541), and SH-5 (ab141442) were purchased from Abcam Trading Co., Ltd. (Shanghai, China). The mouse (CD68) ELISA Kit (ZY-E6759M) was purchased from Shanghai ZeYe Biotechnology Co., Ltd. (China).Abstract
The objective of this study was to investigate the effects of alginate oligosaccharides on wound cell proliferation, migration, apoptosis, macrophage polarisation, and wound healing. The results of the Cell Counting Kit-8, Transwell method, and caspase-3 immunofluorescence showed that alginate oligosaccharides effectively promoted keratinocyte proliferation, migration, and reduced apoptosis by activating the PI3K/AKT1 signalling pathway. The polarisation of macrophages was detected using iNOS and Arg-1 immunofluorescence. Alginate oligosaccharides induced M2 polarisation. This was negated after using an AKT1 inhibitor. In vitro cell experiments showed that alginate oligosaccharides did not affect macrophage proliferation but showed a significant reduction in macrophage numbers in in vivo animal wound models, accompanied by a trend in M2 polarisation. Although alginate oligosaccharides had no antibacterial effect, their use in vivo modulated the composition of wound microbiota by inducing macrophage polarisation and altering the inflammatory response. The combined effects of alginate oligosaccharides on keratinocytes and macrophages ultimately promoted wound healing and altered microbiota composition, thereby providing a new, potential treatment option for wound healing.
Keywords:
alginate oligosaccharides
anti-inflammatory
macrophage polarisation
PI3K/AKT1 pathway
wound healing
Abbreviations
ANOSIM, analysis of similarities; AO, alginate oligosaccharide; ARG-1, arginase-1; CCK-8, Cell Counting Kit-8; DAPI, 4’,6-diamidino-2-phenylindole; ECM, extracellular matrix; HaCat, human keratinocyte; H&E, hematoxylin and eosin; LefSe, linear discriminate analysis effect size
1. Introduction
Wounds are a common occurrence in daily activities that damage the integrity of the skin. A more profound comprehension of the intricacies governing wound healing has precipitated the development of diverse strategies for wound management. Within this intricate healing process, the inflammatory responses within the microenvironment hold pivotal significance. During the initial stages, hemostasis is followed by the recruitment of macrophage cells, serving a dual role in microbial eradication and the clearance of cellular debris 1,2. Excessive and prolonged inflammation proves detrimental, notably contributing to the protracted unhealing evident in chronic wounds such as diabetic ulcers 3,4. Subsequently, macrophage cells undergo polarisation and activation, thereby orchestrating critical pathophysiological functions, including cell proliferation and migration during the overlapping phases of wound healing 5. Consequently, an equilibrium between M1 and M2 macrophage phenotypes assumes paramount importance in regulating the intricacies of the healing process 5–7.
Biological wound dressings, including foam, film, patches, hydrogels, and hydrocolloids, have been the subject of extensive development and application in wound care 8. Presently, a diverse spectrum of functional healing wound materials rooted in hydrogel formulations has emerged, displaying promising potential. Hydrogels possess attributes such as biodegradability, cellular compatibility, and tunable mechanical properties, closely mirroring the porous architecture of the extracellular matrix (ECM). Within this context, numerous biopolymers were successfully harnessed for the creation of hydrogel matrices, encompassing materials such as collagen 9, chitosan 10, alginate 11, hyaluronic acid 12, carrageenan 13, polyvinyl alcohol 11, and cellulose 14,15. The distinctive attributes of hydrogels encompass superior permeability, absorptive capacity, adjustable mechanical resilience, notable antibacterial properties, biocompatibility, and biodegradability 16. Consequently, their wide-ranging application within the realm of wound healing has become increasingly prominent.
Alginate oligosaccharides (AOs) constitute the degradation byproducts of alginate. Divergent from polymeric alginate, AOs lack the attributes of thickening or emulsification; however, their oligomeric nature imbues them with notable bioactivity 17. Empirical investigations have unveiled the immunomodulatory and anti-oxidative stress properties of AOs, with a burgeoning focus on their role in modulating inflammation 17–21. This study consisted of a comprehensive exploration employing in vitro cellular experiments in conjunction with in vivo investigations utilising a wound-healing rats model.
