人参皂苷 Rg1 通过调控缝隙连接拮抗铅致大鼠心肌细胞损伤的作用机制

目的 观察醋酸铅染毒大鼠经人参皂苷 Rg1(ginsenoside Rg1,以下简称Rg1)干预前后心肌缝隙连接蛋白Cx43(connexin 43,Cx43)及其磷酸化蛋白(p-Cx43)的表达变化,探究Rg1通过缝隙连接对铅致心肌细胞损伤的保护作用。方法 将无特定病原体级的Sprague Dawley雄性大鼠按随机数字表法分为对照组,以二甲基亚砜(DMSO)溶解辛醇,作为辛醇对照组,辛醇为Cx43特异性阻滞剂,设置DMSO组及辛醇组,且不做干预;低、中、高剂量铅染毒组(90、180、360 mg/kg),Rg1干预组分为不同剂量铅染毒+Rg1组、辛醇+Rg1组、高剂量铅染毒+辛醇+Rg1 组(Pb360 mg/kg+辛醇+Rg1),共11组,每组8只。对照组给予等量蒸馏水,DMSO组、辛醇组按照体质量进行腹腔注射染毒(5 mmol/kg),不同剂量铅染毒组和铅染毒+Rg1 组经口灌胃醋酸铅溶液,每天染毒1次,每周连续5d,造模4周后,各Rg1(20 mg/kg)干预组每天给药1次,每周连续 5 d,共4周;对照组及不同剂量铅染毒组每天经口灌胃蒸馏水。4 周后处死大鼠收集心脏组织,计算心脏脏器系数,观察心肌组织病理学变化,检测全血血铅浓度,Cx43、p-Cx43、Bcl-2、Bax、Cleaved caspase-3、LC3-Ⅰ和 LC3-Ⅱ蛋白表达水平。结果 与对照组相比,染毒组血铅水平显著升高(P<0.05);高剂量铅染毒组心脏脏器系数显著降低,Rg1干预后脏器系数升高(P<0.05)。HE染色显示,对照组、DMSO组及辛醇组心肌结构无明显变化;低、中剂量铅染毒组出现心肌纤维排列紊乱;高剂量铅染毒组心肌纤维断裂。与铅染毒组、辛醇组相比,Rg1 干预组心肌组织结构明显改善,心肌纤维排列相对整齐紧密,无明显炎症。Western blot检测结果显示,铅染毒组Cx43、p-Cx43、Bcl-2及LC3-Ⅰ蛋白表达低于对照组(P<0.05),Bax、Cleaved caspase-3 及 LC3-Ⅱ蛋白表达高于对照组(P<0. 05);Rg1干预组Cx43、p-Cx43、Bcl-2及LC3-Ⅰ蛋白表达高于各剂量铅染毒组(P<0.05),Bax、Cleaved caspase-3及LC3-Ⅱ蛋白表达低于各剂量铅染毒组(P<0.05)。辛醇组与对照组、辛醇+Rg1 组相比,Cx43蛋白表达差异无统计学意义。结论 人参皂苷Rg1对铅致心肌细胞损伤的保护作用可能与其升高Cx43、p-Cx43 蛋白表达有关。

Mechanism of ginsenoside Rg1 antagonising lead-induced cardiomyocyte injury in rats by modulating gap junctions

Objective To observe the changes in the expression of myocardial gap junction protein Cx43 (Cx43) and its phosphorylated protein (p-Cx43) before and after the intervention of ginsenoside Rg1 (Rg1) in lead acetate-contaminated rats,and to investigate the protective effect of Rg1 on lead-induced cardiomyocyte injury through gap junctions.Methods Sprague Dawley male rats without specific pathogen grade were divided into control groups by random number table method,and octanol was dissolved in DMSO as a control group of octanol,which is a Cx43-specific blocker,setting DMSO group,octanol (Oct) group, low,medium and high dose of Pb trained and poisoned group (90,180 and 360 mg/kg) and Rg1 intervention group as low,medium and high dose of Pb poisoning+Rg1 intervention group,Oct+Rg1 intervention group,and high dose lead poisoning+ Oct+Rg1 intervention group (Pb 360 mg/kg+Oct+Rg1),totalling 11 groups of 8 animals each.The control group was given an equal amount of distilled water,the DMSO group and the octanol group (5 mmol/kg) were stained by peritoneal injection according to their body mass,and different doses of lead-stained groups (90,180,360 mg/kg) and each dose of lead-stained+ Rg1 group were stained by oral gavage of the lead acetate solution once a day for 5 d consecutive per week.After 4 weeks of mod-eling, Rg1 (20 mg/kg) intervention was administered once daily for 5 d consecutive per week for 4 weeks in each group;distilled water was orally gavaged daily in the control group and the low,medium,high dose lead-stained groups.Cardiac tis-sues were collected from rats after 4 weeks of execution, cardiac apparatus coefficients were calculated, myocardial histopatho-logical changes were observed, and whole blood lead concentration, Cx43, p-Cx43, Bcl-2,Bax,Cleaved caspase-3,LC3-Ⅰand LC3-Ⅱprotein expression levels were detected. Results Blood lead levels were significantly higher in all tainted groups compared to the control group (P<0.05);cardiac apparatus coefficient levels were significantly lower in the high-dose tainted group (360 mg/kg) and higher after Rg1 intervention (P<0.05).HE staining showed no significant change in myocardial struc-ture in the control,DMSO,and octanol groups;myocardial fiber arrangement disorders were observed in the low and medium-dose lead-stained groups;and myocardial fibers were broken in the high-dose lead-stained group.Compared with the lead-stained and octanol groups,the myocardial tissue structure of the Rg1 intervention group was significantly improved, and the my-ocardial fibers were arranged relatively neatly and tightly without obvious inflammation.The results of Western blot showed that the protein expression of Cx43,p-Cx43,Bcl-2,and LC3-Ⅰ in the lead-tainted group was lower than that in the control group (P<0.05),and that the protein expression of Bax,Cleaved caspase-3 and LC3-Ⅱ was higher than that in the control group (P<0.05);in the Rg1 intervention group, the protein expression of Cx43, p-Cx43, Bcl-2, and LC3-Ⅰ protein expression was higher than that in the different dose lead-tainted group (P<0.05),and Bax,Cleaved caspase-3 and LC3-Ⅱ protein expression were lower than that with the different dose lead-tainted group (P<0.05).There were no statistically significant difference in Cx43 protein expressed in the octanol group compared to the control group and the Oct+Rg1 group.Conclusion Ginsenoside Rg1 effectively protects against lead-induced cardiomyocyte injury,possibly related to its elevated Cx43 and p-Cx43 protein secretion.