Experimental Study Of S100A8 And S100A9 In Skin Regeneration During Tissue Expansion

BackgroundAs one of the main repair methods in plastic surgery,skin soft tissue expansion is widely used in the repair of skin defects and the treatment of scar contractures.Compared with other methods,skin expansion is safer,and the obtained new skin is similar to the recipient skin in texture and color with the better effects.But there are still some shortcomings,such as complications,long treatment periods,and the large psychological burden on patients.Among them,the exposure of the expanders may be related to the rupture of skin collagen and the dermal thinning of the expanded skin caused by the continuous stretch of the skin.Therefore,to facilitate the synthesis of collagen and increase the thickness of the expanded dermis becomes the key to address the complication including the exposure of the expanders.The synthesis and decomposition of collagen in the skin are often regulated by a series of cytokines.But under expanded conditions,the regulated factors and their regulatory mechanisms are still unclear and needed to be explored.S100A8 and S100A9 are low-molecular-weight proteins of 10-14 KD,belonging to the calcium-binding protein S100 protein family,and play important roles in regulating inflammation and fibrosis in traumas.In wounded skin and other damaged tissues,the expressions of S100A8 and S100A9 were up-regulated and they were involved in the regulation of tissue repair.For example,some scholars have found that S100A8 and S100A9 were abundant in psoriatic keratinocytes and promoted the differentiation and proliferation of keratinocytes.Similarly,S100A8 and S100A9 could also promote fibroblast proliferation,which might be an important reason for promoting collagen synthesis.However,it is unclear whether S100A8 and S100A9 are continuously expressed and play regulatory roles in expanded skin.Therefore,exploring the expression of S100A8 and S100A9 during the expansion process and the mechanism of their involvement in the expansion of skin regeneration is of great significance for us to further understand the mechanism of skin regeneration during expansion and to find new targets to improve expansion efficiency and reduce expansion complications.ObjectiveTo study the characteristics of S100A8 and S100A9 in skin and investigate the roles of S100A8 and S100A9 and the mechanisms during tissue expansion.Methods1)In the first part,we embedded a 1 m L circular expander to the SD rat head subcutaneously to construct a rat head expansion model.16 SD rats were randomly divided into experimental group and control group,and the control group was sham expansion group.The expander was embedded subcutaneously between the top of the rat’s head and the periosteum,and normal saline was injected 1 week after the operation,twice a week,1 m L once.After 4 weeks of expansion to 9 m L,the skin samples were collected,and transcriptome sequencing was performed on the skins of each group.The expression levels and distribution characteristics of S100A8,S100A9 and their corresponding receptors in expanded skin and control skin were detected by real-time quantitative polymerase chain reaction(q PCR),Western Blot and immunofluorescence staining,and the immunofluorescencestaining were photographed under a Nikon C2 confocal microscope.In addition,we collected skin samples from 11 patients before and after skin expansion,and detected the expression levels,distribution characteristics of S100A8 and S100A9 in the skin by immunofluorescence staining.2)The second part was in vitro experiments.Firstly,we treated keratinocytes Ha Ca T cells by in vitro stretching experiments to simulate the stretched state of expanding skin,and detected the expression levels of S100A8 and S100A9 by real-time quantitative polymerase chain reaction(q PCR).The protein levels of S100A8 and S100A9 in the supernatant of stretched Ha Ca T cells were measured with ELISA kit.Secondly,the S100A8 and S100A9 recombinant proteins were used to conduct experiments related to the proliferation,migration,and collagen synthesis of skin fibroblasts.Since S100A9 could specifically bind to TLR-4,we used the receptor inhibitor TAK-242 competitively binding to the TLR-4 receptor to block the binding of S100A9 to the receptor,and detected the level of fibrosis-related protein synthesis in fibroblasts and the phosphorylation level of related proteins in the ERK1/2 signaling pathway,further exploring the roles and mechanisms of S100A9.Then si RNA was used to silence the S100A9 gene in Ha Ca T cells at the m RNA level.si RNA-transfected keratinocytes were used for stretching experiments.Then,fibroblasts were co-cultured with normal Ha Ca T cell supernatant,stretched Ha Ca T cell supernatant,and stretched Ha Ca T cell supernatant after different si RNA transfection to explore the synthesis of collagen and fibrosis-related proteins in fibroblasts.3)In the third part,we used the rat head expansion model exogenously injected with S100A9 and the inhibitor of its receptor TLR-4 to explore the effects of S100A9 on skin regeneration during expansion in vivo.Since S100A9 could specifically bind to its receptor,we used the receptor inhibitor TAK-242 