2. Materials and Methods
2.1 Cell culture
AOs were added to the cell culture medium at concentrations of 0.1%, 0.05%, and 0.01%. Each resulting mixture was added for cell culture under 5% CO2 at 37°C. A control group was prepared and subjected to the same culture procedure in the absence of the AO solution.
2.2 Cell proliferation
The effects of different concentrations of AO solutions on HaCat proliferation were examined according to the process of Cell Counting Kit-8 (CCK-8) method, and a conventional culture without the AO solution was used as the control. HaCat cells were added to a cell suspension at 2 × 103 cells/100 µL, and 100 µL aliquots of the suspension were transferred to a 96-well cell culture plate. This was followed by pre-culturing at 37°C for 2–6 h in an incubator to induce cell adhesion to the culture plate wall. The AO solution was added to the wells at different concentrations (0.10%, 0.05%, and 0.01%), and the culture was incubated for another 24 h. Subsequently, 10 µL of CCK-8 solution was added to each well, and the resulting mixtures were incubated at 37°C for 3 h. The absorbance of the samples was measured at 450 nm to calculate cell viability, and this procedure was performed in eight replicates.
2.3 Cell migration
The effect of different concentrations of AO solutions on HaCat migration was examined using the Transwell assay, with a conventional culture without AO solution as the control. Specifically, 800 µL of macrophage culture medium was added to the bottom of a 24-well plate, and 150 µL of macrophage suspension containing 2 × 105 cells/mL was added to the upper chamber; the setup was incubated for 24 h. The cells were fixed, stained, air-dried, sealed, and observed under an optical microscope; cells in five randomly selected fields of view were counted and averaged. The procedure was performed in eight replicates. Relative cell migration (%) was calculated.
2.4 Apoptosis
The effect of AO solution at different concentrations on HaCat apoptosis was examined using caspase-3 immunofluorescence. A conventional culture was used as the control. An immunofluorescence assay was performed as follows.
Fixing cells with 4% paraformaldehyde for 15 min, and phalloidin (5 µg/mL) was added for cell skeleton staining after incubation with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 5 min. The samples were observed under a fluorescence microscope and analysed quantitatively according to the fluorescence intensity.Cells were fixed using 4% paraformaldehyde for a duration of 15 minutes. Subsequently, phalloidin at a concentration of 5 µg/mL was added for cell skeleton staining, after incubating with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 5 minutes. The samples were then observed under a fluorescence microscope, and a quantitative analysis was carried out based on the fluorescence intensity. Using the fluorescence microscope, the fluorescence intensity of cells in five randomly selected fields of view was measured and averaged; relative fluorescence intensity (%) was calculated.
2.5 PI3K/AKT1 level determination
Based on the cell proliferation, migration, and apoptosis assay results, the concentrations of AOs solution were selected for the treatment of HaCat; subsequently, PI3K and AKT1 levels among the experimental group and the control group were determined. The immunofluorescence sample preparation procedure and measurement conditions were the same as those of the apoptosis assay.
2.6 Macrophage polarisation
M1 macrophages were labelled with CD68, a cluster of leukocyte differentiation antigens, along with iNOS, whereas M2 macrophages were labelled with CD68 and arginase 1 (Arg-1). After being incubated for 24 h under the conditions of 37°C and 5% CO₂, double-labelled macrophages were detected in the experimental and control culture media; the immunofluorescence sample preparation procedure and measurement conditions were the same as those of the apoptosis assay. Five random fields were selected during microscopy to count the positive cells. The number of CD68/iNOS-positive cells was compared with that of CD68/Arg-1-positive cells to evaluate the polarisation trend of macrophages. The IL-1β level was detected by immunofluorescence using the same immunofluorescence sample preparation and measurement conditions as that of the apoptosis test.
