基于MAPK信号通路探讨荔枝核总黄酮对HSC-T6细胞的作用

Total flavonoids of litchi seed inhibit HSC-T6 cells via MAPK signaling pathway

目的观察荔枝核总黄酮对HSC-T6细胞中丝裂原活化蛋白激酶(MAPK)信号通路相关蛋白表达的影响,探讨荔枝核总黄酮是否可通过调控MAPK信号通路影响肝纤维化的进程。方法设置荔枝核总黄酮20μg/mL、40μg/mL、80μg/mL、160μg/mL、320μg/mL组和细胞对照组、空白组,CCK-8法检测各组细胞吸光度,计算细胞增殖抑制率,选择最佳的荔枝核总黄酮浓度进行后续实验。后续实验设置荔枝核总黄酮组、秋水仙碱组、细胞对照组及空白组,加入相应培养基培养24 h、48 h、72 h后,CCK-8法检测各组细胞吸光度,计算细胞增殖抑制率;各组细胞培养72 h后,透射电镜观察细胞的超微结构,ELISA法检测细胞中基质金属蛋白酶-2(MMP-2)、Ⅰ型胶原(Col-Ⅰ)、Ⅲ型胶原(Col-Ⅲ)水平,RT-PCR法检测细胞中c-Jun氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)、p38 mRNA表达情况,免疫组化法检测细胞中JNK、ERK、p38蛋白及其相应磷酸化蛋白(p-JNK、p-ERK、p-p38)表达情况。结果荔枝核总黄酮的最佳实验浓度为160μg/mL,最佳干预时间为72 h;与细胞对照组比较,荔枝核总黄酮组和秋水仙碱组培养24 h、48 h、72 h后的吸光度均明显降低(P均<0.05)。培养72 h后,荔枝核总黄酮组细胞之间间隙变大,细胞膜四周纤毛脱落,细胞浆内线粒体嵴断裂或消失,部分线粒体呈空泡状,粗面内质网扁池扩张,附着的核糖体脱粒,细胞核内染色质浓缩凝结,部分染色质边聚,核膜皱缩,可见凋亡小体形成;荔枝核总黄酮组、秋水仙碱组MMP-2、Col-Ⅰ、Col-Ⅲ水平均明显低于细胞对照组(P均<0.05),秋水仙碱组MMP-2水平明显低于荔枝核总黄酮组(P<0.05);荔枝核总黄酮组JNK、ERK、P38 mRNA相对表达量和p-JNK、p-ERK、p-P38蛋白相对表达量均明显低于细胞对照组(P均<0.05),与秋水仙碱组比较差异均无统计学意义(P均>0.05)。结论荔枝核总黄酮具有抗肝纤维化的生物学效应,其作用机制可能与调控MAPK信号通路相关。

Objective It is to observe the effects of total flavonoids of litchi seed(TFLS)on the expression of MAPK signaling pathway related proteins in HSC-T6 cells,and to explore whether TFLS can reverse the process of liver fibrosis via regulating MAPK signal pathway.Methods The TFLS groups of 20μg/mL,40μg/mL,80μg/mL,160μg/mL,320μg/mL,cell control group and blank groups were set up,the absorbance of the cells in each group was detected by CCK-8 method to calculate cell proliferation inhibition rate,and the optimal concentration of TFLS was selected to carry out the subsequent experiments.Then TFL group,colchicine group,cell control group and blank group were set up and cultured in respective culture medium.After 24 h,48 h,72 h of culture,the absorbance of of cells in each group was detected by CCK-8 method,and the cell proliferation inhibition rate was calculated;after 72 h of culture,the ultrastructure of cells was observed by transmission electron microscopy,the levels of matrix metalloproteinase-2(MMP-2),collagen typeⅠ(Col-Ⅰ),and collagen typeⅢ(Col-Ⅲ)were detected by ELISA,the expressions of c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),and p38 mRNA were detected by RT-PCR,and the expressions of JNK,ERK,p38 proteins and their corresponding phosphorylated kinase(p-JNK,p-ERK,and p-p38)in cells were detected by immunohistochemistry.Results The optimal concentration of TFLS was 160μg/mL and the optimal intervention time was 72 h.Compared with the cell control group,the OD values of TFLS group and colchicine group after 24 h,48 h and 72 h of culture were significantly decreased(all P<0.05).After 72 h of culture in the TFLS group,the cell gap became larger,the cilia around the cell membrane dropped,the mitochondrial cristae in cytoplasm were broken or disappeared,some mitochondria were vacuolated,the flat pool of rough endoplasmic reticulum was dilated with attached ribosomes degranulated,the chromatin in nucleus was condensed with some margination and crumpled nuclear membrane,and formed apoptotic vesicles were found;the levels of MMP-2,Col-Ⅰand Col-Ⅲin the TFLS group and colchicine group were significantly lower than those in the cell control group(all P<0.05),and the level of MMP-2 in the colchicine group was significantly lower than that in the TFLS group(P<0.05);the relative expressions of JNK,ERK and P38 mRNA,and the relative expressions of p-JNK,p-ERK,p-P38 proteins in the TFLS group were significantly lower than those in the cell control group(all P<0.05),and none of the differences were statistically significant when compared with the colchicine group(all P>0.05).Conclusion TFLS has biological effects on liver fibrosis,and its mechanism may be related to regulating MAPK signaling pathway.