Zheng Chuiyang, Nie Huan, Pan Maoxing, Fan Wen, Pi Dajin, Liang Zheng, Liu Dongdong, Wang Fengzhen, Yang Qinhe, Zhang Yupei
School of Traditional Chinese Medicine, Jinan University, Guangzhou, China.
Accreditation Center of TCM Physician State Administration of Traditional Chinese Medicine, Beijing, China.
J Ethnopharmacol. 2024 Jan 10;318(Pt A):116967. doi: 10.1016/j.jep.2023.116967. Epub 2023 Jul 26.
Chaihu Shugan powder (CSP) plays an important role in the prevention and treatment of nonalcoholic fatty liver disease (NAFLD) through a variety of biological mechanisms. However, whether the mechanism involves microRNA (miRNA) regulation remains unknown.
To investigate the effects of CSP on the miRNA expression profile of rats with NAFLD induced by high-fat diet (HFD), and to explore the mechanism of CSP in the treatment of NAFLD.
NAFLD rat models were established by an 8-week HFD. The therapeutic effects of CSP on NAFLD were evaluated by physiological, biochemical and pathological analysis and hepatic surface microcirculation perfusion test. MicroRNA sequencing was used to study the effect of CSP on the miRNA expression profile of NAFLD rats, and the target genes of differentially expressed (DE) miRNAs were predicted for further function enrichment analysis. Next, targets of CSP and NAFLD were collected by a network pharmacological approach, and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis were performed for the common target genes of CSP, NAFLD and DE miRNAs, and the expression levels of key genes and proteins were verified by quantitative Real-time PCR and Western blot. Finally, a network among formula-herb-compound-miRNA-target-biological processes-disease was established to explained the complex regulation mechanism of CSP on NAFLD.
The results showed that CSP significantly improved liver lipid accumulation, serum lipid and transaminase levels and liver surface microcirculation disturbance in HFD-induced NAFLD rats. The intervention of CSP reversed the high expression of 15 miRNAs in liver tissues induced by HFD, including miR-34a-5p, miR-146a-5p, miR-20b-5p and miR-142-3p. The results of pathway and functional enrichment analysis showed that, CSP might play an anti-NAFLD role via regulating DE miRNAs related to fatty acid metabolic process. Combined with the network pharmacological analysis, it was found that the DE miRNAs might affected the fatty acid biosynthesis pathway in the treatment of NAFLD by CSP. Molecular biology experiments have conformed the decreased the gene and protein levels of acetyl-CoA carboxylase alpha (ACACA), fatty acid synthase (FASN) and other fatty acid biosynthesis related enzymes on NAFLD rats after intervention of CSP.
CSP can significantly reduce hepatic lipid accumulation of NAFLD rat model induced by HFD, and its mechanism may be through the action of 15 miRNAs such as miR-34a-5p, miR-146a-5p, miR-20b-5p and miR-142-3p. Reduce the gene and protein expression levels of ACACA, FASN and other fatty acid biosynthesis related enzymes, thus reducing fatty acid biosynthesis. Based on an epigenetic perspective, this study explains the key anti-NAFLD mechanism of CSP via combination of microRNA sequencing and network pharmacological analysis, providing a new reference for the modernization of traditional Chinese medicine.
柴胡疏肝散（CSP）通过多种生物学机制在非酒精性脂肪性肝病（NAFLD）的防治中发挥重要作用。然而，其机制是否涉及微小RNA（miRNA）调控尚不清楚。
探讨CSP对高脂饮食（HFD）诱导的NAFLD大鼠miRNA表达谱的影响，以及CSP治疗NAFLD的机制。
通过8周的HFD建立NAFLD大鼠模型。通过生理、生化和病理分析以及肝脏表面微循环灌注试验评估CSP对NAFLD的治疗效果。采用miRNA测序研究CSP对NAFLD大鼠miRNA表达谱的影响，并预测差异表达（DE）miRNA的靶基因以进行进一步的功能富集分析。接下来，通过网络药理学方法收集CSP和NAFLD的靶点，对CSP、NAFLD和DE miRNA的共同靶基因进行京都基因与基因组百科全书（KEGG）和基因本体论（GO）分析，并通过定量实时PCR和蛋白质印迹法验证关键基因和蛋白质的表达水平。最后，建立方剂-草药-化合物-miRNA-靶点-生物过程-疾病网络，以解释CSP对NAFLD的复杂调控机制。
结果表明，CSP显著改善了HFD诱导的NAFLD大鼠的肝脏脂质蓄积、血脂和转氨酶水平以及肝脏表面微循环障碍。CSP的干预逆转了HFD诱导的肝脏组织中15种miRNA的高表达，包括miR-34a-5p、miR-146a-5p、miR-20b-5p和miR-142-3p。通路和功能富集分析结果表明，CSP可能通过调节与脂肪酸代谢过程相关的DE miRNA发挥抗NAFLD作用。结合网络药理学分析发现，DE miRNA可能在CSP治疗NAFLD中影响脂肪酸生物合成途径。分子生物学实验证实，CSP干预后NAFLD大鼠乙酰辅酶A羧化酶α（ACACA）、脂肪酸合酶（FASN）等脂肪酸生物合成相关酶的基因和蛋白水平降低。
CSP可显著降低HFD诱导的NAFLD大鼠模型的肝脏脂质蓄积，其机制可能是通过miR-34a-5p、miR-146a-5p、miR-20b-5p和miR-142-3p等15种miRNA的作用。降低ACACA、FASN等脂肪酸生物合成相关酶的基因和蛋白表达水平，从而减少脂肪酸生物合成。本研究从表观遗传学角度，通过miRNA测序与网络药理学分析相结合，阐释了CSP抗NAFLD的关键机制，为中药现代化提供了新的参考。