2.7 In vitro antimicrobial assay
Bacterial suspensions (105 cfu/mL, 100 µL) were transferred to wells 1–10 of a 96-well plate. The AO solutions were added to wells 1–10 at concentrations of 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, and 0.0078125%, respectively. Subsequently 200 µL of sterile Mueller–Hinton broth was added into well 11, and 200 µL of the bacterial suspension was added to well 12. The plates were then incubated at 37°C for 22 hours. A spectrophotometer was utilized to measure the optical density of the samples in every well. The minimum inhibitory concentration for each bacterial strain was defined as the lowest concentration at which there was no change in turbidity. All bacterial strains and disinfectants were tested in three replicates.
2.8 Animal experiments
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We used 40 male Sprague–Dawley rats aged 8–10 weeks and weighing 180–220 g. We randomly divided the rats into positive experimental and control groups (n = 20 rats per group). A circular full-thickness skin defect model was constructed with an outer diameter of 3.0 cm. An AO hydrogel was used for dressing changes in the experimental group (the concentration was determined based on the results of the cell experiment), whereas 0.9% sodium chloride solution was used for the control group. The dressing was changed daily. At 6 and 12 d, five rats were randomly selected, sacrificed, and sampled, and the wound healing rate was recorded. Wound tissues were sampled and examined for macrophage polarisation, PI3K, AKT1, and IL-1β expression, and apoptosis indices using immunofluorescence, followed by a between-group comparison.
Compositional changes in the microbiota of samples within 14 d of sampling were detected using a 16S rRNA assay. DNA from normal or wound tissues was isolated and sequenced using the MetaVTM Library Construction Kit (GENEWIZ, Inc., South Plainfield, NJ, USA). Follow-up sequencing was conducted using a previously described sequencing method 22.
2.8 Ethical considerations
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All animal studies (including the rat euthanasia procedure) were performed in compliance with the regulations and guidelines of Shanghai Depeac Biotechnology Co., Ltd’s institutional animal care and conducted according to the IACUC guidelines(SH2022-090906).2.9 Statistical analysis
Data are expressed as the mean ± standard deviation. All data were statistically analysed using SPSS software (version 21.0; IBM Corp., Armonk, NY, USA). The paired samples Student’s t-test was used if the data were normally distributed; otherwise, the Wilcoxon signed-rank sum test of paired samples was used. Differences were considered significant at P < 0.05.
3. Results
3.1. Cell proliferation, migration, and apoptosis
When the concentration of AOs was 0.05% and 0.01%, it was found that HaCat cell proliferation and migration were enhanced, and the promoting effect was more remarkable at 0.05% (P < 0.05). Conversely, when the concentration of AOs reached 0.1%, there was no significant change in cell proliferation and migration (P > 0.05).The AO solutions at 0.1%, 0.05%, and 0.01% reduced HaCat apoptosis compared to 0.05% AOs + 10 µM SH-5 solution and the 10 µM SH-5 solution alone (P < 0.05). HaCat cell proliferation and migration under 0.05% AO solution and 10 µM SH-5 treatments were not significantly different from those in the conventional culture (P > 0.05, Fig. 2); moreover, SH-5 inhibited the effect of the AO solutions as a inhibitor of AKT(P < 0.05). Based on the cell proliferation, migration, and apoptosis results, the 0.05% AO concentration was chosen for co-culture with HaCat cells. After 24 h of co-culture, immunofluorescence testing revealed a significant increase in PI3K and AKT1 levels in HaCat cells (P < 0.05, Fig. 2).
3.2. Activation of the PI3K/AKT1 signalling pathway
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Based on the cell proliferation, migration, and apoptosis assay results, the 0.05% AO solution was selected for the treatment of HaCat cells. Following 24 h of treatment, an immunofluorescence assay revealed an increase in the PI3K and AKT1 levels in HaCat cells (P < 0.05, Fig. 1).
