| Hypertrophic scars(HS),is a kind of pathological scar,which is caused by the excessive repair of skin tissue after various kinds of trauma,such as surgery.It is also called protuberant scar or hypertrophic scar.At present,hypertrophic scar formation has many causes,which are much complex.Excessive proliferation of human fibroblasts,which leads to excessive increased deposition and reduced degradation of extracellular matrix(ECM)and increased secretion of collagen,are the main histological reason of hypertrophic scar formation.There is no exact pathogenesis of HS formation.The course in the occurrence and development of hypertrophic scar is complicated,in which inflammation and endoplasmic reticulum stress(ERS)are considered as two key factors.Endoplasmic reticulum stress(ERS)has close ties with inflammation reported by large number of recent studies.Endoplasmic reticulum stress(ERS)can be coupled with the inflammatory response pathway through unfolded protein response(UPR)transmembrane receptor signaling pathway,thus participating in the pathophysiological process of a wide range of diseases related to inflammation.The interrelated pathways between endoplasmic reticulum stress and inflammation may perform a vital role in hypertrophic scar.Therefore,a new concept for the treatment of hypertrophic scar can be found,when we actively explore and regulate the interrelated pathways between endoplasmic reticulum stress and inflammation in the procdure of hypertrophic scar formation.Tumor necrosis factor αstimulated gene 6(TSG-6)is an inflammatory related secretory protein with important anti-inflammatory properties and tissue protection.The inflammatory reaction was inhibited and the expression of IL-6 and IL-1β was decreased by TSG-6 in the many former experimental studies in vitro,which leads to the significantly reduced formation of hypertrophic scar in rabbit ears.In vitro,TSG-6 can also induce apoptosis,reduce fibroblast fibrosis and promote scar healing.However,the potential molecular mechanism of TSG-6 in anti-scar formation is still unclear due to the complexity of the pathogenesis of hypertrophic scar.Based on the above theory,the effect of TSG-6 on human hypertrophic scar fibroblasts was observed,moreover,its possible mechanism was also explored,including endoplasmic reticulum stress,inflammatory response and the interrelated IRE1α pathway between endoplasmic reticulum stress and inflammation.Our research complies with relevant standards and requirements of medical ethics.Part 1 Effect of TSG-6 on fibroblasts in hypertrophic scar1.TSG-6 inhibits the proliferation of human hypertrophic scar fibroblasts(HSFs)in vitroObjective:To observe the effect of TSG-6 on the proliferation of hypertrophic scar fibroblasts(HSFs)in vitro.Methods:Human hypertrophic scar fibroblasts(HSFs)and Human normal skin fibroblasts(HFF)were cultured in vitro and treated with exogenous TSG-6.The viability of HSFs and HFF were detected by Cell Counting Kit-8(CCK-8)assay.The effects of different concentrations of TSG-6(0,10,100,200,400 ng/ml)on cell proliferation were observed at different time points(24,48,72,96 h).Results:TSG-6 significantly inhibited the proliferation of human hypertrophic scar fibroblasts(HSFs)in vitro.TSG-6 treatment reduced the cell viability in a dose-dependent and time-dependent manner.It was found that the 200 ng/ml dose group had the strongest inhibitory effect on the proliferation of human hypertrophic scar fibroblasts(HSFs)after 48 h of TSG-6 treatment.However,TSG-6 had no significant inhibitory effect on the proliferation of HFFs(P>0.05).2.TSG-6 induces apoptosis of human hypertrophic scar fibroblasts(HSFs)in vitroObjective:To observe the effect of TSG-6 on the apoptosis of human hypertrophic scar fibroblasts(HSFs).Methods:Human hypertrophic scar fibroblast and and Human normal skin fibroblast line were cultured in vitro,and exogenous TSG-6 was applied to the cells;flow cytometry was used to detect the effect of TSG-6(200 ng/ml)on cell apoptosis at the time point(48 h).Results:Compared with HSFs group,the poptosis rate in HSFs+TSG-6(TSG)group were significantly increased(P<0.05),suggesting that TSG-6 can significantly induce the apoptosis of human hypertrophic scar fibroblasts(HSFs)in vitro.FCM revealed that there was no significant change in apoptosis rate between TSG+HFF and HFF groups(P>0.05),indicating that TSG-6 have no obvious inducing effect on the apoptosis of HFFs.3.TSG-6 inhibits the proliferation of collagen Ⅰ,collagen Ⅲ,α-SMA and proliferating cell nuclear antigen(PCNA)in hypertrophic