Study On The Mechanism Of Action Of Panax Notoginseng Saponins In Inhibiting Ferroptosis And Preventing Cerebral Ischemia-reperfusion Inflammatory Injury By Activating Nrf

Objective:1.To clarify the regulatory effects of Panax notoginseng saponins（PNS）on ferroptosis and inflammatory response in cerebral ischemic-reperfusion injury（CIRI）.2.To investigate the mechanism of PNS anti-inflammatory injury against cerebral ischemia-reperfusion based on negative regulation of ferroptosis by Nrf2 pathway.Methods:1.We determined the effect of PNS on Erastin-induced ferroptosis in SH-SY5Y cells.Ferroptosis was induced in SH-SY5Y cells by stimulating with erastin and oxygen/glucose deprivation-reperfusion（OGD/R）in vitro.We divided the cells into sham group,model group,low,medium,and high-PNS group,ferroptosis inhibitor（Fer-1）group and Deferoxamine（DFO）group.The degree of cell damage in each group was detected by CCK-8 assay and lactate dehydrogenase（LDH）leakage rate.The changes in key indicators of ferroptosis including Fe²⁺,GSH,MDA,and ROS contents were detected.2.We determined the effect of PNS on ferroptosis and inflammatory response in brain tissue of middle cerebral artery occlusion and reperfusion（MCAO/R）rats.We constructed the MCAO/R-induced CIRI model in rats and divided them into sham group,MCAO/R group,low,medium,and high-PNS group,Fer-1 group and DFO group.After ischemia for 2h and reperfusion for 24h,the degree of brain injury was detected by TTC staining and behavioral tests including neurologic deficit scoring,beam balance test,grip test,time to remove tape test,and asymmetry test.Fe²⁺,GSH,MDA and Lipid hydroperoxide（LPO）kits were used to detect the contents of key indicators of ferroptosis.And Western blot was used to detect the expression of ferroptosis regulatory proteins glutathione peroxidase 4（GPX4）,light chain subunit of the cystine/glutamate antiporter xc system（SLC7A11）,and Acyl-Co A synthetase long chain family ember 4（ACSL4）.IL-6,IL-1β,TNF-αand myeloid peroxidase（MPO）kits were used to detect the expression of inflammation-related indicators.3.We revealed the mechanism of PNS on inhibiting ferroptosis by activating Nrf2 to attenuate inflammatory injury in cerebral ischemia-reperfusion.We constructed OGD/R-induced cellular ischemia-reperfusion model and MCAO/R-induced CIRI model in rats.SH-SY5Y cells and rats were divided into sham group,model group,Nrf2 activator group,PNS high-dose group,Nrf2inhibitor group,and PNS high-dose+Nrf2 inhibitor group.The Nrf2 nuclear entry level in cells of each group was detected by immunofluorescence.Western blot was used to detect the expression of Nrf2 total protein,nuclear protein,and cytoplasmic protein in the brain tissue of rats.The expression of inflammation and ferroptosis-related indicators were detected by kits.The expression levels of key proteins of ferroptosis and regulatory pathways were detected by Western blot.Neurologic deficit scoring,behavioral tests and TTC staining were used to evaluate the degree of brain injury in rats.Results:1.The results of CCK-8 and the LDH leakage rate showed that,compared with the sham group,Erastin-treated or OGD/R-treated cells experienced a significant decrease in viability,a significant increase in the LDH leakage rate.In contrast,PNS,Fer-1,and DFO blocked these changes.The results of the ferroptosis kits showed that,compared with the sham group,the Fe²⁺,MDA,and ROS content were significantly increased and the GSH content was significantly decreased after Erastin treatment in SH-SY5Y cells.Compared with the Erastin group,the content of Fe²⁺,MDA,and ROS significantly reduced,and the content of GSH significantly increased in the PNS groups,Fer-1 group and DFO group in SH-SY5Y cells.2.The results of TTC staining showed that,compared with the sham group,the cerebral infarct volume increased in the MCAO/R group.Compared with the MCAO/R group,the cerebral infarct volume decreased in the PNS groups,Fer-1group,and DFO group.The neurologic deficit scores and behavioral tests results showed that,compared with the sham group,the neurologic deficit scores in the MCAO/R group were significantly increased,the time to remove tape was significantly increased,the beam balance test scores,asymmetry scores,and grip strength were significantly increased.Compared with the MCAO/R group,the difference in the PNS groups