P38MAPK Silencing Attenuates Scar Proliferation After Cleft Palate Repair Surgery In Rats Via MRTF-A/SRF Pathway

BACKGROUND AND OBJECT Palate cleft repair surgery causes scarring on the hard palate,which restricts the development of maxilla.p38 mitogen-activated protein kinase(p38MAPK)promotes fibrosis in multiple organs.However,its role in scarring after hard palate repair surgery is not fully understood.The p38 mitogen-activated protein kinase(p38MAPK)promotes fibrosis in multiple organs.However,its role in scarring after hard palate surgery is not fully understood.The purpose of this study was to investigate the regulation and mechanism of p38 MAPK gene in rat palatal scar model and rat fibroblasts.The p38 MAPK was silenced by a recombinant adenovirus vector,the effect of p38 MAPK silencing on rat palatal scar fibroblasts was determined through Western blot,Ed U assay,RT-PCR,and Immunofluorescence.This study aims to investigate the therapeutic effect and molecular mechanism of adenovirus vector-mediated p38 MAPK gene silencing in rat palatal scar model,and provide new ideas and methods for the treatment of scarring on hard palate.METHODS 1.Create a rat palatal scar model and isolate primary scar fibroblasts.2.Construct an effective adenovirus vector to silence p38 MAPK in rat palatal fibroblasts.3.The effect of P38 MAPK silencing on cell proliferation was determined by Ed U assay.The effects of P38 MAPK silencing on the expression of COL I,COL III,and α-smooth muscle actin(α-SMA)and other scar-related genes were determined by RT-PCR,Western blot and immunofluorescence staining.4.Preliminary exploration of the regulatory mechanism of P38 MAPK silencing on myocardial-related transcription factor-A/serum response factor(MRTF-A/SRF).5.To verify the inhibitory effect of silencing p38 MAPK gene on scarring in animal models.Rats were randomly divided into the following groups: 1)non-operated group,2)untreated group,3)negative control virus group,and 4)experimental group.Except for the non-operated group,the left hard palate mucosa of all rats was surgically removed.Local injection of adenoviral vector was started two weeks after surgery and injected weekly until the rats were 3 months old.After 14 days of adenovirus vector treatment,immunohistochemical analysis was used to evaluate the histological changes of the hard palate mucosa in each group,and to explore the regulatory effect of P38 silencing on α-SMA,collagen fibers and MRTF-A.The width of the hard palate was measured at the age of 3 months and the ratio of the maxillary width was calculated.RESULTS 1.The rat palatal scar model was successfully created.Compared with normal mucosa,the scar tissue had disorganized collagen fiber structure and expressed more p38 MAPK and α-SMA.2.The recombinant adenovirus vector can effectively silence the expression of p38 MAPK in scar fibroblasts,and the PCR results show that the silencing efficiency is about 69%(P<0.05).3.p38 MAPK gene did not affect the proliferation of scar fibroblasts(P>0.05),but p38 MAPK silencing effectively inhibit the expression of collagen I,III and α-SMA in scar fibroblasts(P<0.05).4.p38 MAPK silencing regulated MRTF-A/SRF-dependent α-SMA transcription by directly inhibiting the expression of MRTF-A and changing the ratio of G-action/Factin to inhibit MRTF-A into the nucleus(P<0.05).5.In animal experiments,the expressions of p38 MAPK,α-SMA and collagen fibers in the scar tissue of the experimental group were significantly decreased,and the ratio of maxillary width was higher than that of the untreated group and the negative control virus group(P<0.05).CONCLUSION Scarring caused by palate cleft surgery limits the development of the maxilla.Local injection of Ad-P38MAPK-1 in vivo reduce the expression of p38 MAPK and scar-related genes,and attenuate the effect of scarring on the development of hard palate.Mechanistically,p38 MAPK silencing inhibited the expression of α-SMA expression through mediating MRTF-A production and nuclear localization in fibroblasts.These results reveal molecular pathways involved in p38MAPK/MRTF-A pathway.p38 MAPK silencing is a promising way to treat postoperative scarring of hard palate surgery.