scar fibrosisObjective:To investigate the effect of TSG-6 on fibrosis-related molecules and fibroblast proliferation in HSFs.Methods:Human hypertrophic scar fibroblasts were cultured in vitro and treated with exogenous TSG-6(200 ng/ml)for 48 h.The experiment was divided into two groups:HSFs group and HSFs+TSG-6(TSG)group.The expressions of collagen Ⅰ,collagenⅢ,α-SMA and proliferating cell nuclear antigen(PCNA)were detected by Western blot and Real time PCR.Results:Compared with HSFs group,the the protein levels and mRNA expressions of collagen Ⅰ,collagen Ⅲ,α-SMA and proliferating cell nuclear antigen(PCNA)in HSFs+TSG-6(TSG)group were significantly decreased(P<0.05),suggesting that TSG-6 can significantly repress fibrosis-related molecules and fibroblast proliferation in HSFs.Summary:1.TSG-6 can significantly inhibit the proliferation of human hypertrophic scar fibroblasts(HSFs)in vitro.2.TSG-6 can significantly induce the apoptosis of human hypertrophic scar(HS)fibroblasts(HSFs)in vitro.3.TSG-6 can effectively inhibitfibrosis-related molecules and fibroblast proliferation in HSFs.Part 2 Effect of TSG-6 on endoplasmic reticulum stress and inflammatory response in HSFs1.Expression of Bip and p-IRE1α protein and mRNA of endoplasmic reticulum stress(ERS)in hypertrophic scar fibroblastsObjective:To observe the expression of endoplasmic reticulum stress related mRNA and proteins Bip and p-IRE1α in human hypertrophic scar fibroblasts.Methods:Human normal skin fibroblasts(HFF)and hypertrophic scar fibroblasts(HSFs)were cultured in vitro and divided into two groups:HFF group and HSF group.The expression of endoplasmic reticulum stress-related mRNA and proteins Bip and p-IRE1αwere detected by Realtime PCR and Western blot.Results:Compared with HFF group,the expression of endoplasmic reticulum stress-related proteins Bip and p-IRE1α and Bip mRNA in HSF group were significantly increased(P<0.05),suggesting that ER stress is obviously activated on HSFs.2.TSG-6 inhibits endoplasmic reticulum stress(ERS)response induced by hypertrophic scar fibroblastsObjective:To investigate the effect of TSG-6 on the expression of endoplasmic reticulum stress(ERS)related mRNA and proteins Bip and p-IRE1α induced by hypertrophic scar fibroblasts(HSFs).Methods:Human hypertrophic scar fibroblasts(HSFs)were cultured in vitro and treated with exogenous TSG-6(200 ng/ml);the hypertrophic scar fibroblasts(HSFs)were randomly divided into HSFs group and HSFs+TSG-6(TSG)group.Realtime PCR and Western blot was used to detect the expression of endoplasmic reticulum stress-related mRNA and proteins Bip and p-IRE1α,and immunofluorescence staining was used to detect the expression of p-IRE1α.Results:Realtime PCR and Western blot showed that the expression levels of endoplasmic reticulum stress proteins Bip and p-IRE1α,and Bip mRNA in HSFs+TSG-6(TSG)group were significantly lower than those in HSFs group(P<0.05);immunofluorescence staining showed that the fluorescence intensity of endoplasmic reticulum stress protein p-IRE1α in HSFs+TSG-6(TSG)group was significantly lower than that in HSFs group(P<0.05);it suggested that TSG-6 could effectively inhibit the endoplasmic reticulum stress(ERS)response induced by hypertrophic scar fibroblasts.3.TSG-6 inhibits the expression of IRE1α signal transduction pathway related proteins induced by endoplasmic reticulum stress(ERS)in hypertrophic scar fibroblasts(HSFs)Objective:To investigate the effect of TSG-6 on the expression of IRE1α signal transduction pathway related proteins induced by endoplasmic reticulum stress(ERS)in hypertrophic scar fibroblasts(HSFs).Methods:Firstly,Human normal skin fibroblasts(HFF)and human hypertrophic scar fibroblasts(HSFs)were cultured in vitro and human hypertrophic scar fibroblasts(HSFs)treated with exogenous TSG-6(200 ng/ml),and then the experiment were divided into HFFgroup,HSFs group and HSFs+TSG-6(TSG)group.The expression levels of TRAF2 and NF-κB p65 were detected by Realtime PCR and Western blot.Results:Compared with HFF group,the expression of TRAF2 and NF-κB p65 in HSF group were significantly increased(P<0.05),while the expression levels of TRAF2 and NF-κB p65 mRNA and proteins in HSFs+TSG-6(TSG)group were significantly lower than those in HSFs group(P<0.05),suggesting that the IRE1α signal transduction pathway(IRE1α-TRAF2-NF-κB p65)in HSFs was activated,and TSG-6 can effectively inhibit the downstream signal transduction pathway of IRE1α induced by hypertrophic scar fibroblasts.4.TSG-6 inhibits the inflammatory response induced by hypertrophic scar