3.3. Macrophage polarisation and inflammatory cytokine secretion
As macrophages were the target cells, both experimental and control cell groups were CD68-positive, as observed using immunofluorescence, indicating no contamination of the target cells. The iNOS-positive immunofluorescence showed the presence of M1 macrophages and revealed a significant reduction in the iNOS positivity of macrophages in the experimental group (P < 0.05). In contrast, Arg-1-positive immunofluorescence showed the presence of M2 macrophages and revealed a significant enhancement of macrophage Arg-1 positivity in the experimental group (P < 0.05). The M1-to-M2 macrophage ratio was significantly lower in the treated group compared with the control group (P < 0.05). In macrophage culture, there was no significant difference in CD68, iNOS, and Arg-1 positive immunofluorescence between the 0.05% AO and 10 µ M SH-5 treatment groups and the control group (P > 0.05). Among the inflammatory cytokines, the mean intensity of IL-1β immunofluorescence suggested that the levels of proinflammatory cytokines were significantly declined in the treated group than in the control group (P < 0.05) (Fig. 3).
3.4. In vitro antibacterial activity
AOs did not exhibit inhibitory or bactericidal activity against either Escherichia coli or Staphylococcus aureus in vitro; that is, there was no detectable minimum inhibitory concentration. Bacterial growth was still observable at the highest concentration of AOs.
3.5. Wound-healing animal experiments
The wounds exhibited rosy granulated tissues in the experimental group, and neither of the groups showed significant infection. The wound-healing rate of the treated group at each stage was significantly higher than that of the control group (P < 0.05), and the results of hematoxylin and eosin (H&E) staining suggested no significant difference in histology between the groups. The results of caspase-3 staining suggested that the apoptosis rate was significantly lower in the experimental group than in the control group (P < 0.05), whereas the mean intensity of PI3K and AKT1 immunofluorescence was higher in the experimental group than in the control group (P < 0.05). Regarding macrophage polarisation indicators, the number of macrophages with CD68-positive immunofluorescence was significantly lower in the experimental group than in the control group (P < 0.05). Furthermore, the number of M1 macrophages with iNOS-positive immunofluorescence was significantly lower in the experimental group than in the control group, and the number of M2 macrophages with Arg-1-positive immunofluorescence was significantly higher in the experimental group than in the control group (P < 0.05). The mean immunofluorescence intensity of inflammatory cytokine IL-1β was significantly decreased in the treated group than in the control group (P < 0.05; Figs. 4 and 5).
The composition of wound microbiota showed significant changes after 12 d following treatment with AOs. The principle component analysis showed that the wound microbiota usually showed a homogenous composition at the levels of PC1 and PC2. The analysis of similarities (ANOSIM) results suggested that long-term (i.e. 12 d) treatment with AOs resulted in significant between-group differences in wound microbiota composition (P < 0.05), but the results of 6d treatment showed no significant difference (P > 0.05). The linear discriminate analysis effect size (LefSe) results suggested that AOs increased the proportion of cocci and bacilli, such as Staphylococcus spp. and decreased that of Ralstonia and Burkhoideriaceae (Fig. 6).
4. Discussion
AOs can be produced from a variety of raw materials, and they have been used widely in the medical and food fields 23. However, it is difficult for AOs to pass through various biological barriers or to enter cells owing to their high DP; thus, their biological activities are rarely reported. However, AOs with a low DP that possess several biological properties such as apoptosis-inducing, antibacterial, immunomodulatory, and inflammatory-modulatory effects can be formed through degradation. The latter two properties are useful in the treatment of digestive and cardiovascular system diseases 18,19,21. However, based on our knowledge, no studies have reported the effects of AO treatment on wound healing, although clinical practices with AOs have confirmed that they play a positive role in promoting wound healing. Therefore, this study explored the effect of AOs on wound healing through cellular and animal experiments. The selected commercial AOs had a low DP but a high PDI owing to the production process.
The proliferation and migration of wound cells are essential for wound healing, which is especially true for keratinocytes, whose proliferation and migration ensure epithelialization 24–26. This study confirmed that AOs effectively promoted the proliferation and migration of HaCat, which are typical epidermis cells. Furthermore, this study confirmed that AOs reduced HaCat apoptosis, suggesting that the early use of AOs after wound formation can effectively reduce secondary damage to wound cells. Moreover, the PI3K/AKT1 signalling pathway is important because the AKT1 inhibitor abolished the above effects.
