The Protective Effect Of Ginkgo Biloba Extract On Cardiomyocytes Injured With Hypoxia/reoxygenation And Its Mechanism Based On Keap1/Nrf2/ARE Signaling Pathway

ObjectiveTo explore the protective effect of Ginkgo biloba extract on cardiomyocytes injured with oxidative stress resulted from hypoxia/reoxygenation and its mechanism based on Keap1/Nrf2/ARE signaling pathway.Method1. The hypoxia/reoxygenation cardiomyocytes models were established with the primary cultured rats cardiomyocytes, and the cardiomyocytes were divided into four groups: ① the control group, include the blank control group(Con group) i.e. the primary cultured rats cardiomyocytes and the high dose of Ginkgo biloba extract control group(C160 group), ②hypoxia/reoxygenation cardiomyocytes group(H/R group), ③EGb pretreated group, include low dose of Ginkgo biloba extract on hypoxia/reoxygenation cardiomyocytes group(EGb40 group), medium dose of Ginkgo biloba extract on hypoxia/reoxygenation cardiomyocytes group(EGb80 group) and high dose of Ginkgo biloba extract on hypoxia/reoxygenation cardiomyocytes group(EGb160 group), ④Nrf2 inhibitor-ATRA group(ATRA group).2. Cell viability was measured with Methyl thiazolyl tetrazolium(MTT) assay, in each group MDA content, SOD activity, GSH and GSSG content were detected with enzyme-linked immunosorbent method. Then the GSH/GSSG ratio and the redox potential of GSH/GSSG redox couple( i.e. Eh(GSH/GSSG)) were calculated.3. The m RNA and protein expression of Keap1, Nrf2, GCLC and GSTP1 were,respectively, elucidated with real-time PCR and Western-blot(WB) analysis.Results1. Compared with control group, MDA content, the value of Eh(GSH/GSSG) in H/R group were significantly higher(P<0.01), cell viability, GSH/GSSG ratio and SOD activity were significantly lower(P<0.01). MDA content, the value of Eh(GSH/GSSG) in EGb40 、 EGb80 and EGb160 group were significantly lower than the H/R group(P<0.01), cell viability, GSH/GSSG ratio and SOD activities were significantly higher(P<0.01), with a concentration-dependent manner, and there were significant differences among groups(P<0.01). Compared with EGb160 group, MDA content, the value of Eh(GSH/GSSG) in ATRA group were significantly higher(P<0.01), cell viability,GSH/GSSG ratio and SOD activity were significantly lower(P <0.01). But there were not significant difference in MDA content, GSH/GSSG ratio, the value of Eh(GSH/GSSG),SOD activity and cell viability between ATRA group and H/R group(P>0.05).2. Real-time PCR and WB show: Compared with control group, the m RNA and protein expression of Keap1 in H/R group was significantly decreased(P<0.05).Compared with H/R group, the m RNA and protein expression of Keap1 in EGb40、EGb80 and EGb160 group were significantly decreased(P<0.01), with a concentration-dependent manner, and there was a significant differences among groups(P<0.01). Compared with H/R group, the m RNA and protein expression of Keap1 in ATRA group were significantly decreased(P<0.01), but there were not significant difference between ATRA and EGb160 group(P>0.05). Compared with control group,the m RNA and protein expression of Nrf2, GCLC and GSTP1 in H/R group were significantly increased(P<0.05). Compared with H/R group, the m RNA and protein expression of Nrf2, GCLC and GSTP1 in EGb40、EGb80 and EGb160 group were significantly increased(P<0.01), with a concentration-dependent manner, and there was a significant differences among groups(P<0.01). Compared with EGb160 group,the m RNA and protein expression of Nrf2, GCLC and GSTP1 in ATRA group were significantly decreased(P<0.01), but there were not significant difference between ATRA group and H/R group(P>0.05).Conclusion1. Keap1/Nrf2/ARE signaling pathway may be involved in the pathophysiological mechanisms of rat cardiomyocytes subjected to oxidative stress in the process H/R.2. EGb pretreatment can significantly attenuated H/R-induced oxidative damage of cardiomyocytes by upregulating expression of antioxidant enzymes and enhancing the cellular antioxidant capacity.3. EGb possibly through Keap1/Nrf2/ARE signaling pathway in cardiomyocytes play a protective anti-oxidative damage which induced by H/R.