天香丹胶囊通过调节G蛋白偶联雌激素受体保护心肌缺血再灌注损伤

Tianxiangdan capsule protects myocardial ischemia-reperfusion injury by regulating G protein-coupled estrogen receptor

目的探讨天香丹胶囊(TXD)对预处理去卵巢雌性合并心肌缺血再灌注(I/R)大鼠心肌损伤的保护作用与其可能机制。方法将60只SD雌性大鼠摘去卵巢后随机分为假手术组、模型组、阳性对照组、低、中、高3个剂量实验组,每组10只。阳性对照组灌胃0.8 mg·kg^(-1)雌二醇溶液,低、中、高剂量实验组分别灌胃给予0.2、0.4和0.8 g·kg^(-1)天香丹溶液,假手术组、模型组灌胃等量双蒸水。连续处理60 d。末次给药12 h后建立心脏缺血再灌注模型;其中假手术组只穿线不结扎,另取10正常SD雌性大鼠为正常组(不做任何处理)。用酶联免疫吸附试验法检测血清中大鼠心肌肌酸激酶同工酶(CK-MB)活性、三磷酸肌醇(IP3)的含量;用微量法检测血清中乳酸脱氢酶(LDH);用MTB微板法检测心肌组织中Ca^(2+)浓度;用苏木精-伊红染色检测大鼠心肌组织病理变化;用蛋白质印迹法检测心肌组织中G蛋白偶联雌激素受体(GPER)、磷脂酶C(PLC-β)、三磷酸肌醇受体(IP3R)以及肌浆网钙ATP酶(SERCA2)蛋白表达水平。结果正常组、假手术组、模型组、阳性对照组和高剂量实验组的血清中CK-MB活性表达分别为(64.88±4.70)、(65.86±4.40)、(161.40±10.97)、(78.31±6.64)和(79.62±7.43)pg·mL^(-1);Ca^(2+)浓度分别为(3.20±0.11)、(3.41±0.12)、(5.05±0.15)、(4.10±0.12)和(4.18±0.18)mmol·L^(-1);模型组与假手术组比较,高剂量实验组与模型组比较,差异均有统计学意义(均P<0.05)。正常组、假手术组、模型组、阳性对照组和高剂量实验组的GPER蛋白相对表达水平分别为0.74±0.06、0.73±0.04、0.36±0.03、0.67±0.06和0.65±0.05;模型组与假手术组比较,差异均有统计学意义(P<0.05或P<0.01);阳性对照组、高剂量实验组与模型组比较,差异均有统计学意义(P<0.05或P<0.01)。结论TXD对I/R大鼠心肌损伤有保护作用,可能与调节GPER/PLC/IP3R通路有关。

Objective To explore the protective effects of Tianxiangdan capsule(TXD)on myocardial injury in pretreated ovariectomized female rats with myocardial ischemia/reperfusion(I/R)and its possible mechanism.Methods A total of 60 female SD rats after ovariectomy were randomly divided into sham-operation group,model group,positive control group and experimental-L,-M,-H groups,10 rats in each group.The positive control group was given 0.8 mg·kg^(-1) estradiol solution.The experiment-L,-M,-H groups were given 0.2,0.4,0.8 g·kg^(-1) Tianxiangdan solution,respectively.The sham-operation group and the model group were given the same amount of double distilled water.These 6 groups of rats were administered for 60 days.Cardiac ischemia-reperfusion model was established 12 h after the last administration.Among them,the sham-operated group was only threaded without ligation,and 10 normal SD female rats were taken as the control group(without any treatment).Serum creatine kinase isoenzyme(CK-MB)activity and inositol 1,4,5-trisphosphate(IP3)content were detected by enzyme-linked immunosorbent assay.The activities of lactate dehydrogenase(LDH)in serum was detected by micro-assay.The concentration of Ca^(2+)in myocardial tissue was detected by MTB microplate method.Hemotoxylin and eosin staining was used to detect pathological changes of myocardial tissue in rats.The levels of G protein-coupled estrogen receptor(GPER),phospholipase C(PLC-β),inositol triphosphate receptor(IP3R)and sarcoplasmic reticulum calcium ATPase(SERCA2)proteins in the myocardial tissue were detected by Western blot.Results The active expression levels of CK-MB in serum of control group,sham-operation group,model group,positive control group and experimental-H group were(64.88±4.70),(65.86±4.40),(161.40±10.97),(78.31±6.64)and(79.62±7.43)pg·mL^(-1) respectively;the Ca^(2+)concentrations were(3.20±0.11),(3.41±0.12),(5.05±0.15),(4.10±0.12)and(4.18±0.18)mmol·L^(-1),respectively.The differences between the model group and the sham-operation group or between the experimental-L,-M,-H groups and the model group were statistically significant(all P<0.05).The relative expression levels of GPER proteins in the control group,the sham-operation group,the model group,the positive control group and the experimental-H group were 0.74±0.06,0.73±0.04,0.36±0.03,0.67±0.06 and 0.65±0.05,respectively.Compared with the sham-operation group,the differences in model group were statistically significant(P<0.05,P<0.01);the differences between the positive control group and the experimental-H group and the model group were statistically significant(P<0.05,P<0.01).Conclusion Tianxiangdan capsule has a protective effect on myocardial injury in I/R rats,which may be related to the regulation of GPER/PLC/IP3R pathway.