was consistent with the Fer-1 group and the DFO group,showing a significant reduction in neurologic deficit scores,and tape removal time,respectively beam balance test scores,asymmetry scores,and grip strength increased significantly.The results of the ferroptosis kits showed that,compared with the sham group,the contents of Fe²⁺,MDA,and LPO in the brain tissue of rats in the MCAO/R group were significantly increased,and the contents of GSH were significantly decreased.Compared with the MCAO/R group,the contents of Fe²⁺,MDA,and LPO significantly reduced,and the contents of GSH significantly increased in the PNS groups,Fer-1 group,and DFO group.The results of Western blot showed that,compared with the sham group,the protein level of ACSL4 in the brain tissue of rats in the MCAO/R group was significantly increased,and the protein level of GPX4 and SLC7A11were significantly decreased.Compared with the MCAO/R group,the protein level of ACSL4 was significantly decreased,and the protein level of GPX4 and SLC7A11 were significantly increased in the PNS groups,the Fer-1 group,and the DFO group.The results of the inflammation-related kits showed that,compared with the sham group,the contents of IL-1β,IL-6,TNF-α,and MPO in the brain tissue of rats in the MCAO/R group were significantly increased.Compared with the MCAO/R group,the contents of IL-1β,IL-6,TNF-α,and MPO in the brain tissue of rats were significantly reduced in the PNS groups,the Fer-1 group,and the DFO group.3.Immunofluorescence results in cells showed that,compared with the sham group,the Nrf2 nuclear entry level increased significantly in OGD/R-induced SH-SY5Y cells.Compared with the OGD/R group,the Nrf2 nuclear entry level was further increased after the intervention of PNS and Nrf2 activators.And compared with the PNS group,when PNS and Nrf2 inhibitors are used in combination,this promoting effect of PNS can be blocked by Nrf2-specific inhibitors.In addition,the results of Western Blot of rats brain tissue showed that compared with the sham group,the level of Nrf2 total protein and nuclear protein in the brain tissue of rats in the MCAO/R group were significantly increased,and the level of Nrf2 cytoplasmic protein was significantly decreased.Compared with the MCAO/R group,after the intervention of PNS and Nrf2agonists,the levels of Nrf2 total protein and nuclear protein further increased,and the level of Nrf2 cytoplasmic protein further decreased.And compared with the PNS group,when PNS and Nrf2 inhibitors were used in combination,the effect of PNS was blocked by Nrf2 inhibitors.The results of the ferroptosis kits showed that,compared with the MCAO/R group,the contents of Fe²⁺,MDA,and LPO decreased significantly after the intervention of PNS and Nrf2 activator,and the content of GSH significantly increased.When PNS and Nrf2 inhibitor were used in combination,the above effects were reversed.In addition,the results of Western blot showed that,compared with the MCAO/R group,the protein levels of GPX4,SLC7A11,transferrin（FPN）and ferritin heavy chain（FTH）were significantly increased after PNS and Nrf2 activator intervention.And compared with the PNS group,when PNS and Nrf2 inhibitor were used in combination,the level of ferroptosis-related proteins decreased significantly.The inflammation-related kits showed that compared with the MCAO/R group,the contents of IL-1β,IL-6,TNF-a and MPO were significantly reduced in the PNS+Nrf2 activator group.And compared with the PNS group,when PNS and Nrf2 inhibitor were used in combination,the content of inflammatory factors and MPO increased significantly.Finally,the results of brain injury showed that compared with the MCAO/R group,the intervention of PNS and Nrf2 activator significantly reduced the cerebral infarct volume,significantly reduced the neurologic deficit scores,and relieve motor dysfunction.And compared with the PNS group,when PNS and Nrf2 inhibitor were used in combination,the cerebral infarct volume increased as well as neurological deficit symptoms and motor sensory dysfunction aggravated.Conclusion:1.PNS inhibit ferroptosis induced by erastin and OGD/R in SH-SY5Y cells.2.PNS inhibit ferroptosis and inflammatory response in the brain tissue of MCAO/R rats.3.Panax notoginseng saponins activate Nrf2 to inhibit ferroptosis and attenuate inflammatory injury in cerebral ischemia-reperfusion.