清润方含药血清通过支链α-酮酸脱氢酶调控BCAAs/mTOR信号通路改善HepG2细胞胰岛素抵抗的作用机制探讨  

Mechanism of Qingrun Prescription-containing Serum Improving Insulin Resistance in HepG2 Cells via Branched-chainα-keto Acid Dehydrogenase Regulation of Branched-chain Amino Acids(BCAAs)/mTOR Pathway

作　　者：卜祥伟 郝晓晖 张润云[1] 张美珍 王泽 王皓朔 王洁 倪青[1] 林兰[1] BU Xiangwei;HAO Xiaohui;ZHANG Runyun;ZHANG Meizhen;WANG Ze;WANG Haoshuo;WANG Jie;NI Qing;LIN Lan(Guang'anmen Hospital,China Academy of Chinese Medicine Sciences,Beijing 100053,China;First Teaching Hospital of Tianjin University of Traditional Chinese Medicine(TCM),Tianjin 300381,China;Shunyi Hospital,Beijing TCM Hospital,Beijing 101300,China)

机构地区：[1]中国中医科学院广安门医院,北京100053 [2]天津中医药大学第一附属医院,天津300381 [3]北京中医医院顺义医院,北京101300

出　　处：《中国实验方剂学杂志》2025年第9期90-98,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81973685,82305212)。

摘　　要：目的:探讨清润方含药血清对HepG2细胞胰岛素抵抗的改善作用及其潜在作用机制。方法:采用1×10^(-6)mol·L^(-1)胰岛素诱导HepG2细胞建立胰岛素抵抗模型,利用siRNA沉默支链α-酮酸脱氢酶(BCKDH)基因,分为8组。分别为正常组,模型组(1×10^(-6) mol·L^(-1)胰岛素),二甲双胍组(1 mmol·L^(-1)二甲双胍),清润方高、中、低剂量组(20%、10%、5%清润方含药血清),清润方+si-BCKDH组(10%清润方含药血清+si-BCKDH),清润方+si-NC组(10%清润方含药血清+si-NC)。葡萄糖试剂盒检测细胞上清液葡萄糖含量。糖原检测试剂盒检测糖原含量。采用超高效液相色谱-质谱串联法(UHPLC-MS/MS)检测支链氨基酸(BCAAs)、支链酮酸(BCKAs)水平,实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)分别检测BCKDH、含DEP结构域的哺乳动物雷帕霉素靶蛋白(mTOR)相互作用蛋白(DEPTOR)、mTOR、核糖体蛋白S6激酶1(S6K1)的mRNA转录及蛋白表达水平。结果:与正常组比较,模型组葡萄糖消耗量、糖原含量显著降低,BCAAs及BCKAs水平显著升高,BCKDH、DEPTOR表达显著下调,mTOR、S6K1表达显著上调(P<0.01)。与模型组比较,清润方各剂量组葡萄糖消耗量及糖原含量显著升高,BCAAs及BCKAs水平显著降低(P<0.01),清润方高、中剂量组BCKDH mRNA转录及蛋白表达水平均显著上升,DEPTOR mRNA转录水平均明显上升,清润方各剂量组DEPTOR蛋白表达水平均显著上升,mTOR、S6K1 mRNA转录及蛋白表达水平均明显下调(P<0.05,P<0.01)。与清润方+si-NC组比较,清润方+si-BCKDH组BCAAs及BCKAs明显升高,BCKDH mRNA转录及蛋白表达水平明显降低,DEPTOR mRNA转录及蛋白表达明显下调,mTOR、S6K1 mRNA转录及蛋白表达明显上调(P<0.05,P<0.01)。结论:清润方可能通过上调BCKDH抑制BCAAs和BCKAs,进而抑制mTOR通路激活,改善BCAAs代谢重编程介导的胰岛素抵抗。Objective:To investigate the effect of Qingrun prescription(QRP)-containing serum on improving insulin resistance in HepG2 cells and its potential mechanisms.Methods:An insulin resistance model was established in HepG2 cells with 1×10^(-6) mol·L^(-1) insulin.Branched-chainα-keto acid dehydrogenase(BCKDH)gene silencing was achieved using siRNA,and the cells were divided into 8 groups:normal group,model group(1×10^(-6) mol·L^(-1) insulin),metformin group(1 mmol·L^(-1) metformin),high-,medium-,and low-dose QRP groups(20%,10%,and 5%QRP-containing serum,respectively),QRP+siRNA-silenced BCKDH(si-BCKDH)group(10%QRP-containing serum+si-BCKDH),and QRP+si-NC group(10%QRPcontaining serum+si-NC).Glucose levels in the supernatant were measured with a glucose assay kit,while glycogen content was assessed using a glycogen assay kit.Levels of branched-chain amino acids(BCAAs)and branched-chain keto acids(BCKAs)were determined using ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).mRNA transcription and protein expression levels of BCKDH,dishevelled,Egl-10,and pleckstrin(DEP)domain-containing mammalian target of rapamycin(mTOR)-interacting protein(DEPTOR),mTOR,and ribosomal protein S6 kinase 1(S6K1)were detected using real-time quantitative polymerase chain reaction(Real-time PCR)and Western blot.Results:Compared to the normal group,the model group exhibited significantly decreased glucose consumption and glycogen content,increased levels of BCAAs and BCKAs,downregulated expression of BCKDH and DEPTOR,and upregulated mTOR and S6K1 expression(P<0.01).In comparison to the model group,QRP treatment at all doses significantly enhanced glucose consumption and glycogen content while reducing BCAAs and BCKAs levels(P<0.01).The high-and medium-dose QRP groups demonstrated significant upregulation of BCKDH mRNA transcription and protein expression,as well as DEPTOR mRNA transcription.Moreover,the DEPTOR protein expression level was significantly increased in high-,medium-,and low-dose QRP groups,whi

关 键 词：清润方 2型糖尿病 胰岛素抵抗 代谢重编程 支链氨基酸 

分 类 号：R243[医药卫生—中医临床基础] R285[医药卫生—中医学] R289