6-Hydroxygenistein Improved Hypoxia-Induced Injury By Activating The Nrf-2/OH-1Pathway In PC12 Cells

Objective:1.We designed and synthesized 6-hydroxygenistein(6-hydroxygenistein,6-OHG)and its four methylated derivatives.And we investigated their anti-hypoxic activity and structure-activity relationship.2.We elucidated the protective mechanism of 6-OHG against hypoxia-induced injury in PC12 cells.Methods:1.Using biochanin A as raw material,6-OHG and its four methoxylated derivatives were synthesized through methylation,bromination,methoxylation and demethylation.We detected the free radical scavenging activity by DPPH free radical scavenging experiment,investigate the anti-hypoxic activity using the PC12 cell hypoxic injury model,and summarized the structure-activity relationship based on the results.2.We divided PC12 cells into normal control group,hypoxia model group,rutin group and 6-OHG group.We measured the contents of MDA,ROS and GSH,the activities of SOD and CAT and other oxidative stress-related indicators in the cells of each group,as well as the expressions of HIF-1αand VEGF proteins.We measured the content of ATP,the activity of Na~+-K~+-ATPase and the energy metabolism indexes such as mitochondrial membrane potential in the cells.We measured the expression of inflammatory response-related indicators such as TNF-α,IL-6 and IL-10 in cells,and the expression of inflammatory-related proteins such as NF-κB and TNF-α.We used flow cytometry to detect the apoptosis of PC12 cells in each group,and measured the activity of Caspase-3 and Caspase-9 and the protein expression of Bcl-2,Bax,Cleaved Caspase-3.3.PC12 cells were divided into 4 groups:normal control group,hypoxia model group,6-OHG group and 6-OHG+ML385(Nrf-2 blocker)group,and the cells in each group were treated accordingly.We measured the expression of Nrf-2/HO-1 protein,ROS content,inflammatory factors and cell apoptosis in each group of cells.Results:1.6-OHG was prepared by this synthetic route,and the total yield was 49.37%.the 6-OHG and compound 7 exhibited excelent DPPH free radical scavenging activities,effectively improved,the morphology of hypoxia PC12 cells,significantly increased the viability of hypoxia PC12 cell.2.Compared with the normal control group,the cell membrane integrity of the cells in the hypoxic model group was destroyed,the cell morphology changed,and the cell viability decreased significantly;the contents of ROS and MDA were significantly increased,the levels of CAT,SOD and GSH were significantly reduced,and the protein expression of HIF-1α/VEGF was significantly increased;The concentration of IL-10 was significantly reduced,the concentration of TNF-αand IL-6 was significantly increased,and the protein expression levels of NF-κB and TNF-αwere significantly increased;the content of ATP and the activity of Na~+-K~+-ATPase decreased significantly;the activity of Caspase-3 and Caspase-9,the ratio of Bax/Bcl-2 and the expression of Cleaved Caspase-3 protein were significantly increased.Compared with the hypoxic model group,after 6-OHG administration,the cell morphology was good,the cell membrane was relatively complete,and the cell viability could be significantly improved.After 6-OHG administration,it can significantly reduce the content of ROS and MDA in cells,increase the levels of antioxidant enzymes CAT,SOD and GSH,and inhibit the HIF-1α/VEGF signaling pathway.6-OHG can significantly increase the concentration of anti-inflammatory factor IL-10 in cells,reduce the concentration of pro-inflammatory factors TNF-αand IL-6,and inhibit the NF-κB/TNF-αsignaling pathway.After6-OHG administration,the content of ATP and the activity of Na~+-K~+-ATPase increased significantly in the hypoxic PC12 cells.In addition,the apoptosis rate,the activity of Caspase-3 and Caspase-9,the ratio of Bax/Bcl-2 and the expression of Cleaved Caspase-3 protein were significantly down-regulated in the 6-OHG administration group.3.Compared with the 6-OHG administration group,the integrity of the cell membrane was destroyed,the cell morphology changed,and the cell viability decreased significantly;the contents of ROS and MDA were significantly increased,and the levels of antioxidant enzymes CAT,SOD and GSH were significantly decreased;the protein expression and apoptosis rate of NF-κB and TNF-αwere significantly increased,the ratio of Bax/Bcl-2 and the expression of Cleaved Caspase-3 protein were significantly increased in Nrf-2 blocker co-treated with 6-OHG group.Conclusion:1.Ortho-triphenol hydroxyl group was the main group of 6-OHG for exerting anti-oxidation and anti-hypoxia effects.6-OHG has better anti-hypoxic and antioxidant activities than the other four methylated derivatives.2.6-OHG could relieve hypoxia-induced oxidative stress and energy metabolism disorder in PC12 cells,reduce inflammatory response and apoptosis.3.6-OHG alleviated hypoxia-induced injury in PC12 cells possibly by activating Nrf-2/HO-1 signaling pathway.