Effects Of Tetramethylpyrazine On Neuclear Factor-κb And Transforming Growth Factor-β₁ In Human Hepatic Stellate Cells

Object:To investigate the effects of tetramethylpyrazine on signal transduction induced by neuclear factor-κB and transforming growth factor-β₁ in human hepatic stellate cells.To explore the anti-hepatic fibrosis mechanisms of tetramethylpyrazine by studying the expressions ofα-smooth muscle actin and proteins of Neuclear factor -κB p65,and transforming growth factor-β₁ mRNA,typeⅠand typeⅡTGF-βreceptor mRNA,Smad 3 and Smad 7 mRNA.Method:1,Cells Culture:Used human hepatic stellate cells(Lx-2) which the phenotype activates to carry on vitro culture.They were cultured in DMEM supplemented with 4mmol/L L-glutamine,10%fetal calf serum,100 U/mL penicillin,and 100 U/mL streptomycin at 37℃in a humidified atmosphere of 5%CO₂ and 95%air.2,Experimental groups:The experiment is divided into different concentrations of TMP groups,the positive control groupand the negative control group.3,Lx-2 cells were seeded onto culture slides and cultured in medium with different concentrations of TMP.48 hours later,the expression ofα-SMA in Lx-2 cells was detected by the method of S-P immunohistochemistry stain.The concentration of antibody for immunohistochemistry was 1:100.4,Lx-2 cells were seeded onto culture slides and cultured in medium with different concentrations of TMP.48 hours later,NF-κB expression translocation was detected by the method of S-P immunohistochemistry stain.The concentration of antibody for immunohistochemistry was 1:100.5,Lx-2 cells were seeded in the culture plates and were treated with different concentrations of TMP.RNA was extracted from Lx-2 cells using total RNA isolation reagent.RNA integrity was assessed and purity was analyzed.Then RT-RNA was performed.Results:1,Compared with negative control group,the level ofα-SMA expression in TMP groups and positive control group were lower.The difference had significant means (P＜0.01);Compared with positive control group,the level ofα-SMA expression in TMP groups(200 mg/L,400 mg/L,800 mg/L) were lower.The difference had significant means(P＜0.05 or P＜0.01).This effection had good dose dependablity.2,The level of NF-κB expression in TMP groups and positive control group were significantly decreased compared with negative control group(P＜0.01);Compared with positive control group,TMP groups(200 mg/L,400 mg/L,800 mg/L)decreased NF-κB level significantly(P＜0.05 or P＜0.01).3,Compared with the negative control group,TMP groups and the positive control group of TGF-β₁ mRNA expression of both significant difference(P＜0.01);Compared with the positive control group,TMP 800mg/L concentration of TGF-β₁ mRNA expression of a obvious reduction,there were significant differences(P＜0.01).4,Compared with the negative control group,TMP 200 mg/L,800 mg/L groups of TβRⅠmRNA expression was decreased,the difference was significant(P＜0.05, P＜0.01);Compared with the positive control group,TMP 800 mg/L concentration of TβRⅠmRNA expression with significant differences(P＜0.01).5,Compared with the negative control group,the positive control group and TMP 200 mg/L and TMP 800 mg/L groups of TβRⅡmRNA relative expression of both significant difference(P＜0.01);Compared with the positive control group,TMP 200 mg/L and 800 mg/L groups of TβRⅡmRNA expression of a significant difference (P＜0.05 or P＜0.01). 6,Compared with the negative control group,the positive control group of Smad 3 mRNA expression levels were significantly decreased(P＜0.05),TMP 200 mg/L,800 mg/L groups of Smad 3 mRNA relative expression of both significant difference(P＜0.01);Compared with the positive control group,TMP 800 mg/L group of Smad 3 mRNA expression of the difference was significant(P＜0.01).7,Compared with the negative control group,TMP 200 mg/L and 800 mg/L groups of Smad 7 mRNA expression were significantly increased,the difference was significant(P＜0.01);Compared with the positive control group,TMP 800 mg/L group of Smad 7 mRNA expression of the difference was significant(P＜0.01).Conclusion:TMP reduces the expression ofα-SMA,a hepatic stellate cell activation symbol,it also reduces the expression of NF-κB and inhibits its nuclear transfer,reduces TGF-β₁ andⅠ,Ⅱreceptor and Smad 3 mRNA to express,simultaneously promotes Smad 7 mRNA to express.These results indicates that TMP may through blocking NF-κB and TGF-β/Smad signal transduction pathways to inhibit the activation of hepatic stellate cells.