地黄多糖对乳鼠心肌细胞缺氧/复氧损伤后氧化应激和细胞凋亡的影响

目的:研究地黄多糖(polysaccharides from Rehmanniae Radix,PRR)对乳鼠心肌细胞缺氧/复氧损伤后氧化应激和细胞凋亡的影响,并探讨其可能的作用机制。方法:分离并培养乳鼠心肌细胞72 h后随机分为空白组,缺氧/复氧(H/R)模型组,PRR 10,20,40 mg·L~(-1)干预组和舒血宁注射液(SXN,100 mg·L~(-1))干预组,每组设10个复孔。各组细胞经药物干预6 h后,采用噻唑蓝(MTT)检测细胞存活率;检测培养基中天门冬氨酸氨基转移酶(AST),肌酸激酶(CK),乳酸脱氢酶(LDH)活性;测定细胞中超氧化物歧化酶(SOD),过氧化氢酶(CAT)活性和丙二醛(MDA)含量;采用流式细胞仪检测细胞凋亡状况并计算凋亡率,通过实时荧光定量-聚合酶链式反应(Real-time PCR)检测凋亡相关基因B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax)mRNA表达并计算Bcl-2/Bax表达,通过蛋白质免疫印迹(Western blot)方法检测细胞中凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)表达并进行半定量分析。结果:与正常组比较,H/R模型组细胞存活率显著降低(P0.01),培养基中AST,CK,LDH活性显著升高(P0.01),细胞中SOD,CAT活性显著降低,MDA含量显著升高(P0.01),细胞凋亡率显著升高(P0.01),细胞中Bcl-2和Bax mRNA表达明显升高(P0.05,P0.01),Bcl-2/Bax表达显著降低(P0.01),Caspase-3蛋白表达显著升高(P0.01);与H/R模型组比较,PRR 20,40 mg·L~(-1)干预组乳鼠心肌细胞存活率明显升高(P0.05,P0.01),培养基中AST,CK,LDH活性明显降低(P0.05,P0.01),细胞中SOD,CAT活性明显升高,且MDA含量明显降低(P0.05,P0.01),细胞凋亡率显著降低(P0.01),细胞中凋亡相关基因Bcl-2 mRNA明显上调而Bax mRNA明显下调,Bcl-2/Bax显著升高(P0.05,P0.01),Caspase-3蛋白表达量明显降低(P0.05,P0.01)。结论:PRR能够通过抑制氧化应激损伤和细胞凋亡而对H/R损伤乳鼠心肌细胞起到一定的保护作用;作用机制可能与其改善抗氧化酶活性,上调抑凋亡基因Bcl-2表达、下调促凋亡基因Bax表达,提高Bcl-2/Bax表达,以及抑制促凋亡蛋白Caspase-3表达有关。

Effect of Polysaccharides from Rehmanniue Radix on Oxidative Stress and Apoptosis of Neonatal Rat Cardiomyocytes Impaired by Hypoxia-Reoxygenation

Objective: To investigate the effects of polysaccharides from Rehmanniae Radix(PRR) on oxidative stress and apoptosis of neonatal rat cardiomyocytes impaired by hypoxia-reoxygenation (H/R), and its potential mechanism. Method: Cardiomyocytes of neonate rat were isolated, cultured for 72 hours, and then randomly divided into six groups:normal control group, model control group, PRR (10, 20, 40 mg·L^-1) treatment groups and SXN (100 mg·L^-1)treatment group (n=10). Six hours after the drugs were given, the survival rate was detected by MTT; the activities of aspartate aminotransferase(AST), creatine kinase(CK) and lactic dehydrogenase(LDH) in culture medium were detected; the activities of superoxide dismutase(SOD) and catalase(CAT) and the content of malondialdehyde(MDA) in cardiomyocytes were determined; the apoptosis was observed by flow cytometry, and the apoptosis rate was calculated; the mRNA expressions of B cell lymphoma/leukemia-2(Bcl-2) and Bcl-2 associated X protein(Bax) were detected by Real-time PCR, and the ratio of Bcl-2/Bax was calculated; the protein expression of Caspase-3 was detected by Western blot. Result: Compared with the model control group, the survival rate of H/R model group was significantly decreased (P<0.01), the activities of AST, CK, LDH in culture medium were significantly increased (P<0.01), the activities of SOD and CAT were significantly decreased (P<0.01), the apoptosis rate was significantly increased (P<0.01), Bcl-2 and Bax mRNA expressions were significantly increased (P<0.05, P<0.01), Bcl-2/Bax expression was significantly decreased (P<0.01), and Caspase-3 protein expression was significantly increased (P<0.01). Compared with the H/R model group, the survival rate of PRR (20, 40 mg·L^-1) treatment group was significantly increased (P<0.05, P<0.01); the activities of CK, AST, LDH in culture medium were significantly decreased (P<0.05, P<0.01); the activities of SOD, CAT in cardiomyocytes were significantly increased, and the content of MDA was significantly decreased (P<0.05, P<0.01); the apoptosis rate was significantly decreased (P<0.01); the expression of Bcl-2 mRNA was significantly up-regulated, whereas the expression of Bax mRNA and caspase-3 protein were significantly down-regulated, and the ratio of Bcl-2/Bax was significantly increased (P<0.05, P<0.01). Conclusion: PRR had an inhibitive effect on oxidative stress and apoptosis of neonatal rat cardiomyocytes impaired by H/R, which may be related to PRR'' capacities of improving the activity of antioxidase, raising the oxygen radical scavenging effect, up-regulating the expression of Bcl-2, down-regulating the expression of Bax, increasing the ratio of Bcl-2/Bax, and inhibiting the expression of Caspase-3 protein.