枳实薤白桂枝汤通过保护缝隙连接干预心肌缺血再灌注钙超载的研究
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摘要
目的:探讨枳实薤白桂枝汤能否通过保护细胞缝隙连接干预钙超载,进而减轻心肌细胞缺血再灌注损伤。方法:大鼠随机分假手术组、模型组、枳实薤白桂枝汤2.30g/kg、4.59g/kg、9.18g/kg组,给药14天后结扎冠状动脉前降支造模,观察各组心电图、心肌酶(CK-MB、LDH、ALT、AST)、病理及超微结构的改变,并以免疫组织化学染色法观察闰盘重构程度及缝隙连接蛋白43(connexin43,Cx43)表达,Western blot法检测Cx43、磷酸化缝隙连接蛋白43(phospho-connexin43,p-Cx43)、蛋白激酶C(protein kinase C,PKC)及钠钙交换体1(Na~+/Ca~(2+) exchanger 1,NCX1)的表达。结果:与模型组相比,枳实薤白桂枝汤组心电图及心肌酶改变明显减轻、细胞超微结构改变及闰盘重构程度也明显减轻,Cx43、PKC、p-Cx43的表达增加, NCX1的表达降低。结论:枳实薤白桂枝汤在发生心肌缺血再灌注损伤时能有效保护细胞缝隙连接,减少NCX1的表达,进而干预心肌细胞钙超载的过程,产生心肌保护作用,且4.59g/kg、9.18g/kg剂量组效果较为明显。
Objective: To investigate whether ZhishiXieBaiGuizhi Decoction can interfere with calcium overload by protecting cell gap junction, thereby reducing myocardial ischemia-reperfusion injury. Methods: SD rats were randomly divided into 5 groups: the sham operation group(SHAM), the model group(IR), 2.30 g/kg, 4.59 g/kg and 9.18 g/kg ZhishiXieBaiGuizhi Decoction groups. After 14 days' administration, the anterior descending coronary artery was ligated to observe the changes of electrocardiogram, myocardial enzyme(CK-MB, LDH, ALT, AST), pathology and ultrastructure changes. The degree of remodeling and expression of connexin43(Cx43) were observed by immunohistochemical staining. Expressions of Cx43, phospho-connexin43(p-Cx43),protein kinase C(PKC) and sodium-calcium exchanger 1(Na~+/Ca~(2+) exchanger 1, NCX1) were detected by Western blot. Results: Compared with IR group, the changes of electrocardiogram and myocardial enzymes in all doses of drug groups were significantly reduced, the ultrastructural changes of the cells and the degree of disc remodeling were also significantly reduced, the expressions of Cx43, PKC and p-Cx43 were increased. The expression of NCX1 was reduced. Conclusion: ZhishiXieBaiGuizhi Decoction can effectively protect the cell gap junction and reduce the expression of NCX1 in myocardial ischemia-reperfusion injury, and then intervene in the process of myocardial calcium overload, resulting in myocardial protection. 4.59 g/kg and 9.18 g/kg ZhishiXieBaiGuizhi Decoction show better effect.
Objective: To investigate whether ZhishiXieBaiGuizhi Decoction can interfere with calcium overload by protecting cell gap junction, thereby reducing myocardial ischemia-reperfusion injury. Methods: SD rats were randomly divided into 5 groups: the sham operation group(SHAM), the model group(IR), 2.30 g/kg, 4.59 g/kg and 9.18 g/kg ZhishiXieBaiGuizhi Decoction groups. After 14 days' administration, the anterior descending coronary artery was ligated to observe the changes of electrocardiogram, myocardial enzyme(CK-MB, LDH, ALT, AST), pathology and ultrastructure changes. The degree of remodeling and expression of connexin43(Cx43) were observed by immunohistochemical staining. Expressions of Cx43, phospho-connexin43(p-Cx43),protein kinase C(PKC) and sodium-calcium exchanger 1(Na~+/Ca~(2+) exchanger 1, NCX1) were detected by Western blot. Results: Compared with IR group, the changes of electrocardiogram and myocardial enzymes in all doses of drug groups were significantly reduced, the ultrastructural changes of the cells and the degree of disc remodeling were also significantly reduced, the expressions of Cx43, PKC and p-Cx43 were increased. The expression of NCX1 was reduced. Conclusion: ZhishiXieBaiGuizhi Decoction can effectively protect the cell gap junction and reduce the expression of NCX1 in myocardial ischemia-reperfusion injury, and then intervene in the process of myocardial calcium overload, resulting in myocardial protection. 4.59 g/kg and 9.18 g/kg ZhishiXieBaiGuizhi Decoction show better effect.
引文
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[6]李楠楠,石月萍.加减枳实薤白桂枝汤通过AC/cAMP通路对缺血再灌注大鼠心肌组织水通道蛋白表达的影响.中国动脉硬化杂志,2018,26(3)∶237～244.