competitively binding to the TLR-4receptor to block the binding of S100A9 to the receptor.Firstly,the rats were anesthetized with isoflurane,and 0.5 cm on both sides of the midpoint of the line connecting the front of the two ears was used as the bottom edge,and a square area of 1cm×1 cm was marked forward with tattoo fluid.The nodes of the tattoo line were the subcutaneous injection points,which were above the expander.The drug and saline were injected at the same time,and the water was injected twice a week with 1 m L once.The concentrations of each drug were as follows: PBS and S100A9 were injected in equal volumes;TAK-242 was dissolved in 0.05% DMSO,and then diluted with appropriate PBS.The injection concentration of TAK-242 was 20 μg/μL;S100A9+TAK-242 group was injected with S100A9 six hours after TAK-242 injection.The area of the expanded skin in each group was recorded;Image J software was used to measure the thickness of the expanded skin of HE staining in each group;Masson staining and Sirius red staining were performed on the skin of each group,and Image-Pro Plus 6.0 software was used to analyze the results.The collagen staining area was measured;the total RNA of the expanded skin in each group were extracted,and q PCR was used to detect collagen I,collagen III,TGF-β.Western Blot was used to detect the effects of S100A9 and its receptor inhibitors on fibrosis-related proteins such as collagen I,collagen III and TGF-βin expanded skin.Results1)The rat head expansion model was successfully constructed in this experiment.We found significantly elevated gene expressions of S100A8 and S100A9 of the rat skin in expansion model by transcriptome sequencing、q PCR and Western Blot.Meanwhile,the study found the expressions of their receptors were also significantly increased.We also found in human expanded skin,the expression levels of S100A8 and S100A9 were significantly up-regulated,and were mainly distributed in the epidermis.2)We found that the expressions of S100A8 and S100A9 in Ha Ca T cells were up-regulated at both m RNA and protein levels after stretching in vitro;and the expression levels of S100A8 and S100A9 in the supernatant of stretched Ha Ca T cells also increased.This suggested that S100A8 and S100A9 could be secreted out of the cells to play a role in binding with receptors on fibroblast membranes,which may be the mechanism of S100A8 and S100A9 in expanded skin.Through cell proliferation and cell migration experiments,it was found that S100A8 and S100A9 promoted the proliferation and migration of fibroblasts,and promoted the expression of collagen I,collagen III,TGF-β and TLR-4 in fibroblasts.And S100A9 could elevate phosphorylation level of ERK1/2 in fibroblasts.The use of TLR-4 receptor inhibitors hindered the synthesis of collagen I,collagen III and fibrosis-related proteins in fibroblasts by S100A9.In addition,we successfully transfected Ha Ca T cells with si RNA silencing the expression of S100A9 gene in Ha Ca T cells.After co-culturing the Ha Ca T cell supernatant under different treatments with fibroblasts,it was found that the stretched supernatant of normal Ha Ca T cells could promote the synthesis of collagen and fibrosis-related proteins in fibroblasts,while si RNA-transfected Ha Ca T cell supernatant had no significant effects on the synthesis of collagen and fibrosis-related proteins in fibroblasts,which further verified the paracrine mode of S100A9 protein.3)Using the rat head expansion model,we exogenously injected with PBS,S100A9 recombinant protein,S100A9 receptor inhibitor TAK-242 and S100A9+TAK-242.We found that the expanded skin area injected with S100A9 was significantly larger than other treatment groups,and there was no significant differences between other groups;expanded skin injected with S100A9 was significantly thicker than that of other groups,and there was no significant differences between other groups;it was found by Masson staining that the proportion of collagen,especially the collagen I,in the expanded skin injected with S100A9 was higher;and m RNA levels of collagen I,collagen III,TGF-βwere higher in the expanded skin injected with S100A9.Western Blot results showed that the expressions of collagen I and other fibrosis-related proteins such as collagen III and TGF-β were significantly up-regulated.At the same time,the expression levels of its receptor TLR-4 and the phosphorylation level of ERK1/2 were up-regulated activated by S100A9.Conclusions1)The expressions of S100A8 and S100A9 were significantly up-regulated in expanded skin of rat and human,and were mainly distributed in the epidermis;2)After being stretched,Ha Ca T cells could express and secrete large amounts of S100A8 and S100A9.S100A9 might promote the proliferation and migration of fibroblasts,regulate the synthesis of collagen and TGF-β proteins in fibroblasts through paracrine mode.At the same time,S100A9 could also promote the phosphorylation of ERK1/2 protein of fibroblasts;3)The rat expanded skin injected with S100A9 was manifested as increased area,increased thickness and increased collagen content.At the same time,the expression levels of TLR-4 and the phosphorylation levels of ERK1/2 in the expanded skin also increased significantly.