Preliminary Study On The Mechanism Of HDAC5-Mediated Fibroblasts-to-myofibroblasts Transformation In Oral Submucosal Fibrosis

Objective: Oral submucosal fibrosis(OSF)is a chronic,progressive precancerous disease of the oral mucosa.The purpose of this study was to investigate the role of histone deacetylase 5(HDAC5)in the transition of fibroblast to myofibroblast phenotype in OSF.Method:(1)Immunohistochemical staining(IHC)and polymerase chain reaction(PCR)were used to detect the expression level of HDAC5 in OSF tissues,and the relationship between HDAC5 expression and clinicopathological characteristics of OSF patients was analyzed.(2)Primary normal buccal mucosal fibroblasts(BMFs)and human fibrotic buccal mucosal fibroblasts(f BMFs)were obtained by tissue block digestion.Morphological observation of BMFs and f BMFs,and immunofluorescence to identify markers of BMFs and f BMFs;CCK-8screened the optimal concentration range of arecoline acting on BMFs;PCR and Western blot(WB)were used to detect the expression of arecoline-induced BMFs activation markers,f BMFs markers and target gene(HDAC5).Wound healing test was used to detect the effect of arecoline on the migration ability of BMFs,and the difference of the migration ability of BMFs and f BMFs was also detected.This is to verify whether the cells derived from the two methods are myofibroblasts(MFBs).(3)The expression of HDAC5 in MFBs was silenced,and the expression changes of MFBs markers,secreted proteins of MFBs and TGF-β signaling pathway node molecule Smad2/3 and its phosphorylation levels were detected by WB and PCR;Collagen contraction assay and wound healing test to detect changes in the contraction and migration capacity of MFBs after silencing HDAC5.Result:(1)IHC and PCR results showed that compared with normal oral mucosa,HDAC5 was highly expressed in OSF tissue;The expression level of HDAC5 in OSF patients was significantly correlated with pathological stage and chewing betel quid;(2)BMFs were long spindle-shaped under light microscope,and immunofluorescence showed that BMFs highly expressed Vimentin protein;Screening the concentration range(0-60μg/m L)of arecoline acting on BMFs by CCK-8experiment;The results of WB and PCR indicated that arecoline could induce the up-regulation of α-SMA and HDAC5 in BMFs;The wound healing test results show that arecoline can promote the cell migration ability of BMFs.Arecoline-induced BMFs fits the characteristics of MFBs;(3)FBMFs showed polymorphic changes under the light microscope,and immunofluorescence showed that f BMFs highly expressed α-SMA;WB and PCR results showed that f BMFs highly expressed α-SMA and HDAC5;The wound healing test results also showed that the migration ability of primary f BMFs was significantly enhanced than that of BMFs.f BMFs fits the characteristics of MFBs;(4)After silencing HDAC5,the secretion,contraction and migration functions of MFBs were significantly decreased,and the phosphorylation level of Smad2/3 protein was significantly decreased.Conclusion:(1)HDAC5 is highly expressed in OSF tissues and f BMFs,and targeting HDAC5 can inhibit the function of f BMFs,suggesting that HDAC5 is related to the pathogenesis of OSF;(2)Arecoline can induce the transdifferentiation of BMFs to MFBs,and the optimal induction concentration is 20μg/ml;(3)Down-regulation of HDAC5 expression level can significantly inhibit the pro-fibrotic function of MFBs induced by arecoline;(4)HDAC5 may exert its fibrogenic effect by acting on the TGF-β/Smad signaling pathway.