However, the biological effects of AOs were not concentration dependent. When AO concentration reached 0.05%, further concentration increase led to a decrease in HaCat proliferation and migration and failed to show a significant improvement in the anti-apoptotic effect. This observation is consistent with our previous study that showed that AOs can cause skin irritation and sensitization at high concentrations. Such concentration dependence on the biological effects of AOs above a certain concentration may be attributed to the adverse influence of high AO concentrations on biocompatibility and potential impurities in commercial AOs introduced during the manufacturing process; these hypotheses remain to be verified with further experimentation.
Animal experiments also confirmed that AOs promote wound healing in vivo, significantly reduce apoptosis in tissues, and promote PI3K and AKT1 levels in wound tissues. This observation confirmed that AOs accelerate wound healing by promoting wound cell proliferation and migration and inhibiting apoptosis by activating the PI3K/AKT1 signalling pathway. These results are consistent with the conclusion of Fang et al. 27, who revealed that AOs could activate RAW264.7 macrophages through the PI3K/AKT1 signalling pathway. However, the mechanism of how AOs can activate the PI3K/AKT1 signalling pathway to promote cell proliferation and migration, although the inhibition of apoptosis remained to be explored.
The effect of AOs on macrophage polarisation was also verified in this study. Further exploration confirmed that this biological effect lasted a long time, significantly altering the composition of wound microbiota. Cell experiments showed that the total number of CD68-positive macrophages did not change after co-culture with AOs, suggesting that AOs did not promote macrophage proliferation. However, 24 h co-culture led to reduced iNOS-positive M1 macrophage expression, suggesting a decrease in the level of M1 macrophages. Whereas Arg-1-positive M2 macrophage expression increased, suggesting an increase in the level of M2 macrophages and, in turn, a decrease in M1-to-M2 macrophage ratio, namely, an alteration in macrophage polarisation status 6,28. This alteration is also related to the PI3K/AKT1 signalling pathway, and the aforementioned effects were abolished by AKT1 inhibitors 27. The in vivo animal wound model showed a decrease in M1 macrophages, an increase in M2 macrophages, a decrease in the M1-to-M2 macrophage ratio, and a decrease in the secretion of proinflammatory cytokines, consistent with previous reports 29–31. However, the model also showed a significant decrease in the total number of macrophages in wound tissue and a significant decline in the level of IL-1β, which was different from the observations in other studies 32–37. The reasons for the differences in the findings among different studies may depend on different animal models; our application was for healing wounds, whereas others were mostly for immunoregulation or anti-tumour effects. Moreover, the M1-to-M2 macrophage ratio in the in vivo wound model was reversed compared to the ratio in the cell experiments. A possible reason for the discrepancy is that the wound is a complex environment where macrophages act on not only macrophages but also other cells, and such action would lead to changes in the wound microenvironment, causing differences in the experimental results between in vitro cell experiments and in vivo animal wound models. However, these controversial results remain to be further explored and verified.
Here, the alteration in macrophage polarisation status by AOs had a significant effect on the composition of wound microbiota, and this was independent of the antimicrobial activity of AOs. In vitro antimicrobial experiments have verified that AOs do not possess antimicrobial properties, and opposite results were obtained in the present study 38–40; however, the in vivo animal wound model experiments showed that long-term AO application exerted a significant effect on wound microbiota composition by increasing the compositional homogeneity. This observation and the accelerated wound healing suggest that the homogenization and directed changes in microbiota composition are beneficial to healing wounds, which is consistent with our previous study results 22.
In summary, this study explored the effects of AOs on wound healing and confirmed that they could promote the proliferation and migration of keratinocytes, reduce their apoptosis by activating the PI3K/AKT1 signalling pathway, and induce changes in macrophage polarisation status. AOs improved the local inflammatory response while inducing changes in the composition of the local microbiota, thereby promoting wound healing. These findings provide a new direction for wound treatment, but further research is needed to gain deeper insights into the underlying mechanisms.
