基于miR-23a-3p的扶正通络法抗肝纤维化的机制

Mechanism of anti-liver fibrosis by reinforcing the healthy qi and promoting blood circulation method based on miR-23a-3p

目的:研究扶正通络法验方芪甲柔肝方抗肝纤维化的疗效与机制。方法:采用皮下注射四氯化碳(CCl4)花生油+10%乙醇代替饮水的方式建立大鼠肝纤维化模型,随机将大鼠分为空白组,模型组,芪甲柔肝方高、中、低剂量组及鳖甲煎丸组,分别以0.9%氯化钠溶液,高、中、低剂量芪甲柔肝方水煎剂,鳖甲煎丸混悬液灌胃,疗程为6周。检测肝功能、肝指数与血清纤维化标志物以判断疗效,并用qRT-PCR检测大鼠肝miR-23a-3p的表达,结合生物信息学分析其调控机制。结果:(1)与空白组比较,模型组大鼠ALT、AST、肝指数及血清纤维化标志物(PⅢP、PCⅣ、HA、LN)显著升高(P<0.05),肝组织显示明显的炎症与纤维化,并出现大量假小叶。与模型组比较,芪甲柔肝方高、中、低剂量组及鳖甲煎丸组均能在一定程度上改善上述指标,其中芪甲柔肝方高剂量组与鳖甲煎丸组疗效最为显著;(2)与空白组比较,模型组miR-23a-3p表达显著降低(P<0.05),芪甲柔肝方高剂量组与鳖甲煎丸组能显著升高miR-23a-3p的表达(P<0.05);(3)miR-23a-3p的靶基因主要参与调控p53、磷脂酰肌醇等通路。结论:芪甲柔肝方治疗CCl4+乙醇的复合造模诱导的大鼠肝纤维化,可显著改善肝功能,降低血清纤维化标志物,减轻肝组织的炎症及纤维化程度,其作用机制可能与上调miR-23a-3p的表达,调控p53、磷脂酰肌醇等通路有关。

Objective: To study the efficacy and anti-fibrosis mechanism of Radix Astragali and Carapax Trionycis Formula(RCF) by method of reinforcing the healthy qi and promoting blood circulation. Methods: The model of liver fibrosis in rats was established by subcutaneous injection of CCl4 peanut oil+10% alcohol in place of water. After judging the success of modeling, the rats were divided into control group, model group, RCF high-dose, medium-dose, low-dose group and Biejiajian Wan group. Gavage treatment was performed separately. The control and the model group were given saline, RCF highdose, medium-dose and low-dose group were treated with RCF decoction, and the Biejiajian Wan group was given Biejiajian Wan suspension. After 6 weeks of treatment, the rats were sacrificed, and liver function, serum liver fibrosis markers and were measured, and the expression of miR-23 a-3 p in liver tissue of each group was detected by RT-qPCR technology. The bioinformatics technique was used to predict the target genes of miR-23 a-3 p and analyze their functions. Results:(1)Compared to the control group, the model group displayed significant higher index of ALT, AST, liver index, serum fibrosis markers(PⅢP, PCIV, HA, LN)(P<0.05) and sever hepatic inflammation and fibrosis. A large number of pseudo lobules were observed in the model group. While all of the RCF high-dose, medium-dose, low-dose group and the Biejiajian Wan group could improve the above index to a certain extent compared with the model group, above which the efficacy of the RCF high-dose group and the Biejiajian Wan group were found the most significant.(2)In comparison with the control group, the expression of miR-23 a-3 p was significantly decreased in the model group(P<0.05), whereas its expression was notably increased in the RCF high-dose group and the Biejiajian Wan group(P<0.05).(3)The results of bioinformatics analysis suggest that miR-23 a-3 p is mainly involved in the regulation of p53, phosphatidylinositol signal pathway. Conclusion: RCF-HD can significantly reduce liver tissue inflammation and fibrosis, and reduce collagen fiber percentage. The anti-fibrosis effect may related to upregulating miR-23 a-3 p and regulate p53, phosphatidyl inositol signals.