miR-19b参与山楂叶总黄酮调控缺氧复氧PC12细胞损伤和缺血性大鼠脑损伤的研究

Study on miR-19b Involved in the Regulation of Hawthorn Leaf Total Flavonoids in the Regulation of Hypoxia-reoxygenation PC12 Cell Injury and Ischemic Brain Injury in Rats

目的研究miR-19b参与山楂叶总黄酮对氧化损伤途径的作用机制研究。方法构建缺氧复氧(H/R)PC12细胞模型,给予山楂叶总黄酮处理,细胞计数(CCK-8)法检测细胞增殖,流式细胞术检测细胞凋亡,蛋白质印迹(Western blot)检测B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达,试剂盒测定乳酸脱氢酶(LDH)、丙二醛(MDA)、活性氧(ROS)、超氧化物歧化酶(SOD)水平,实时荧光定量聚合酶链反应(qRT-PCR)检测miR-19b表达水平。在PC12细胞中转染miR-19b inhibitor,给予缺氧复氧和山楂叶总黄酮处理,同样检测细胞增殖、凋亡和LDH、MDA、ROS、SOD水平。同时,构建脑缺血性模型大鼠,用不同剂量山楂叶总黄酮处理,连续4周后,对各组大鼠的神经功能缺损进行评估,并对各组大鼠海马组织CA1区锥体细胞层进行染色,在光学显微镜下观察其细胞学形态。再通过qRT-PCR检测各组大鼠脑细胞海马组织中miR-19b的表达。结果缺氧复氧处理以后的PC12细胞增殖能力下降,LDH漏出率升高,细胞凋亡率升高,细胞中Bax蛋白表达增多,Bcl-2蛋白表达减少,MDA、ROS水平升高,SOD水平降低,miR-19b表达水平减少。山楂叶总黄酮处理后的缺氧复氧PC12细胞增殖能力升高,LDH漏出率降低,细胞凋亡率降低,细胞中Bax蛋白表达减少,Bcl-2蛋白表达增多,MDA、ROS水平降低,SOD水平升高,miR-19b表达水平升高。转染miR-19b inhibitor可以逆转山楂叶总黄酮对缺氧复氧PC12细胞增殖、凋亡和LDH、MDA、ROS、SOD水平影响。脑缺血模型大鼠实验结果发现,山楂叶总黄酮能够明显改善脑缺血模型大鼠的神经功能缺损症状,减轻脑神经元海马组织的细胞损伤,qRT-PCR检测各组大鼠脑组织中miR-19b表达,结果与PC12细胞中一致。结论miR-19b参与山楂叶总黄酮对氧化损伤途径的作用。

Objective To study the mechanism of miR-19b involved in the mechanism of hawthorn leaf total flavonoids on oxidative damage pathways.Methods The hypoxia-reoxygenation(H/R)PC12 cell model was constructed,and the hawthorn leaves were treated with total flavonoids.Cell counting(CCK-8)method was used to detect cell proliferation,and flow cytometry was used to detect cell apoptosis.Western blot was used to detect B lymphoma-2 related protein(Bax)and B lymphocytic tumor-2(Bcl-2)protein expression.The kit was used to measure lactate dehydrogenase(LDH),malondialdehyde(MDA),reactive oxygen species(ROS),superoxide dismutase(SOD)level.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression level of miR-19b.The PC12 cells were transfected with miR-19b inhibitor,treated with hypoxia-reoxygenation and hawthorn leaf total flavonoids,and cell proliferation,apoptosis and LDH,MDA,ROS,SOD levels were also detected.At the same time,rat with cerebral ischemia were constructed and treated with different doses of total flavonoids from hawthorn leaves.After 4 weeks,the neurological deficits of rats in each group were evaluated.The somatic cell layer was stained and its cytological morphology was observed under an optical microscope.Then,the expression of miR-19b in the hippocampus of rat brain cells of each group was detected by qRT-PCR.Results After hypoxia-reoxygenation,the proliferation of PC12 cells decreased,LDH leakage rate increased,apoptosis rate increased,Bax protein expression increased,Bcl-2 protein expression decreased,MDA,ROS levels increased,SOD levels reduced,miR-19b expression level decreased.After the treatment of total flavonoids in hawthorn leaves,the proliferation capacity of hypoxia-reoxygenated PC12 cells increased,the LDH leakage rate decreased,the apoptosis rate decreased,Bax protein expression decreased,Bcl-2 protein expression increased,MDA,ROS levels decreased,SOD level increased and the expression level of miR-19b increased.Transfection of miR-19b inhibitor could reverse the effects of hawthorn leaves total flavonoids on the proliferation,apoptosis and LDH,MDA,ROS,SOD levels of hypoxia-reoxygenation PC12 cells.The experimental results of cerebral ischemia model rat found that hawthorn leaves total flavonoids significantly improve the symptoms of neurological deficits in rats with cerebral ischemia,and reduce the cell damage of hippocampal tissue of brain neurons.QRT-PCR was used to detect miR-19b expression,and the results were consistent with PC12 cells.Conclusion MiR-19b is involved in the effect of hawthorn leaves flavonoids on oxidative damage pathway.