| Objective: To establish the effect of MFA on the expression of nicotinamide ade-nine dinucleotidephosphate oxidase 4,reactive oxygen species level and expression of ?-smooth muscle actin and procollagen?in the HSC-LX2 cells stimulated with transforming growth factor ?1 in vitro.Methods:HSC-LX-2 were cultured in vitro as blank group and using 1 ?g/L TGF-?1 to stimulate HSC-LX-2 as model group.According to MTT,the different concentrations of MFA were detected by HSC-LX-2 proliferation and human hepatocytes(L-O2).Then HSC-LX-2 were stimulated by TGF-?1 and treated with different concentrations of MFA(1.25,2.5,5 mg/L)for 72 hours.The mRNA expressions of Nox4 and ?-SMA were determined by Real-time quantitative PCR and the protein expressions of Nox4 and ?-SMA were determined by Western blot.ROS levels were tested by in situ loading probe method.The mRNA expressions of PC? were determined by Real-time PCRand the protein expressions of PC? were determined by ELISA.Results: 1.Using the MTT assay MFA effect on HSC-LX-2 cell proliferation and L-O2 activity.When the MFA concentration range(0.3125-5 mg/ L)between the concentration with the MFA,HSC-LX-2 cell growth inhibition rate gradually increased,while L-O2 absorptiometry value than the normal group elevated,MFA namely low concentration can promote the growth of L-O2.When the MFA concentration range(10-40 mg/L)between the concentration increased as the MFA,HSC-LX-2 inhibition rate gradually decreased absorption spectrophotometry value L-O2 decreased significantly compared with the normal group,that may be a high concentration of MFA L-O2 growth inhibition.So we use a relatively small dose MFA,MFA inhibition rate is relatively high treatment group(1.25mg/L,2.5 mg/L,5.0mg/L),between the groups was statistically significant difference(P <0.05).2.Detected by RT-PCR and Western blot method Nox4 mRNA and protein expression results of HSC-LX-2cells,with the MFA concentration gradually increased,mRNA and protein expression of Nox4 substantially decreased.The mRNA and protein expression of Nox4 on MFA of 5.0 mg/L were lower than 1.25mg/L group,2.5mg/L group,the difference was statistically significant(P <0.05).3.Using the situ loading probe to test the ROS level of HSC-LX-2.As the MFA concentration gradually increased,the fluorescence intensity of HSC-LX-2 within waning,the number of cells containing green fluorescence decreases.Compared with normal controlgroup,model group stronger fluorescence.4.Detected by RT-PCR and Western blot method ?-SMA mRNA and protein expression results of HSC-LX-2 cells,with the MFA concentration gradually increased,mRNA and protein expression of ?-SMA substantially decreased.The mRNA and protein expression of ?-SMA on MFA of 5.0 mg/L were lower than 1.25mg/L group,2.5mg/L group,the difference was statistically significant(P<0.05).5.The PC? mRNA and protein expression of HSC-LX-2 cells were detected by RT-PCR and ELISA.While the MFA affected HSC-LX-2 cells after 72 hours,with the MFA concentration gradually increased,PC?mRNA and protein expression of HSC-LX-2 cells substantially decreased.The mRNA and protein expression of PC? in the concentration of 5.0 mg/L MFA group were lower than the group of1.25mg/L,2.5 mg/L,the difference was statistically significant(P <0.05).Conclusion: 1.MFA can inhibit the proliferation of HSC-LX-2 cells,which from 0.3125 mg/L to 5 mg/L with the concentration showed dose-dependent inhibition was significantly increased,from 10 mg/L to 40 mg/L inhibition decreased with increasing concentration.2.Under the intervention of MFA in HSC-LX-2 cells maybe downregulate the expression of Nox4,?-SMA and PC?.In the concentration of 5.0 mg/L,the expression of the Nox4,?-SMA and PC?were lower than other groups.MFA maybe down-regulate the mRNA and protein expression of Nox4,?-SMA and PC?.And down-regulate the level of ROS.Thereby inhibiting the synthesis of HSC-LX2 ECM,inhibiting thetransformation of HSC-LX-2 phenotype.Ultimately inhibiting the occurrence of and development of hepatic fibrosis. |
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