5. Conclusions
To our knowledge, this was the first study to confirm that AOs promote wound healing and regulate the structure of wound microbiota by altering the polarisation of macrophages. Furthermore, we found that AOs promote the proliferation, migration, and apoptosis of keratinocytes and improve local inflammatory reactions by inducing M2-type polarisation of macrophages, thereby promoting wound healing. The wound microflora structure also significantly changed following M2 polarisation under AO treatment. Overall, we showed that AOs have the potential to be developed as a new strategy to improve wound healing with high biocompatibility.
Figure legends
Figure 1. Mechanism of alginate oligosaccharides (AOs) promoting wound healing.
PI3K/AKT1 signalling pathway. They induced M2 polarisation. This was counteracted by an AKT1 inhibitor. However, in vivo, animal wound models showed a significant reduction in the total number of macrophages, accompanied by a trend in M2 polarisation, which resulted in decreased secretions of IL-1β. Although AOs had no antibacterial effect, their use in vivo modulated the composition of wound microbiota by inducing macrophage polarisation and altering the inflammatory response. The combined effects of AOs on keratinocytes and macrophages ultimately promoted wound healing and altered microbiota composition. Thus, this study provides a new potential treatment option for wound healing.
Fig. 2
Impact of different alginate oligosaccharide concentrations on HaCat in vitro. (a) Caspase-3 immunofluorescence reflecting apoptosis (200×, red fluorescence indicates that the apoptosis index Caspase3 is positive). Effect of alginate oligosaccharides on HaCat (b) apoptosis; Effect of alginate oligosaccharides on HaCat (c) proliferation and (d) migration. (e) PI3K and (g) AKT1 immunofluorescence (200×, red fluorescence indicates that the expression of PI3K(e) or AKT1(g) are positive). Effect of alginate oligosaccharides on (f) PI3K and (h) AKT1 expression. * Statistically significant difference relative to the control group (P < 0.05).
Fig. 3
Effects of alginate oligosaccharides on macrophage polarisation. CD68 (a), IL-1β (c), iNOS (e), and Arg-1 (g) immunofluorescence (200×). (a) Images of CD68 immunofluorescence. (b) M1-to-M2 macrophage ratio. (d) Mean intensity of IL-1β immunofluorescence. (f) Number of M1 macrophages with inducible nitric oxide synthase (iNOS)-positive immunofluorescence. (h) Number of M2 macrophages with arginase-1 (Arg-1)-positive immunofluorescence. * Statistically significant difference relative to the control group (P < 0.05).
Fig. 4
Animal experiments. (a, b) Gross photographs of wounds and wound-healing rate. (c) Hematoxylin-eosin staining of wound sections. (d, g) Caspase-3 immunofluorescence of wound tissue to reflect apoptosis. (e, h) AKT1 immunofluorescence of wound tissue. (f, i) PI3K immunofluorescence of wound tissue. *Statistically significant difference relative to the control group (P < 0.05).
Fig. 5
Macrophage polarisation of wound tissue. (a, b) Number of macrophages with CD68-positive immunofluorescence. (c, d) Number of M1 macrophages with inducible nitric oxide synthase-positive immunofluorescence. (e, f) Number of M2 macrophages with arginase 1-positive immunofluorescence. (g, h) IL-1β immunofluorescence of wound tissue. *Statistically significant difference relative to the control group (P < 0.05).
Fig. 6
Changes in wound microbiota composition. (a) Alginate oligosaccharides induced a homogenous composition of wound microbiota at the levels of PC1 and PC2. (b, c) Analysis of similarity showed no significant between-group differences in wound microbiota composition after 6 d of treatment with alginate oligosaccharides. However, there were significant between-group differences after long-term treatment (12 d; P < 0.05). (d) Linear discriminate analysis effect size revealed the composition changes of various bacterial species in wound microbiota.
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