缝隙连接蛋白43及其S368位点磷酸化水平与甲基苯丙胺诱导的心肌毒性的关系
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摘要
目的通过构建SD大鼠的甲基苯丙胺(METH)中毒模型与心肌细胞中毒模型,检测缝隙连接蛋白43(Cx43)及其S368位点磷酸化(p-Cx43)表达水平;检测吸食METH的人心肌组织内Cx43及其S368位点p-Cx43的表达情况,分析其与METH诱导的心肌毒性的关系,探讨Cx43及S368位点p-Cx43水平在METH诱导的心肌毒性中的作用。方法建立SD大鼠METH慢性中毒动物模型和新生SD大鼠心肌原代METH中毒细胞模型;提取蛋白,采用Western blot检测Cx43、p-Cx43蛋白的表达情况;分析Cx43及其S368位点p-Cx43水平与METH诱导的心肌毒性的关系。收集贵州医科大学法医司法鉴定中心中经毒化检验确认吸食METH的人心肌组织为实验组,无吸食任何毒品者为对照组;常规石蜡切片,HE染色观察2组心肌的结构改变;免疫组织化学染色法、Western blot检测Cx43及其S368位点p-Cx43的表达水平。结果 (1)在METH慢性中毒动物模型中Cx43及S368位点p-Cx43表达较对照组明显下降(P<0.05);(2)在METH中毒细胞浓度、时间梯度模型中,Cx43及其S368位点p-Cx43表达量较对照组显著下降(P<0.05);(3)与对照组比较,实验组人心肌细胞呈现萎缩、坏死及局灶性出血等病理改变;(4)与对照组相比,实验组人心肌组织中Cx43及S368位点p-Cx43表达水平降低,主要表现为心肌细胞之间闰盘处的棕黄色着色减少,部分呈现侧膜化改变;(5)实验组人心肌组织Cx43及S368位点p-Cx43蛋白表达较对照组下降(P<0.05)。结论 METH能通过减少心肌中Cx43及其S368位点p-Cx43的表达,从而破坏心肌的组织结构,影响心脏的正常功能。
Aim By establishing methamphetamine(METH) poisoning model and myocardial cell poisoning model in SD rats, to detect the expressions of gap junction protein connexin 43(Cx43) and its phosphorylation(p-Cx43) at S368 site. By examining the expressions of Cx43 and its S368 site p-Cx43 in human myocardial tissues after taking METH, to analyze the relationship between the expressions of Cx43, p-Cx43 and METH-induced myocardial toxicity, and to investigate the role of Cx43 and its S368 site p-Cx43 in myocardial toxicity induced by METH. Methods METH chronic poisoning animal model in SD rats and primary METH poisoning cell model of cardiac muscle in neonatal SD rats were established. The proteins were extracted and the expressions of Cx43 and p-Cx43 proteins were detected by Western blot. The relationship between the levels of Cx43 and its S368 site p-Cx43 and METH-induced myocardial toxicity was analyzed. The myocardial tissues of METH users confirmed by toxicity test in Forensic Judicial Appraisal Center of Guizhou Medical University were collected as experimental group and those without any drugs as control group. After routine paraffin section, HE staining was used to observe the structural changes of myocardium in two groups. Immunohistochemical staining and Western blot were used to detect the expressions of Cx43 and its S368 site p-Cx43. Results(1)Expressions of Cx43 and its S368 site p-Cx43 in METH chronic poisoning animal model was significantly lower than those in control group(P<0.05).(2)Expressions of Cx43 and its S368 site p-Cx43 in METH poisoning cell concentration and time gradient model were significantly lower than those in control group(P<0.05).(3)Compared with the control group, the atrophy, necrosis and focal hemorrhage of human cardiac myocytes were observed in the experimental group.(4)Compared with the control group, the expression levels of Cx43 and S368 site p-Cx43 in human myocardium in the experimental group were lower than those in the control group, mainly expressed as the decrease of brown-yellow staining at intercalated disc between cardiac myocytes, and some of them showed lateral membrane changes.(5)Expressions of Cx43 and S368 site p-Cx43 proteins of human myocardium in experimental group were lower than those in control group(P<0.05). Conclusion METH can reduce the expressions of Cx43 and its S368 site p-Cx43 in myocardium, thus destroying the structure of myocardium and affecting the normal function of heart.
