| Objective:Nonalcoholic fatty liver disease(NAFLD) is a kind of clinical metabolic syndrome of liver steatosis. Its pathogenesis is not yet clear. Now it is thought that it is associated with "secondary".C-Jun amino terminal kinase(c-Jun N-terminal kinase, JNK) is also called as stress-activated protein kinase(SAPK)and JNK/SAPK pathway is a member of the MAPK signal transduction pathway.It is an important pathway in the pathogenesis of NAFLD. Exendin- 4 is glucagon peptide 1(the incretin hormone glucagon- like peptide 1, glp-1) analogues. In recent years, many studies have shown that exendin- 4 can delay the development of NAFLD to some extent, but so far, its mechanism is not known yet. The purpose of this paper is to investigate the intervention of glp-1 analogues exendin-4 to the toxicity of BRL 3A cells. As well as,to observe the influences to the expression of the cell split original activated protein kinase kinase(MKK4/7) and the changes of c-jun amino terminal kinase(JNK) in the phosphorylation level.Then it provides possible new targets for prevention and control of NAFLD.Methods: The BRL 3A cells were trained with DMEM medium containing10% fetal bovine serum in vitro under appropriate condition.Then we inoculated in six orifice plate at a concentration of some 1 x 105 / hole. After entering the logarithmic phase medium, the cells were experimentally intervened after being absorbed the medium and washed twice by PBS.1.Fat toxicity test :The cells were assigned to normal control group(DMEM broth only containing 10% FBS), palmitic acid(PA) intervention group(0.25mmol/L、0.5mmol/L、1.0mmol/L), respectively intervened for 24 h. At the same time,the cells were cultured with 1.0mmol/L PA for 12 h,24 h, 48 h. The cell apoptosis rate was detected by Annexin V- FITC/PI apoptosis kit.2.The effects of Exendin-4 intervention lipid toxic: We grouped the cells into1.0mmol/L PA intervention group, 10nmol/L Exendin-4 +1.0mmol/L PA intervention group, 50nmol/L Exendin-4 +1.0mmol/L PA intervention group,100nmol/L Exendin-4 +1.0mmol/L PA intervention group, respectively intervened for 24 h. We applied Annexin V- FITC/PI apoptosis kit to detect apoptosis rate.3.The effects of JNK activity to Exendin-4 intervention :The cells were divided into four groups for blank control group(DMEM broth only containing 10% FBS),1.0mmol/L PA intervention group, 1.0mmol/L PA+50nmol/L Exendin-4 group and1.0mmol/L PA+50nmol/L Exendin-4+0.25mmol/L SP600125 group. Triglyceride kits were applied to detect triglyceride levels in the cells; Annexin V-FITC/PI apoptosis kit was used to detect apoptosis rate; We adopted Reverse Transcription Polymerase Chain Reaction(rt-pcr) method to detect the expression of MKK4,MKK7, JNK mRNA at the genetic level and used Western Blot method to detect the expression of MKK4, MKK7, p-JNK protein at the molecular level.Statistical analysis: Statistical analyses were proceeded using SPSS statistical software(version13.0 SPSS Inc).All data was detected for normality test and F test.Numerical variables with a normal distribution were presented as the mean ? standard deviation. Comparisons among groups were conducted with One-Way ANOVA. The variance of the normal distribution data were followed by a SNK-q test to compare two individual groups and none of variance of the normal distribution data were followed by a game-Howell test. Data with non-normal were performed with non-parametric Kruskal-wallis test.Results:(1) Different concentration of PA intervened BRL 3A cells for 24 h,with the increase of PA concentration, BRL 3A cell apoptosis rate showed a trend of rise. Differences between the groups’ apoptosis had statistical significance(P <0.05). However when BRL 3A cell were treated with 1mmol/L PA for 12 h,compared with the blank control group, cell apoptosis rate did not see significant change(P > 0.05). When the intervention time prolonged to 24h、48h, the apoptosis rate was significantly higher. Compared with the blank control group, the difference was statistically significant(P < 0.05).(2)The cellular apoptosis rate wasgradually decreased when BRL 3A cells were stimulated with higher concentration of Exendin-4.Compared with the group intervened by PA, the difference was statistically significant(P < 0.05).(3) Compared with the normal group, the apoptosis rate and TG concentration of the group intervened by PA increased.The expression of MKK4、MKK7、JNK mRNA and MKK4、MKK7protein expression level both raised. JNK phosphorylation level also increased. TG concentration were improved and reduced respectively after addition of Exendin-4or the mixture of Exendin-4 and SP600125. The expression of MKK4、MKK7、JNK mRNA and MKK4 、 MKK7 protein expression level,JNK phosphorylation level both partly decreased in the presence of Exendin-4 or Exendin-4+SP600125( P<0.05).The improvement effect was more obvious in PA+Exendin-4+SP600125 group than PA+Exendin-4 group(P<0.05).Conclusion: Unnormal concentration palmitic acid has a certain nocuous effect to BRL 3A cells and increases cell apoptosis rate time-dose-dependently.Glucagon like peptide-1 analogue, Exendin-4, to a certain extent, can reduce BRL3 A cell fatty toxicity induced by palmitate and improve the function of cells to protect liver cells. This effect is also dose dependent. Its mechanism may be finished through influencing JNK signal transduction pathway. |
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