基于肝细胞lncRNA-miRNA-mRNA调控网络和“核-线粒体”信号传递探讨解毒化瘀颗粒拮抗肝衰竭的机制研究

Liver failure is the most serious type of liver disease in clinic，marked by a high mortality. Previous research suggests that Jie Du Hua Yu Granule have significant mitochondrial protection effect and its efficacy in treating liver failure have been achieved in multiple aspects. Therefore, the investigation of the antagonistic mechanism of Jie Du Hua Yu Granule on Liver Failure based on the global regulation and multi-target theory of traditional Chinese medicine treatment is crucial. This project intends to establish D-GalN+LPS rat model of liver failure for exploring the genetic mechanism of Jie Du Hua Yu Granule on the treatment of liver failure. Three aspects will be investigated: ⑴ Multi-angle and comprehensive evaluation of Jie Du Hua Yu Granule on mitochondrial function improvement of liver cells;⑵Identification of lncRNA-miRNA-mRNA interaction network through high-throughput whole transcriptome sequencing and systematic bioinformatic analysis;⑶The types and expression levels of lncRNA and miRNA in the mitochondria of rat liver cells were detected by high-throughput whole transcriptome sequencing. Differentially expressed RNA among groups and possible RNA molecule signal transmission between "nucleus-mitochondria" will be explored using systematic bioinformatic analysis by comparing sequencing results from liver cell total RNA and mitochondria RNA extracts. This project is expected to comprehensively explore the target of Jie Du Hua Yu Granule for liver failure intervention from the perspective of genetic regulation and genetic signal transmission between"nucleus-mitochondria", and provide new perspective for prevention and treatment of severe liver diseases such as liver failure by traditional Chinese medicine.

肝衰竭是临床上严重的肝病类型，死亡率高，前期研究发现，解毒化瘀颗粒具有显著的肝细胞线粒体保护作用，并从多层面验证其拮抗肝衰竭具有满意疗效，因此基于中药的全局和多靶点治疗理论探寻解毒化瘀颗粒治疗肝衰竭的作用机制至关重要。本研究拟建立D-GalN+LPS急性肝衰竭大鼠模型，以解毒化瘀颗粒干预肝衰竭的遗传学作用机制为切入点开展如下研究：⑴综合评估解毒化瘀颗粒对肝衰竭大鼠肝细胞线粒体功能的影响；⑵利用高通量全转录组测序技术和生物信息学分析技术完成大鼠肝细胞内lncRNA-miRNA-mRNA调控网络分析；⑶利用高通量测序检测肝衰竭大鼠肝细胞线粒体内RNA的种类和表达水平，结合肝脏组织总RNA的测序数据，分析是否存在“核-线粒体”之间的RNA分子信号传递。旨在从遗传调控和“核-线粒体”之间的遗传分子信号传递的角度探讨解毒化瘀颗粒拮抗肝衰竭新的作用靶点，为中医药防治肝衰竭提供一种新的思路与方法。

肝衰竭是临床上严重的肝病类型，死亡率高，至今尚缺乏确切有效的治疗药物，深入探究治疗机制及药物至关重要。前期研究发现解毒化瘀颗粒具有显著的肝细胞线粒体保护作用，并从多层面验证其拮抗肝衰竭具有满意疗效，因此基于中药的全局和多靶点治疗理论探寻解毒化瘀颗粒治疗肝衰竭的作用机制至关重要。本研究采用建立D-GalN+LPS急性肝衰竭大鼠模型，以解毒化瘀颗粒干预肝衰竭的遗传学作用机制为切入点开展研究。通过检测临床常用指标AST、ALT等、大鼠肝脏组织HE染色、TUNEL染色改变作为评价大鼠肝功能指标。结果提示：解毒化瘀颗粒干预组大鼠MDA、AST、ALT、胆红素水平较模型组大鼠显著下调（p＜0.05）；SOD、GSH-XP、ATP水平较模型组显著上调（p＜0.05）。结合肝组织高通量全转录组测序结果及生物信息学分析，构建lncRNA-miRNA-mRNA-miRNA与“核-线粒体”的调控网络，并进行Western-blot验证，得出解毒化瘀颗粒可能通过调控lncRNA-miRNA-mRNA互作网络，促进线粒体结合达到改善肝衰竭情况起到治疗作用的结论。本项目揭示了中药解毒化瘀颗粒的干预肝衰竭的可能靶点，为肝衰竭的防治提供了新思路、新方法。