川芎嗪通过激活AMPK改善高糖损伤心肌细胞H9c2的分子机制

目的：探讨川芎嗪对高糖诱导损伤H9c2细胞的保护作用。方法：采用CCK8法检测细胞活力；葡萄糖试剂盒考察40 mmol·L^-1高糖培养H9c2心肌细胞48 h后，川芎嗪对葡萄糖代谢消耗量的影响。采用浓度和时间依赖性试验确定川芎嗪对高糖刺激H9c2细胞保护作用的最佳处理时间和浓度。Western-blot检测细胞内能量感受器磷酸化腺苷酸活化蛋白激酶（phospho-adenosine monophosphate-activated protein kinase，p-AMPK）/腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase，AMPK）及胰岛素信号通路磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）、磷酸化蛋白激酶B（phospho-protein kinase B，p-Akt）/蛋白激酶B（protein kinase B，Akt）及自噬抑制子磷酸化-雷帕霉素靶蛋白（phospho-mammalian target of rapamycin，p-mTOR）/雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）的表达水平。结果：40 mmol·L^-1高糖刺激H9c2心肌细胞超过48 h，细胞活力明显下降（P<0.05）；葡萄糖消耗量显著减少（P<0.05）；使用0.1 mmol·L^-1川芎嗪预处理4 h后，与胰岛素作用相当，葡萄糖消耗量明显增加。同时，高糖诱导下调的p-AMPK/AMPK、PI3K及p-Akt/Akt的蛋白表达水平被0.1 mmol·L^-1川芎嗪显著逆转（P<0.05）；高糖诱导升高的mTOR的磷酸化水平也显著抑制（P<0.05），显示紊乱的自噬功能得到有效控制。结论：高糖下心肌细胞发生葡萄糖代谢紊乱，川芎嗪激活AMPK，一方面改善葡萄糖代谢紊乱，另一方面，通过激活胰岛素信号通路及自噬途径来缓解高糖对心肌细胞的损伤作用。  

Objective:To investigate the protective effects of tetramethylpyrazine on H9c2 cells injured by high glucose. Methods:Cell viability was measured by CCK8 assay. The glucose test kit was used to investigate the glucose remainder after H9c2 cells were stimulated with 40 mmol·L^-1 high glucose for 48 h. The concentration and time-dependent experiments were used to determine the optimal treatment time and concentration of tetramethylpyrazine in high glucose-stimulated H9c2 cells. The expression of P-AMPK/AMPK,PI3K,p-Akt/Akt and autophagy suppressor p-mTOR/mTOR were detected by western-blot. Results:The cell activity (P<0.05) and glucose consumption (P<0.05) were significantly decreased after H9c2 cells were treated with 40 mmol·L^-1 high glucose for more than 48 h. After pretreatment with 0.1 mmol·L^-1 tetramethylpyrazine for 4 h,the glucose consumption was significantly increased,which was equivalent to the action of insulin. At the same time,the high glucose-induced down-regulation of p-AMPK/AMPK,PI3K and p-Akt/Akt were significantly inhibited by tetramethylpyrazine (P<0.05). The high glucose-induced up-regulation of mTOR phosphorylation was also significantly inhibited (P<0.05),showing that the disordered autophagy function was effectively controlled. Conclusion:Tetramethylpyrazine can ameliorate the glucose metabolism disorder induced by high glucose by activating AMPK. On the other hand, Tetramethylpyrazine can activates insulin signaling pathway and autophagy pathway to alleviate the damage of on cardiomyocytes induced by high glucose. 

