Mechanism Of Benign Biliary Stricture And Effects Of Tetramethylpyrazine And Dexamethasone

Background: Benign biliary stricture(BBS)includes several different clinical features due to different etiologies.In BBS fibroblasts active proliferation and extracellular matrix active accumulation;fibroblasts differentiate into myofibroblasts featured α-smooth muscle actin(α-SMA);transforming growth factor-β1(TGF-β1)play an important role.P311 gene and tumor necrosis factor-α(TNF-α)play an important role in skin healing;however their roles in progression of BBS are unknown.Tetramethylpyrazine(TMP)has been reported to inhibit liver and kidney fibrosis by inhibiting fibroblasts proliferation and expression of TGF-β1.Dexamethasone(DEX)has been used for therapy in skin keloid disease.However,the function and mechanism of TMP/DEX in BBS have not been elucidated to date.Aims of the study were:(1).Changes of P311,TGF-β1 and α-SMA genes and effects of TMP/DEX on these genes in BBS model fibroblasts(MF)and normal biliary fibroblasts(NF);(2).Effects of TNF-α and TMP/DEX on TGF-β1,α-SMA and P311 genes in NF;(3).Effects of TMP/DEX on TGF-β1,α-SMA and P311 in BBS model tissues(1-6w).Methods: 1.BBS model was made and then MF/NF was cultivated.(1).MF/NF was treated by TMP/DEX/TMP+DEX.(2).Cells proliferation was studied by CCK-8.(3).Relative m RNA expression of TGF-β1,α-SMA and P311 were assayed by RT-PCR.(4).Protein expression of TGF-β1,α-SMA was investigated by western blot.2.NF was treated by TNF-α or TNF-α+TMP /DEX /TMP+DEX.Cells proliferation was studied by CCK-8 assay.Relative m RNA expression of TGF-β1,α-SMA and P311 were assessed by RT-PCR.Relative proteins expressions of TGF-β1,α-SMA were investigated by western blot.3.BBS model was copied in 78 rabbits during 1w,3w and 6w,and then these rabbits were treated by DEX /TMP /DEX +TMP every day except control group.Relative m RNA expression of TGF-β1,α-SMA and P311 were assessed by RT-PCR.Relative proteins expressions of TGF-β1,α-SMA were investigated by western blot.Results: 1.In MF,up-regulation expression of P311 m RNA(P<0.05)was positive correlation to TGF-β1 and α-SMA(R: 0.801 and 0.904,P<0.01).TMP/DEX/TMP plus DEX significantly attenuated cells proliferation and relative m RNA/protein expression of TGF-β1,P311 and α-SMA in MF/NF(P<0.05),and the inhibiting effect in MF was more significant than NF(P<0.05).2.TNF-a significantly promoted cells proliferation and up-regulated m RNA/protein expression of TGF-β1,P311 and α-SMA in NF(P<0.05),and the expressions of TGF-β1,P311 and α-SMA in NF by treated TNF-a reached 58.77%-96.56% of those in MF.However,the promoted effect of TNF-a on NF were significantly inhibited by DEX /TMP(P<0.05).3.In BBS model tissues,up-regulation of P311 was positive correlation to TGF-β1 and α-SMA(R: 0.905 and 0.916,P<0.01).The m RNA/protein expression of TGF-β1,P311 and α-SMA in BBS tissues were significantly up-regulated(P<0.05)from 1w to 6w.TMP/ DEX/TMP +DEX significantly down-regulated the relative m RNA/protein expression of TGF-β1,P311 and α-SMA in BBS model tissues(P<0.05).Conclusions:1.The up-regulation of P311 was positive relation to TGF-β1and α-SMA.It was suggested that the P311 gene was main gene of regulation and relative to BBS.2.TNF-α may be main cytokines stimulating P311 gene etc and relation with pathogenesis of BBS progression.3.The m RNA/protein expression of P311,TGF-β1 and α-SMA in BBS tissues were significantly up-regulated from 1w to 6w,it is suggested that using anti-scar drug in late of wound healing may be useful.4.TMP/DEX/TMP+DEX significantly inhibited TGF-β1,P311 and α-SMA m RNA/protein expression and the fibroblasts proliferation,and may be use for prevent BBS.