Effects And Mechanisms Of N-acetyl-seryl-aspartyl- Lysyl-proline Inhibited The Transformation Of Skin Myfibroblasts Through Rho GTPase Signal

Objectives To investigate whether N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)can inhibit the fibroblast migration and myofibroblast transformation induced by transforming growth factor-?1(TGF-?1)through blocking the Rho GTP Pase.Methods Primary cultured neonatal rat fibroblasts were cultured in the second passage.The experimental groups were: 1 blank control group: fibroblasts were cultured in serumfree medium,2 TGF-?1 induction group: fibroblast treated with TGF-?1 for 48 h,TGF-?1was added at a final concentration of 100 nmol under serum-free culture conditions,3 AcSDKP pretreatment Group: fibroblasts treated with 100 nmol of AC-SDKP for 1h,followed by addition of 100 nnol of TGF-?1 added to serum-free medium for 48 h.Scratch test to detect cell migration ability,immunocytochemical detection of ?-smooth muscle actin(?-SMA),type I collagen(Col I),Ras-related C3 botulinum toxin substrate(Rac1),Rho-associated coiled coiled protein kinase(ROCK),cell division cycle protein 42(CDC42),Ras homolog gene family,member A(Rho A)protein expression.Western-blot assay for ?-SMA,serum response factor(SRF),myocardin related transcription factor A(MRTF-A),type I collagen(Col I),Rac1,ROCK,type III collagen(Col III),Rho A and CDC42 protein expression.Results 1 The sctatch results showed that compared with the control group,TGF-1 can significantly promote the migration of neonatal rat fibroblasts to the scratched blank area.Pretreatment with Ac-SDKP and TGF-?1 induction can significantly reduce cell migration and the difference is statistically significant.2 The results of immunocytochemistry showed that the expression of protein ?-SMA and Rac1,ROCK,Rho A,CDC42 and Col1 were strongly detected intracellularly after TGF-?1-treatment compared with the blank control group,while in the Ac-SDKP pretreatment group the expression of protein ?-SMA,Rac1,ROCK,Rho A,CDC42 and Col1 were significantly attenuated.3 Western-blot results showed that compared with the blank control group,in the TGF-?1 treatment group the expression of myofibroblasts transformation related protein ?-SMA,Col I,Col III,SRF and MRTF-A was up-regulated by 1.8-fold,2.8-fold,2-fold,1.69-fold and 1.76-fold respectively,meanwhile the expression of Rho GTPase-related proteins Rac1,CDC42,ROCK and Rho A was respectively up-regulated by 2-fold,1.43-fold,1.78-fold and 2.21-fold.Whereas in pretreated with Ac-SDKP group,the protein expression was decreased compare with the TGF-?1 treatment group.The expression of the myofiboblast transformation-related proteins ?-SMA,Col I,Col III,SRF and MRTF-A decreased by 57%,36%,71%,67.76% and 52.08% respectively,simultaneouslythe expression of Rho GTPase-related proteins Rac1,CDC42,ROCK and Rho A were also down-regulated by18.75%,51.52%,51.39% and 23.81% respectively(P<0.05).Conclusions Ac-SDKP can inhibit TGF-?1-mediated transformation and migration of skin myofibroblasts by regulating Rho GTPase signaling,and exert its anti-fibrotic effect.Figure10;Table4;Reference 153...