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纸质出版日期:2016
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彭辉, 张华龙. 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[J]. 中国实验方剂学杂志, 2016,22(23):155-160.
PENG Hui, ZHANG Hua-long. Effect of Polysaccharides from Rehmanniue Radix on Oxidative Stress and Apoptosis of Neonatal Rat Cardiomyocytes Impaired by Hypoxia-Reoxygenation[J]. Chinese journal of experimental traditional medical formulae, 2016, 22(23): 155-160.
彭辉, 张华龙. 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[J]. 中国实验方剂学杂志, 2016,22(23):155-160. DOI: 10.13422/j.cnki.syfjx.2016230155.
PENG Hui, ZHANG Hua-long. Effect of Polysaccharides from Rehmanniue Radix on Oxidative Stress and Apoptosis of Neonatal Rat Cardiomyocytes Impaired by Hypoxia-Reoxygenation[J]. Chinese journal of experimental traditional medical formulae, 2016, 22(23): 155-160. DOI: 10.13422/j.cnki.syfjx.2016230155.
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(P<0.01),培养基中AST,CK,LDH活性显著升高(P<0.01),细胞中SOD,CAT活性显著降低,MDA含量显著升高(P<0.01),细胞凋亡率显著升高(P<0.01),细胞中Bcl-2和Bax mRNA表达明显升高(P<0.05,P<0.01),Bcl-2/Bax表达显著降低(P<0.01),Caspase-3蛋白表达显著升高(P<0.01);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(P<0.05,P<0.01),培养基中AST,CK,LDH活性明显降低(P<0.05,P<0.01),细胞中SOD,CAT活性明显升高,且MDA含量明显降低(P<0.05,P<0.01),细胞凋亡率显著降低(P<0.01),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(P<0.05,P<0.01),Caspase-3蛋白表达量明显降低(P<0.05,P<0.01)。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。
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.
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