The Mechanism Of Nrf2-ARE Pathway On Angiogenesis And The Effect Of Naojian Tablet After Cerebral Ischemic Injury

Objectives:Explore the mechanism of Nrf2-ARE Pathway on angiogenesis and the effect of NaoJian tablet after cerebral ischemic injury condition in vivo/in vitro.Methods:(1)In vivo study with MCAO rats as animal model,SD rats were randomly divided into sham operation group, MCAO model group, Buyang Huanwu Decoction group (gavage:3.15g/kg) and the NaoJian tablet group (gavage:2.41g/kg).The mice were given corresponding treatment, in ID,3D,7d three time points were detected in hippocampus, Immunohistochemical method was used to detect the plasma levels of factor VIII related antigen (vWF), determination of microvessel density (MVD).Determination of SOD activity of brain tissue (Huang Piaoling oxidase method), the content of MDA (sparse generation of barbituric acid method), the content of GSH (ELISA) and GSH-Px activity (colorimetric method).Expression of Nrf2, HO-1and gamma γ-GCS gene and protein by Real-Time PCR and Western blot assay in rat hippocampus.(2)In vitro studies with the concentration (300mol L-1) of vascular endothelial cells in rats H2O2for4h to establish an in vitro model of endothelial cell oxidative stress.According to the different treatment were divided into normal group, model group,10%Buyang Huanwu Decoction containing serum group,5%NaoJian tablet containing serum group,10%NaoJian tablet containing serum group,20%NaoJian tablet containing serum group6.Acquisition of cell culture fluid were detected, MTT assay and flow cytometry to detect the proliferation and regulation of vascular endothelial cell apoptosis,Determination of cell SOD activity (Huang Piaoling oxidase method), the content of MDA (sparse generation of barbituric acid method), the content of GSH (ELISA).Expression of Nrf2, HO-1, γ-GCS gene and protein by Real-Time PCR and Western blot assay in rat hippocampus.Results:Animal experimental results:sham operation group of vWF positive expression in microvascular only a few, no significant difference between the vWF model group, positive expression of microvessel density in1D,3D expression was significantly increased (P<0.05),With the animal survival time prolonged, the positive expression of vWF in model group decreased. Treatment group and model group, the expression of rules are basically consistent, which gives the medicine group7d compared with the model group, there was significant difference (P<0.05), compared to the drug group had no difference.Each time point model group and sham operation group than in the brain, the content of SOD, tissue GSH content, GSH-Px activity decreased significantly (P<0.01, P<0.05), MDA (P<0.05), and increased the content of each treated group compared with the model group, which has statistical significance as compared to third days of SOD content, GSH content, GSH-Px activity and the same time point model the difference between groups (P<0.05), there was significant difference between the seventh day of the MDA content, GSH-Px activity and at the same time points in the model group (P<0.05). Real Time-PCR the results showed:the sham operation group in Nrf2, HO-1and y-GCSmRNA showed a small amount of gamma expression at various time points in model group, the expression of Nrf2mRNA was slightly higher compared with the sham operation group, but no significant difference (P>0.05), model group of seventh days of HO-1mRNA, there was statistical significance in first days compared with model group y-GCS mRNA the same time point false differences between the operation group (P<0.05).Each group can be different degree of upregulation of the expression of Nrf2mRNA Nrf2mRNA in seventh days, which raised the most obvious with the same section of the model group with significant difference (P<0.05). HO-1mRNA and y-GCS mRNA is most obvious in the third heaven, adjustable, and at the same time the model group with significant difference (P<0.05). Each time point between the drug groups have no significant difference (P>0.05).Western-blot results showed that Nrf2protein was localized in the68KD, HO-1protein was localized in the32KD, gamma localization of γ-GCS protein in92KD, GAPDH located in the37KD internal control,Sham operation group Nrf2, HO-1and γ-GCS proteins showed a small amount of gamma expression, the expression of Nrf2protein in model group had statistical significance level of third days compared with the sham operation group (P<0.05), the difference of expression of γ-GCS protein increased in model group gamma, first days, third days the most visible expression (P<0.05, P<0.01). Each group can increase the Nrf2protein expression in different degrees, one in1days and7days rise the most obvious, and at the same time the model group with significant difference (P<0.05). Each group can increase the HO-1protein expression in different degrees, one in3days and7days rise the most obvious, and at the same time the model group with significant difference (P<0.05). In each group third days can upregulate the expression of γ-GCS protein and the gamma, the same section of the model group with significant difference (P<0.01, P<0.05).(2) Morphology, flow cytometry and MTT detected10%NaoJian tablet containing serum group compared with5%and20%treatment group cell damage degree is low, the most obvious effect of inhibiting apoptosis. Compared with the normal group, hypoxia group, SOD, GSH content decreased significantly (P<0.01), the content of MDA increased significantly (P<0.01); compared with the hypoxia group, each group of SOD, the contents of GSH increased significantly (P<0.01), MDA content was significantly decreased (P<0.01), three different groups of streamlining the medicated serum group of NaoJian tablet containing serum group inhibit oxidative stress effect is the best (P<0.05). Real Time-PCR and Western-blot the results showed that:HO-1, model group Nrf2, γ-GCSmRNA and protein expression in normal group and high, with different concentrations of NaoJian tablet containing serum groupcontaining serum treatment, improved to different degree of HO-1, Nrf2, Y-GCSmRNA and protein expression of gamma, of which10%NaoJian tablet containing serum group increased most obviously (P<0.05).Conclusion:(1)The Nrf2/ARE pathway is activated in the conditions of early injury of cerebral ischemia and hypoxia, the expression of Nrf2increased its downstream of Ho-1, γ-GCS levels, suggesting that Nrf2-ARE pathway may be involved in the endogenous stress defense mechanisms after cerebral ischemia.(2)EBSPTR(Eliminating bloodStasis and promoting tissue regeneration) is a effective therapeutic principle in TCM,to treat the NaoJian tablet can promote the angiogenesis after cerebral ischemia, this may be through increased antioxidant enzyme activity on the role of vascular protection, reduce the degree of oxidation of the lipid and activation of Nrf2-ARE pathway in HO-1, Nrf2,γ-GCS expression of target genes and inhibiting oxidative stress injury to achieve.(3)10%NaoJian tablet containing serum can significantly reduce the oxidative stress caused by the injury of vascular endothelial cells, inhibit cell apoptosis, which may be related to inhibiting the oxidative stress response pathway and up regulation of Nrf2-ARE expression.