[1] HAYAT SA,PATEL B,KHATTAR RS,et al.Diabetic cardiomyopathy:mechanisms,diagnosis and treatment[J].Clin Sci,2004,107(6):539
[2] FANG ZY,PRINS JB,MARWICK TH.Diabetic cardiomyopathy:evidence,mechanisms,and therapeutic implications[J].Endocr Rev,2004,25(4):543
[3] 潘大彬,汪旻晖,曹蘅.糖尿病心肌病发病机制的研究进展[J].中国临床药理学与治疗学,2013,18(7):831 PAN DB,WANG MH,CAO H.Research progress of pathogenesis of diabetic cardiomyopathy[J].Chin J Clin Pharmacol Ther,2013,18(7):831
[4] XIE Z,HE C,ZOU MH.AMP-activated protein kinase modulates cardiac autophagy in diabetic cardiomyopathy[J].Autophagy,2011,7(10):2
[5] VARGA ZV,GIRICZ Z,LIAUDET L,et al.Interplay of oxidative,nitrosative/nitrative stress,inflammation,cell death and autophagy in diabetic cardiomyopathy[J].BBA-Mol Basis Dis,2015,1852(2):232
[6] 刘春蕾,王莉莉,何昆仑.糖尿病心肌病与自噬[J].中华病理学杂志,2015,44(2):146 LIU CL,WANG LL,HE KL.Diabetic cardiomyopathy and autophagy[J].Chin J Pathol,2015,44(2):146
[7] CAO J,MIAO Q,MIAO S,et al.Tetramethylpyrazine (TMP) exerts antitumor effects by inducing apoptosis and autophagy in hepatocellular carcinoma[J].Int Immunopharmacol,2015,26(1):212
[8] ZHAO H,DAN L,ZHANG W,et al.Protective action of tetramethylpyrazine phosphate against dilated cardiomyopathy in cTnT(R141W)transgenic mice[J].Acta Pharmacol Sin,2010,31(3):281
[9] 朱浩,陈益君,卢子会,等.川芎嗪对脓毒症相关性脑病大鼠海马神经元自噬的影响[J].中华麻醉学杂志,2017,37(10):1278 ZHU H,CHEN YJ,LU ZH,et al.Effect of tetramethylpyrazine on autophagy in hippocampal neurons of rats with sepsis-associated encephalopathy[J].Chin J Anesthesiol,2017,37(10):1278
[10] 王莞,包海花,杨旭芳.实验性糖尿病大鼠心肌磷酸肌酸和肌酸含量的变化及川芎嗪对其影响的研究[J].中国糖尿病杂志,2000,8(5):311 WANG W,BAO HH,YANG XF.Changes of phosphocreatine and creatine contents in myocardium of experimental diabetic rats and the effects of ligustrazine on them[J].Chin J Diabetes,2000,8(5):311
[11] ZHU Y,PEREIRA RO,O'NEILL BT,et al.Cardiac PI3K-Akt impairs insulin-stimulated glucose uptake independent of mTORC1 and GLUT4 translocation[J].Mol Endocrinol,2013,27(1):172
[12] LI X,YU L,GAO J,et al.Apelin ameliorates high glucose-induced downregulation of connexin 43 via AMPK-dependent pathway in neonatal rat cardiomyocytes[J].Aging Dis,2018,9(1):66
[13] ROGACKA D,PIWKOWSKA A,AUDZEYENKA I,et al.Involvement of the AMPK-PTEN pathway in insulin resistance induced by high glucose in cultured rat podocytes[J].Int J Biochem Cell B,2014,51:120
[14] ROFACKA D,AUDZEYENKA I,RYCHLOWSKI M,et al.Metformin overcomes high glucose-induced insulin resistance of podocytes by pleiotropic effects on SIRT1 and AMPK[J].BBA-Mol Basis Dis,2018,1864(1):115
[15] OUWENS DM,DIAMANT M.Myocardial insulin action and the contribution of insulin resistance to the pathogenesis of diabetic cardiomyopathy[J].Arch Physiol Biochem,2007,113(2):76
[16] MI X,HOU J,WANG Z,et al.The protective effects of maltol on cisplatin-induced nephrotoxicity through the AMPK-mediated PI3K/Akt and p53 signaling pathways[J].Sci Rep-UK,2018,8(1):15922
[17] SIMON HU,FRⅡS R,TAIT SWG,et al.Retrograde signaling from autophagy modulates stress responses[J].Sci Signal,2017,10(468):2791
[18] ZUCCO AJ,DAL PV,AFINOGENOVA A,et al.Neural progenitors derived from tuberous sclerosis complex patients exhibit attenuated PI3K/AKT signaling and delayed neuronal differentiation[J].Mol Cell Neurosci,2018,92:149