Quercetin Improves Cerebral Ischemia-Reperfusion Injury By Activating Nrf2 Signaling Pathway In Rats

Objective:To investigate the effect and mechanism of quercetin on cerebral ischemia-reperfusion（I/R）injury by activating Nrf2 signaling pathway,and to provide a basis for reducing cerebral ischemia-reperfusion injury.Methods:1.In vitro experiment:（1）Glucose and oxygen deprivation/reperfusion（OGD/R）of rat adrenal pheochromocytoma cells（PC12 cells,well differentiated）was established by using glucose-free medium and hypoxia workstation（5%CO₂,94.5%N₂,0.5%O₂）.OGD/R was performed for 6-hour OGD followed by 12-hour reperfusion.Intervention was performed with a gradient concentration of quercetin.（2）The Cell viability was detected by cell Counting Kit-8（CCK-8）,and the optimal dose range of quercetin was preliminarily screened.（3）The apoptosis rate and the level of Reactive oxygen species（ROS）in cells were detected by flow cytometry.The tetramethylrhodamine ethyl ester（TMRE）was used to detect the mitochondrial membrane potential level to study the effect of quercetin on mitochondria.（4）Immunofluorescence was used to indicate the location of Nuclear factor erythroid2-related factor 2（Nrf2）in cells.Western Blot to detect total Nrf2,nuclear Nrf2 and related signaling pathway proteins:Kelch like epichlor-ohydrin related protein 1（Keap1）,heme oxygenase-1（HO-1）,glutamate cysteine ligase catalytic subunit（GCLC）,NAD（P）H:quinone oxidoreductase 1(NAD（P）H:quinone oxidoreductase 1（NQO1）and sequestosome 1（P62/SQSTM1）.（5）Small Interfering Ribonucleic Acid（si RNA）was used to silence the expression of Nrf2,and Real-Time quantitative PCR（RT-q PCR）was used to detect the m RNA expression.（6）After Nrf2 expression was inhibited,the expression of Nrf2-related pathway proteins,apoptosis rate,intracellular ROS level,and superoxide anion fluorescence intensity were detected to verify whether Nrf2 was the target of quercetin.2.In vivo experiment:（1）The Middle Cerebral Artery Obstruction（MCAO）model was established by thread occlusion in rats:Blood flow was restored 2 hours after middle cerebral artery occlusion,followed by 72 hours of reperfusion.Different doses of quercetin were injected intraperitoneally at the beginning of reperfusion.（2）Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolium chloride（TTC）staining.The behavior of rats was evaluated by modified Neurological Severity Score（m NSS）.（3）Immunofluorescence Td T-mediated d UTP Nick-End Labeling（TUNEL）method was used to detect neuronal apoptosis on the cerebral infarction side,and Hematoxylin-Eosin（HE）and Nissl staining were used to detect the pathological damage of rat cerebral cortex.（4）Mitochondrial morphology was detected by transmission electron microscopy.（5）The Thibabituric Acid（TBA）method was used to detect the content of Malondialdehyde（MDA）in serum,and the hydroxylamine method was used to detect the content of Total Superoxide dismutas（T-SOD）in serum.（6）Immunofluorescence was used to detect the distribution and nuclear translocation of Nrf2,and Western Blot was used to detect the expression of total Nrf2and its related signaling pathway proteins Keap1,HO-1,GCLC and NQO1.Results:1.In vitro experiment:（1）Under conventional culture conditions,There was no significant change in cell viability in quercetin groups（P>0.05）.Cell viability was significantly decreased in the Glucose and oxygen deprivation/reperfusion（OGD/R）group compared with the control group（Ctrl）,and the difference was statistically significant（P<0.01）.Compared with the OGD/R group,quercetin（1μmol/L,5μmol/L,10μmol/L）significantly increased cell viability,and the difference was statistically significant（P<0.05）.（2）Compared with the Ctrl group,the apoptosis rate in the OGD/R group was significantly increased,and the difference was statistically significant（P<0.01）.Compared with the OGD/R group,the quercetin treatment groups all reduced the apoptosis rate,and the 5μmol/L quercetin treatment group had the most significant reduction in the apoptosis rate,and the difference was statistically significant（P<0.01）.ROS assay showed that compared with the Ctrl group,the intracellular ROS level in the OGD/R group was doubled,and the difference was statistically significant（P<0.01）.Compared with the OGD/R group,the ROS level was decreased in the quercetin intervention group,and the 5μmol/L group had the most significant reduction in ROS level,and the difference was statistically significant（P<0.01）.