[7]徐萍,石月萍.加减枳实薤白桂枝汤对大鼠心肌缺血再灌注过氧化损伤的影响.中药药理与临床,2017,33(3)∶14～18.
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[11]Johnstone S R ,Ross J ,Rizzo M J ,et al.Oxidized Phospholipid Species Promote in Vivo Differential Cx43 Phosphorylation and Vascular Smooth Muscle Cell Proliferation.American Journal of Pathology,2009,175(2)∶916～924.
[12]Schulz R ,Boengler K ,Totzeck A ,et al.Connexin 43 in ischemic pre- and postconditioning.Heart Failure Reviews,2007,12(3-4)∶261～266.
[13]John S A ,Kondo R ,Wang S Y ,et al.Connexin-43 Hemichannels Opened by Metabolic Inhibition.Journal of Biological Chemistry,1999,274(1)∶236～240.
[14]Li F ,Sugishita K ,Su Z ,et al.Activation of Connexin-43 Hemichannels Can Elevate [Ca2+]i and [Na+]i in Rabbit Ventricular Myocytes During Metabolic Inhibition.Journal of Molecular and Cellular Cardiology,2001,33(12)∶2145～2155.
[15]Delmar M ,Michaels D C ,Johnson T ,et al.Effects of increasing intercellular resistance on transverse and longitudinal propagation in sheep epicardial muscle.Circulation Research,1987,60(5)∶780～785.
[16]Dhein S ,K Kr?1/4semann,Schaefer T .Effects of the gap junction uncoupler palmitoleic acid on the activation and repolarization wavefronts in isolated rabbit hearts.Br J Pharmacol,2010,128(7)∶1375～1384.
[2]Kitao T ,Takuma K ,Kawasaki T ,et al.The Na/Ca exchanger-mediated Ca influx triggers nitric oxide-induced cytotoxicity in cultured astrocytes.Neurochemistry International,2010,57(1)∶58～66.
[3]Ottolia M ,Torres N ,Bridge J H B ,et al.Na/Ca exchange and contraction of the heart - Journal of Molecular and Cellular Cardiology.Journal of Molecular & Cellular Cardiology,2013,61(16)∶28～33.
[4]David GarcíaDorado,Antonio RodríguezSinovas,Ruizmeana M .Gap junction-mediated spread of cell injury and death during myocardial ischemia-reperfusion.Cardiovascular Research,2004,61(3)∶386～401.
[5]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.
[6]李楠楠,石月萍.加减枳实薤白桂枝汤通过AC/cAMP通路对缺血再灌注大鼠心肌组织水通道蛋白表达的影响.中国动脉硬化杂志,2018,26(3)∶237～244.
[7]徐萍,石月萍.加减枳实薤白桂枝汤对大鼠心肌缺血再灌注过氧化损伤的影响.中药药理与临床,2017,33(3)∶14～18.
[8]Jongsma H J ,Wilders R .Gap junctions in cardiovascular disease.Circulation Research,2000,86(12)∶1193～1197.
[9]Schulz R ,Heusch G .Connexin 43 and ischemic preconditioning.Cardiovascular Research,2004,62(2)∶335～344.
[10]Shah M M ,Martinez A M ,Fletcher W H .The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C:In vivo,and in vitro,studies.Molecular & Cellular Biochemistry,2002,238(1-2)∶ 57～68.
[11]Johnstone S R ,Ross J ,Rizzo M J ,et al.Oxidized Phospholipid Species Promote in Vivo Differential Cx43 Phosphorylation and Vascular Smooth Muscle Cell Proliferation.American Journal of Pathology,2009,175(2)∶916～924.
[12]Schulz R ,Boengler K ,Totzeck A ,et al.Connexin 43 in ischemic pre- and postconditioning.Heart Failure Reviews,2007,12(3-4)∶261～266.
[13]John S A ,Kondo R ,Wang S Y ,et al.Connexin-43 Hemichannels Opened by Metabolic Inhibition.Journal of Biological Chemistry,1999,274(1)∶236～240.
[14]Li F ,Sugishita K ,Su Z ,et al.Activation of Connexin-43 Hemichannels Can Elevate [Ca2+]i and [Na+]i in Rabbit Ventricular Myocytes During Metabolic Inhibition.Journal of Molecular and Cellular Cardiology,2001,33(12)∶2145～2155.
[15]Delmar M ,Michaels D C ,Johnson T ,et al.Effects of increasing intercellular resistance on transverse and longitudinal propagation in sheep epicardial muscle.Circulation Research,1987,60(5)∶780～785.
[16]Dhein S ,K Kr?1/4semann,Schaefer T .Effects of the gap junction uncoupler palmitoleic acid on the activation and repolarization wavefronts in isolated rabbit hearts.Br J Pharmacol,2010,128(7)∶1375～1384.