Aim By establishing methamphetamine(METH) poisoning model and myocardial cell poisoning model in SD rats, to detect the expressions of gap junction protein connexin 43(Cx43) and its phosphorylation(p-Cx43) at S368 site. By examining the expressions of Cx43 and its S368 site p-Cx43 in human myocardial tissues after taking METH, to analyze the relationship between the expressions of Cx43, p-Cx43 and METH-induced myocardial toxicity, and to investigate the role of Cx43 and its S368 site p-Cx43 in myocardial toxicity induced by METH. Methods METH chronic poisoning animal model in SD rats and primary METH poisoning cell model of cardiac muscle in neonatal SD rats were established. The proteins were extracted and the expressions of Cx43 and p-Cx43 proteins were detected by Western blot. The relationship between the levels of Cx43 and its S368 site p-Cx43 and METH-induced myocardial toxicity was analyzed. The myocardial tissues of METH users confirmed by toxicity test in Forensic Judicial Appraisal Center of Guizhou Medical University were collected as experimental group and those without any drugs as control group. After routine paraffin section, HE staining was used to observe the structural changes of myocardium in two groups. Immunohistochemical staining and Western blot were used to detect the expressions of Cx43 and its S368 site p-Cx43. Results(1)Expressions of Cx43 and its S368 site p-Cx43 in METH chronic poisoning animal model was significantly lower than those in control group(P<0.05).(2)Expressions of Cx43 and its S368 site p-Cx43 in METH poisoning cell concentration and time gradient model were significantly lower than those in control group(P<0.05).(3)Compared with the control group, the atrophy, necrosis and focal hemorrhage of human cardiac myocytes were observed in the experimental group.(4)Compared with the control group, the expression levels of Cx43 and S368 site p-Cx43 in human myocardium in the experimental group were lower than those in the control group, mainly expressed as the decrease of brown-yellow staining at intercalated disc between cardiac myocytes, and some of them showed lateral membrane changes.(5)Expressions of Cx43 and S368 site p-Cx43 proteins of human myocardium in experimental group were lower than those in control group(P<0.05). Conclusion METH can reduce the expressions of Cx43 and its S368 site p-Cx43 in myocardium, thus destroying the structure of myocardium and affecting the normal function of heart.
引文
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[21]Benova T,Viczenczova C,Radosinska J,et al.Melatonin attenuates hypertension-related proarrhythmic myocardial maladaptation of connexin-43 and propensity of the heart to lethal arrhythmias[J].Can J Physiol Pharmacol,2013,91(8):633-639.
[22]Greener ID,Sasano T,Wan X,et al.Connexin 43 gene transfer reduces ventricular tachycardia susceptibility after myocardial infarction[J].J Am Coll Cardiol,2012,60(12):1103-1110.
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[24]徐东宝,王杰,于燕妮,等.联合应激法建立单纯心理应激动物实验模型的研究[J].重庆医科大学学报,2016,40(8):227-229.
[25]Nassal MM,Werdich AA,Wan X,et al.Phosphorylation at connexin43 serine-368 is necessary for myocardial conduction during metabolic stress[J].J Cardiovasc Electrophysiol,2016,27(1):110-119.
[2]Qiao D,Xu J,Le C,et al.Insulin-like growth factor binding protein 5(IGFBP5)mediates methamphetamineinduced dopaminergic neuron apoptosis[J].Toxicol Lett,2014,230(3):444-453.
[3]Giv MJ.Exposure to amphetamines leads to development of amphetamine type stimulants associated cardiomyopathy(ATSAC)[J].Cardiovasc Toxicol,2017,17(1):13-24.
[4]Akhgari M,Mobaraki H,Etemadi-Aleagha A.Histopathological study of cardiac lesions in methamphetamine poisoning-related deaths[J].Daru,2017,25(1):5.