fibroblasts(HSFs)Objective:To observe the effect of TSG-6 on the inflammatory response induced by hypertrophic scar fibroblasts(HSFs).Methods:Firstly,Human normal skin fibroblasts(HFF)and human hypertrophic scar fibroblasts(HSFs)were cultured in vitro and human hypertrophic scar fibroblasts(HSFs)treated with exogenous TSG-6(200 ng/ml),and then the experiment were divided into HFFgroup,HSFs group and HSFs+TSG-6(TSG)group.The activities of IL-1β,IL-6 and TNF-α were detected by ELISA kit.Results:Compared with HFF group,the activities of IL-1β,IL-6 and TNF-α in HSF group were significantly increased(P<0.05).After treatment with TSG-6,the activities of TNF-α,IL-1β and IL-6 in HSFs+TSG-6 group were significantly lower than those in HSFs group(P<0.05),suggesting that TSG-6 can effectively inhibit the inflammatory response induced by hypertrophic scar fibroblasts(HSFs).Summary:1.TSG-6 can significantly inhibit the endoplasmic reticulum stress(ERS)response induced by hypertrophic scar fibroblasts.2.TSG-6 inhibits the IRE1α signal transduction pathway,IRE1α-TRAF2-NF-κB p65,induced by hypertrophic scar fibroblasts(HSFs).3.TSG-6 can inhibit the inflammatory response induced by hypertrophic scar fibroblasts(HSFs).Part 3 TSG-6 plays an important role in HSFs through the inflammatory response mediated by endoplasmic reticulum stress IRE1α signaling pathway1.Endoplasmic reticulum stress(ERS)triggers inflammatory response through IRE1α-TRAF2-NF-κB p65 signaling pathway in hypertrophic scarObjective:To investigate the relationship between endoplasmic reticulum stress(ERS)and inflammation in HSFs.Methods:Human hypertrophic scar fibroblasts were cultured in vitro and treated with IRE1α inhibitor STF083010.The hypertrophic scar fibroblasts(HSFs)were randomly divided into two groups:HSFs group and HSFs+STF(STF)group.The expressions of IRE1α and its downstream signal pathway related mRNA and proteins were detected by Real time PCR and Western blot,and the expression of inflammatory response related factors in hypertrophic scar fibroblasts(HSFs)was detected by ELISA.Results:After STF083010 treatment,the proteins levels of p-IRE1α,TRAF2 and NF-κB p65 and TRAF2 and NF-κB p65 mRNA in HSFs+STF(STF)group were significantly lower than those in HSFs group(P<0.05);the activities of TNF-α,IL-1β and IL-6 in HSFs+STF(STF)group were significantly lower than those in HSFs group(P<0.05);it suggested that endoplasmic reticulum stress(ERS)in HSFs could trigger inflammatory reaction through IRE1α-TRAF2-NF-κB p65 signal pathway.2.TSG-6 alleviates the proliferation of HSFs through endoplasmic reticulum stress IRE1α-mediated inflammatory response signaling pathwayObjective:To further verify whether the inhibitory effect of TSG-6 on hypertrophic scar proliferation is caused by inhibiting the inflammatory response mediated by IRE1α-TRAF2-NF-κB p65.Methods:Human hypertrophic scar fibroblasts were cultured in vitro.Thapsigargin(TG),a specific IRE1α activator,was used in this experiment.The experiment was randomly divided into three groups:HSFs group,HSFs+TSG-6(TSG)group and HSFs+TSG-6+TG(TG)group.The apoptosis of fibroblasts,Collagen Ⅰ,Collagen Ⅲ,smooth muscle actin(a-SMA)and proliferating cell nuclear antigen(PCNA)were observed.Results:(1)Compared with HSFs group,TSG-6 could significantly induce the apoptosis of human hypertrophic scar fibroblasts(P<0.05),and down regulate the protein levels and mRNA expressions of collagen Ⅰ,collagen Ⅲ,smooth muscle actin(α-SMA)and proliferating cell nuclear antigen(PCNA)in hypertrophic scar.(2)Compared with HSFs+TSG-6(TSG)group,the apoptosis rate of fibroblasts in HSFs+TSG-6+TG(TG)group was significantly lower than that in HSFs+TSG-6(TSG)group(P<0.05).The protein levels and mRNA expressions of Collagen Ⅰ,Collagen Ⅲ,smooth muscle actin(α-SMA)and proliferating cell nuclear antigen(PCNA)were up-regulated(P<0.05),suggesting that the anti-proliferation in HSFs of TSG-6 can be reversed by the use of thapsigargin(TG).Summary:1.Endoplasmic reticulum stress(ERS)triggers inflammation through IRE1α-TRAF2-NF-κB p65 signaling pathway in HSFs.2.TSG-6 can also effectively inhibit the endoplasmic reticulum stress IRE1α-TRAF2-NF-κB p65 and its induced inflammatory response in HSFs,which is similar to that of IRE1α inhibitor(STF083010);moreover,the IRE1α activator,Thapsigargin(TG),could reverse the effect of TSG-6 on proliferation of HSFs;it suggested that TSG-6 can suppress the proliferation of HSFs through inhibiting ER stress-mediated inflammation via IRE1α pathways. |
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