温阳通脉方经内质网应激相关通路对H9C2心肌细胞缺氧/复氧损伤的作用
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摘要
目的:观察温阳通脉方含药血清是否在内质网应激相关通路对心肌细胞缺氧/复氧损伤的作用,并探究体外培养时最佳给药剂量。方法:以20%含药血清处理,用四甲基偶氮唑盐(MTT)法测得CoCl_2烫最佳浓度和各组细胞存活率;比色法检测各组半胱氨酸蛋白酶3(Caspase3)酶活性和上清液中乳酸脱氢酶(LDH)活性;Hoechst 33342荧光染色观察各组细胞凋亡形态变化;流式细胞术检测各组细胞凋亡率;Western Blot法检测各组内质网应激相关蛋白GRP78、Caspase12、CHOP表达以及凋亡相关蛋白Bcl-2、Bax表达情况。结果:与空白对照组血清相比,模型组细胞活性明显降低,Caspase3酶活性和LDH活性明显升高,细胞凋亡数量明显增多,凋亡率明显升高, GRP78、Caspase12、CHOP和Bax表达明显增多,Bcl-2表达明显减少;与模型组相比,3.24、6.48、12.96g/kg组含药血清能够不同程度改善细胞活性,Caspase3酶活性和LDH活性明显减少,凋亡率不同程度降低,GRP78、Caspase12、CHOP和Bax表达明显减少,Bcl-2表达不同程度增多。结论:温阳通脉方能减轻缺氧/复氧后H9C2心肌细胞损伤,其机制可能与温阳通脉方抵抗内质网应激介导的细胞凋亡有关,其抗凋亡程度呈剂量依赖性,以温阳通脉方12.96g/kg组效果最佳。
Objective: To observe the effect of Wenyangtongmaifang and its drug-containing serum on the hypoxia/reoxygenation injury of cardiomyocytes induced by endoplasmic reticulum stress-related pathways,and to explore the optimal dose for in vitro culture. Methods: The cells in different groups were treated with 20% blank serum or 20% drug-containing serum respectively. The optimal concentration of CoC l2 was determined and the cell viability in each group was measured by MTT. Caspase3 enzyme activity and supernatant LDH activity was determined by the colorimetric method. Morphological changes of apoptosis were observed by Hoechst 33342 fluorescence staining. Apoptosis rate was detected by flow cytometry. The expressions of GRP78,Caspase12,CHOP,Bcl-2 and Bax were detected by Western Blot. Results: Compared with the blank control group,the survival rates in the model group was significantly reduced,the levels of Caspase3 and LDH were significantly increased,the number of apoptotic cells was significantly increased and the apoptosis rate was increased. The expressions of GRP78,Caspase12,CHOP and Bax were significantly up-regulated while Bcl-2 was significantly down-regulated. Compared with the model group,the levels of Caspase3 and LDH,and the rates of apoptosis were decreased in all doses of Wenyangtongmaifang groups,the expressions of GRP78,Caspase12,CHOP and Bax were decreased while Bcl-2 was increased. Conclusion: Wenyangtongmaifang can alleviate H9 C2 cells injury after hypoxia/reoxygenation,and its mechanism may be related to endoplasmic reticulum stress-mediated apoptosis. Its anti-apoptotic effect is dose-dependent and the 12. 96 g/kg is suggested the optimal dose.
Objective: To observe the effect of Wenyangtongmaifang and its drug-containing serum on the hypoxia/reoxygenation injury of cardiomyocytes induced by endoplasmic reticulum stress-related pathways,and to explore the optimal dose for in vitro culture. Methods: The cells in different groups were treated with 20% blank serum or 20% drug-containing serum respectively. The optimal concentration of CoC l2 was determined and the cell viability in each group was measured by MTT. Caspase3 enzyme activity and supernatant LDH activity was determined by the colorimetric method. Morphological changes of apoptosis were observed by Hoechst 33342 fluorescence staining. Apoptosis rate was detected by flow cytometry. The expressions of GRP78,Caspase12,CHOP,Bcl-2 and Bax were detected by Western Blot. Results: Compared with the blank control group,the survival rates in the model group was significantly reduced,the levels of Caspase3 and LDH were significantly increased,the number of apoptotic cells was significantly increased and the apoptosis rate was increased. The expressions of GRP78,Caspase12,CHOP and Bax were significantly up-regulated while Bcl-2 was significantly down-regulated. Compared with the model group,the levels of Caspase3 and LDH,and the rates of apoptosis were decreased in all doses of Wenyangtongmaifang groups,the expressions of GRP78,Caspase12,CHOP and Bax were decreased while Bcl-2 was increased. Conclusion: Wenyangtongmaifang can alleviate H9 C2 cells injury after hypoxia/reoxygenation,and its mechanism may be related to endoplasmic reticulum stress-mediated apoptosis. Its anti-apoptotic effect is dose-dependent and the 12. 96 g/kg is suggested the optimal dose.
引文
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[18]Wang D, Yuan X, Hu C, et al. Endoplasmic Reticulum Stress Is Involved in Apoptosis of Detrusor Muscle in Streptozocin‐Induced Diabetic Rats. Neurourology and Urodynamics,2015,36(1)∶65～72.
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[23]Zhang Q, Liu J, Chen S, et al. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis,2016,21(4)∶432～442.
