参苓白术散“健脾益气”治疗非酒精性脂肪肝的作用机理研究

The preliminary study in our group found that there are blood stasis syndromes in the early stage of nonalcoholic fatty liver disease of "no symptoms can been diagnostic," and "sputum" is associated with abnormal lipid and mitochondrial energy metabolism. “Strengthen spleen” and “Remove stasis” could be used to treat non-alcoholic fatty liver disease. The curative effect is clear for the treatment of NAFLD with the theory of "treating with liver and spleen together," its mechanism can be explored from the “Strengthen spleen” and “Tonifying primordial qi”. In our plan, to investigate the effect of "Shenling Baizhu powder"(A traditional Chinese medicine for “Strengthen spleen” and “Tonifying primordial qi”) on the intestinal flora and SCFAs in the NAFLD rats induced by an high-fat diet, and observe the changes characteristic of mitochondrial energy metabolism related pathways UCP2/AMPK/ ACC/IF1. Therefore, this subject will carry out 16S rRNA using the technology of high-throughput sequencing in mice intestinal flora, LC-MS detect the SCFAs, tissue oxygen consumption meter measure liver tissue mitochondrial respiration, HPLC detect ATP, ADP, AMP, Rhodamine123 test mitochondrial membrane potential, Western blot determineUCP2, AMPK, ACC and IF1. We believed this study will help us clarify the relation of the "qi" of traditional Chinese medicine and the "energy" of modern medicine. In addition, from the perspective of mitochondrial energy metabolism to discuss the molecular mechanisms of Shenling Baizhu powder treat NAFLD will reveal a scientific connotation, that is "treating with liver and spleen together" and “gut-liver axis”, the purpose is provide new strategies for prophylaxis and treatment of NAFLD.

项目组前期研究结果表明健脾、逐瘀可治疗非酒精性脂肪性肝病（NAFLD）。临床对NAFLD采用“肝脾同治”治法疗效明确，其作用机理可从健脾和益气两方面加以探究。我们拟探讨健脾益气经典方“参苓白术散”对高脂饮食诱导的NAFLD大鼠肠道菌群及其代谢产物短链脂肪酸（SCFAs）的影响（健脾机理），观察线粒体能量代谢相关通路UCP2/AMPK/ACC/IF1的变化情况（益气机理）。本课题将对小鼠肠道菌群16S rRNA进行高通量测序，LC-MS法检测SCFAs，生物组织氧耗仪测定肝脏细胞线粒体呼吸，HPLC法检测ATP、ADP、AMP，Rhodamine123测定线粒体膜电位，Western blot法测定UCP2、AMPK、ACC、IF1。本研究将有助于从微观角度揭示“肝脾同治”的科学内涵，为NAFLD的防治提供新思路和新策略。

临床对脂肪肝采用“肝脾同治”治法疗效明确，中医认为脾为后天之本，固护脾胃亦是日常养生保健的关键。参苓白术散可改善非酒精性脂肪性肝病的症状，然而其作用机制尚不清楚。我们采用高脂饮食建立小鼠非酒精性脂肪性肝病模型，给予不同药物干预，测定血清生化指标：ALT、AST、TC、TG、FBG、LDL-C、HDL-C、TBIL；肝脏病理指标：HE和油红O染色；肠道菌群：用16S rDNA扩增子测序技术分析菌群的多样性和菌群丰度；短链脂肪酸：通过GC-MS法检测肠道内容物短链脂肪酸的含量；取肝组织检测线粒体膜功能指标：线粒体膜电位、ATP；肝脏线粒体呼吸功能指标：Ⅳ态呼吸（ST4）、III态呼吸（ST3）、线粒体呼吸控制率（RCR）、磷氧比（P/O）、氧化磷酸化效率（OPR）、ATP；WB法检测肝脏线粒体相关通路蛋白含量：解偶联蛋白2（UCP2）、腺苷酸活化蛋白激酶 (AMPK)、乙酰辅酶A羧化酶（ACC）、F1Fo ATP合成/水解酶的抑制因子1(IF1)。研究结果表明参苓白术散通过发挥健脾功效，维护肠道菌群稳态，影响菌群代谢产生的短链脂肪酸（乙酸、丙酸和丁酸），调节肝脏线粒体能量代谢的UCP2/AMPK/ACC/IF1信号通路，来改善非酒精性脂肪性肝病小鼠肝脏线粒体膜通透性、呼吸状态、氧化磷酸化效率，增加肝脏ATP含量，起到益气疏肝的作用。项目达到了预期目标，研究成果发表论文4篇，获批实用新型专利1件，培养研究生3名。该研究可为阐释中医中药的用药机理提供一定的理论支持。