[5]王杰,戴佳琳,胡旭初,等.人类Cx43基因的生物信息学分析及其法医学研究意义[J].中国卫生检验杂志,2013,23(3):667-669.
[6]夏冰,刘奇,徐东宝,等.应激对小鼠心肌Cx43表达及磷酸化水平影响[J].中国公共卫生,2015,31(12):1626-1629.
[7]程灵犀,罗斌,林俊莲,等.连接蛋白43与心性猝死的相关性研究进展[J].中国法医学杂志,2009,24(3):179-181.
[8]S9hl G,Willecke K.Gap junctions and the connexin protein family[J].Cardiovasc Res,2004,62(2):228-232.
[9]Kirchhoff S,Kim JS,Hagendorff A,et al.Abnormal cardiac conduction and morphogenesis in connexin 40 and connexin 43 double-deficient mice[J].Circ Res,2000,87(5):399-405.
[10]Bybee KA,Prasad A,Barsness GW,et al.Clinical characteristics and thrombolysis in myocardial infarction frame counts in women with transient left ventricular apical ballooning syndrome[J].Am J Cardiol,2004,94(3):343-346.
[11]王光宇,祝俊英,张庆勇.缝隙连接蛋白43在心血管疾病中的研究进展[J].医学研究杂志,2015,44(1):6-9.
[12]Ekvitorin JF,King TJ,Heyman NS,et al.Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation[J].Circ Res,2006,98(12):1498-1505.
[13]龚一萍,程臻,陈盈.复脉汤预处理对心肌缺血诱导心律失常大鼠心肌细胞蛋白激酶C与Cx43磷酸化信号通路的影响[J].云南中医学院学报,2015,38(4):15-18.
[14]Tomita M,Okuyama T,Katsuyama H,et al.Cardiotoxicity of methamphetamine under stress conditions:comparison of single dose and long-term use[J].Mol Med Rep,2013,7(6):1786-1790.
[15]Chen R,Wang B,Chen L,et al.DNA damage-inducible transcript 4(DDIT4)mediates methamphetamine-induced autophagy and apoptosis through m TOR signaling pathway in cardiomyocytes[J].Toxicol Appl Pharmacol,2016,295:1-11.
[16]Li X,He X,Wang H,et al.Loss of long non-coding RNA ROCR facilitates endogenous cardiac regeneration[J].Cardiovasc Res,2018,114(12):1642-1655.
[17]Niaz K,Pietchman T,Davis P,et al.World Drug Report2017[M].the United States:United Nations,2017:10-14.
[18]Dhein S.Gap junction channels in the cardiovascular system:pharmacological and physiological modulation[J].Trends Pharmacol Sci,1998,19(6):229-241.
[19]Sáez JC,Berthoud VM,Bra1es MC,et al.Plasma membrane channels formed by connexins:Their regulation and functions[J].Physiol Rev,2003,83(4):1359.
[20]Salameh A,Haunschild J,Bruchle P,et al.On the role of the gap junction protein Cx43(GJA1)in human cardiac malformations with fallot-pathology:A study on paediatric cardiac specimen[J].PLoS One,2014,9(4):e95344.
[21]Benova T,Viczenczova C,Radosinska J,et al.Melatonin attenuates hypertension-related proarrhythmic myocardial maladaptation of connexin-43 and propensity of the heart to lethal arrhythmias[J].Can J Physiol Pharmacol,2013,91(8):633-639.
[22]Greener ID,Sasano T,Wan X,et al.Connexin 43 gene transfer reduces ventricular tachycardia susceptibility after myocardial infarction[J].J Am Coll Cardiol,2012,60(12):1103-1110.
[23]Delmar M,Makita N.Cardiac connexins,mutations and arrhythmias[J].Curr Opin Cardiol,2012,27(3):236-241.
[24]徐东宝,王杰,于燕妮,等.联合应激法建立单纯心理应激动物实验模型的研究[J].重庆医科大学学报,2016,40(8):227-229.
[25]Nassal MM,Werdich AA,Wan X,et al.Phosphorylation at connexin43 serine-368 is necessary for myocardial conduction during metabolic stress[J].J Cardiovasc Electrophysiol,2016,27(1):110-119.