[24]Fujita E, Kouroku Y, Jimbo A, et al. Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells. Cell Death and Differentiation,2002,9∶1108～1114.
[25]Liu H, Wang Z, Nowicki M J. Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice. World Journal of Gastroenterology,2014,20(48)∶18189～18198.
[26]Zhang J, Zhang Z, Bao J, et al. Jia-Jian-Di-Huang-Yin-Zi decoction reduces apoptosis induced by both mitochondrial and endoplasmic reticulum caspase12 pathways in the mouse model of Parkinson's disease. Journal of Ethnopharmacology,2017,203∶69～79.
[2]石月萍,马骏,赵建宇.温阳通脉方预处理对再灌注大鼠心肌细胞凋亡的影响.中国中医基础医学杂志,2009,15(12)∶944～945.
[3]刘春晓,石月萍.加减枳实薤白桂枝汤对大鼠心肌缺血再灌注损伤细胞凋亡及Caspase相关蛋白的影响.中药药理与临床,2017,33(1)∶11～14 .
[4]Gao Y, Jia P, Shu W, et al. The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes. European Journal of Pharmacology,2016,774∶71～79.
[5]邹麓,吴楠,李晓岩,等.缺血后处理抑制内质网应激PERK通路减轻大鼠离体心脏缺血/再灌注损伤.解剖科学进展,2017,23(4)∶370～373.
[6]李仪奎,吴健宇.血清药理实验中采血时间的通法方案.中国药理学通报,1999,15(6)∶569～570 .
[7]Jian J, Xuan F, Qin F, et al. The Antioxidant, Anti-Inflammatory and Anti-Apoptotic Activities of the Bauhinia Championii Flavone are Connected with Protection Against Myocardial Ischemia/Reperfusion Injury. Cellular Physiology and Biochemistry,2016,38(4)∶1365～1375 .
[8]Beatriz L H, Valentin C, Inmaculada P. The endoplasmic reticulum stress and the HIF-1 signalling pathways are involved in the neuronal damage caused by chemical hypoxia. British journal of pharmacology,2015,172(11)∶2838～2851 .
[9]Sun L, Liu N, Liu S S, et al. Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chloride-induced hypoxia. American Journal of Cancer Research,2015,5(9)∶2626～2642 .
[10]姚岚,赵赫,王舒,等.血清药理学研究概况及展望.亚太传统医药,2017,13(24)∶52～55 .
[11]Yang H, Wang C, Zhang L, et al. Rutin alleviates hypoxia/reoxygenation-induced injury in myocardial cells by up-regulating SIRT1 expression. Chemico-Biological Interactions,2019,297∶44～49.
[12]Wu L, Xu Y, Yang Z, et al. Hydroxytyrosol and olive leaf extract exert cardioprotective effects by inhibiting GRP78 and CHOP expression. The Journal of Biomedical Research,2018,32(5)∶371～379 .
[13]Hong J, Zhou W, Wang X. Involvement of miR-455 in the protective effect of H2S against chemical hypoxia-induced injury in BEAS-2B cells. Pathology-Research and Practice,2018,214(11)∶1804～1810 .
[14]舒希, 佟晓永.SERCA调控细胞凋亡的研究进展.生命科学仪器,2018,16(3)∶9～15 .
[15]Wu H, Tang Q, Yang J, et al. Atorvastatin ameliorates myocardial ischemia/reperfusion injury through attenuation of endoplasmic reticulum stress-induced apoptosis. International Journal of Clinical and Experimental Medicine,2014,7(12)∶4915～4923 .
[16]Xu C, Bailly-Maitre B, Reed J C. Endoplasmic reticulum stress: cell life and death decisions. Journal of Clinical Investigation,2005,115(10)∶2656～2664 .
[17]Zhang C, Zhang J, Wu Y, et al. Effects of Endoplasmic Reticulum Stress on Pulmonary Hypertension in Rat Induced by Chronic Hypoxia and Hypercapnia. Journal of Biosciences and Medicines,2018,6(6)∶53～67 .
[18]Wang D, Yuan X, Hu C, et al. Endoplasmic Reticulum Stress Is Involved in Apoptosis of Detrusor Muscle in Streptozocin‐Induced Diabetic Rats. Neurourology and Urodynamics,2015,36(1)∶65～72.
[19]Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nature cell biology,2011,13(3)∶184～190 .
[20]Minamino T, Kitakaze M. ER stress in cardiovascular disease. Journal of Molecular and Cellular Cardiology,2010,48(6)∶1105～1110 .
[21]Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death and Differentiation,2004,11(4)∶381～389 .
[22]Youle R J, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology,2008,9(1)∶47～59 .
[23]Zhang Q, Liu J, Chen S, et al. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis,2016,21(4)∶432～442.
[24]Fujita E, Kouroku Y, Jimbo A, et al. Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells. Cell Death and Differentiation,2002,9∶1108～1114.
[25]Liu H, Wang Z, Nowicki M J. Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice. World Journal of Gastroenterology,2014,20(48)∶18189～18198.
[26]Zhang J, Zhang Z, Bao J, et al. Jia-Jian-Di-Huang-Yin-Zi decoction reduces apoptosis induced by both mitochondrial and endoplasmic reticulum caspase12 pathways in the mouse model of Parkinson's disease. Journal of Ethnopharmacology,2017,203∶69～79.