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Effects of Tetramethylpyrazine on Neuclear Factor-κB and Transforming Growth Factor-β1 in Human Hepatic Stellate Cells

Author: FanYuHui
Tutor: ZhuLin；HuaHaiYing
School: Zhengzhou University
Course: Pharmacology
Keywords: Hepatic fibrosis TMP TGF-β1 NF-κB HSC
CLC: R285.5
Type: Master's thesis
Year: 2008
Downloads: 25
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Abstract


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.

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