（3）Compared with the Ctrl group,the fluorescence intensity of mitochondrial membrane potential in the OGD/R group decreased significantly,and the difference was statistically significant（P<0.01）.Compared with the OGD/R group,the fluorescence intensity of the quercetin intervention group（OGD/R+Que）increased,and the difference was statistically significant（P<0.01）.（4）Compared with the Ctrl group,cells in the OGD/R group showed mild nuclear translocation of Nrf2,and cells in the quercetin group showed significant nuclear translocation of Nrf2.The results of WB showed that compared with the Ctrl group,Nrf2 in the nucleus increased slightly after OGD/R,and the OGD/R+Que group increased significantly in the nucleus,and the difference was statistically significant（P<0.05）.（5）WB results of Nrf2-related pathway proteins showed that there was no significant difference in the expression of Keap1 among the three groups（P>0.05）.The changes of HO-1,NQO1 and GCLC were consistent with Nrf2.Compared with the Ctrl group,the three proteins in the OGD/R group increased,but the difference was not statistically significant（P>0.05）.Compared with the Ctrl group,OGD/R+Que group further increased the expression of the above proteins,HO-1 and NQO1increased significantly,and the difference was statistically significant（P<0.01）.（6）Compared with the negative control group（NC）,the expression of Nrf2 in cells was significantly inhibited by si RNA（si Nrf2）,and the difference was statistically significant（P<0.01）.Compared with the OGD/R+Que group,the Nrf2,GCLC,HO-1,NQO1 and P62 in the quercetin treatment plus Nrf2 silencing group（OGD/R+Que+si Nrf2）were significantly decreased,and the difference was statistically significant（P<0.01）.（7）Compared with the OGD/R+Que group,the apoptosis rate of the OGD/R+Que+si Nrf2 group was significantly increased,and the difference was statistically significant（P<0.01）.（8）Compared with the OGD/R+Que group,the ROS level and mitochondrial superoxide superoxide anion in the OGD/R+Que+si Nrf2 group were significantly increased,and the differences were statistically significant（P<0.01）.2.In vivo experiment:（1）There was no cerebral infarction in the Sham group.Compared with the Sham group,the cerebral infarction in the middle cerebral artery occlusion（MCAO）group was more obvious,and there was a significant difference（P<0.01）.Compared with the MCAO group,the quercetin treatment group reduced the cerebral infarction volume in a dose-dependent manner,and the 50 mg/kg·bw group had the most significant reduction in the cerebral infarction volume（P<0.01）.（2）Compared with Sham group,the m NSS score of MCAO group increased significantly,and the difference was statistically significant（P<0.01）.Compared with the MCAO group,the m NSS scores of each quercetin intervention group were decreased,and the 50mg/kg·bw group had the most significant reduction（P<0.01）.（3）Under the transmission electron microscope,compared with the Sham group,the mitochondria of the neurons in the MCAO group were obviously swollen,the double membrane results were seriously damaged,and the crista was broken.Quercetin reduced mitochondrial damage in a dose-dependent manner,and the improvement was most obvious in the 50 mg/kg·bw dose group.（4）Compared with the Sham group,the serum MDA level in the MCAO group was significantly increased（P<0.01）,and the T-SOD level was decreased（P<0.01）.Compared with the MCAO group,the serum MDA level of the 50 mg/kg·bw quercetin group was significantly decreased（P<0.01）,and the T-SOD level was significantly increased（P<0.01）.（5）Compared with the Sham group,the positive rate of TUNEL staining in the neurons of the MCAO group was higher,and the tissue damage was serious.Compared with the MCAO group,the quercetin group reduced the number of TUNEL-positive neurons in the cerebral cortex,reduced neuronal apoptosis,and maintained the normal structure of the tissue（6）WB results showed that compared with Sham group,Keap1 and HO-1 in MCAO group had no significant change,Nrf2,GCLC and NQO1 were significantly decreased（P<0.01）;Compared with the MCAO group,the quercetin group had no significant change in Keap1,and the protein expressions of Nrf2,GCLC and NQO1 were significantly increased（P<0.01）.Conclusions:In vitro,Quercetin slows the damage of PC12 cells caused by OGD/R injury,reduces ROS levels,and protects mitochondrial function.In vivo,quercetin reduced the volume of cerebral infarction caused by MCAO and reduced the level of nerve defect in rats.It can reduce tissue damage and neuronal apoptosis,and reduce oxidative stress level.Nrf2 is the target of quercetin,and quercetin effectively reduces cerebral ischemia-reperfusion injury by regulating Nrf2 signaling pathway.