Protective Effect Of Flavonoid Components Extract From Diopyros Kaki On Cell Injury Induced By OGD/R In HT22 Cells And Its Mechanism

BackgroundStroke is one of the major causes of disability and mortality in the world,in which ischemic stroke accounts for about 87%of all stroke types.At present,the main clinical treatment of ischemic stroke is intravenous thrombolysis therapy as early as possible to restore cerebral tissue reperfusion in ischemic area.However,the time window of intravenous thrombolysis is short(3 h).Reperfusion will aggravate the pathological damage of ischemic brain tissue and cause ischemia reperfusion injury.Therefore,ischemic stroke is still one of the hotspots in cardiovascular and cerebrovascular field.The pathological mechanism of cerebral ischemia reperfusion injury is complex,and the research shows that oxidative stress and apoptosis play an important role in secondary brain injury caused by cerebral ischemia.The occurrence of oxidative stress is mainly related to the imbalance of endogenous antioxidant reduction system,in which the transcription factor NF-E2 related factor 2(NF-E2-related factor 2,Nrf2)and the downstream target acting factor heme oxygenase-1(heme oxygenase-1,HO-1)play an important role in the maintenance of the balance of endogenous antioxidant reduction system.Oxidative stress mediates Nrf2's nuclear translocation and promotes the protein expression of its downstream HO-1 and other endogenous antioxidant enzymes,showing significant antioxidant stress effects.Studies have shown that apoptosis also plays a key role in the pathological process of cerebral ischemia-reperfusion injury.Cerebral ischemia-reperfusion injury may increase the expression of apoptotic protein,decrease the expression of anti apoptotic protein,increase the rate of apoptosis and the volume of cerebral infarction,however,Inhibition of apoptosis can reduce infarct volume and improve neurological function score in rats with cerebral ischemia reperfusion.Therefore,drugs with antioxidant stress and anti-apoptotic activity may provide a potential therapeutic strategy for ischemic stroke.Recent studies have found that Diopyros Kaki have many health care functions such asclearing away heat and detoxicating,moistening lung and strengthening heart,relieving cough and stopping bleeding,preventing cancer and so on.As the main effective component of Diopyros Kaki,the flavonoid components extract from Diopyros Kaki(FLDK)also have many biological effects,such as the occurrence of cancer,the inhibition of oxidative damage,and the improvement and strengthening of the physical and chemical functions of the heart and brain vessels.Our previous results showed that,in vivo,FLDK can reduce the overdeposition of A?,inhibit oxidative stress and inflammatory response,and improve the cognitive function in the brain tissue of APP/PSI transgenic mice.In vitro,persimmon leaf extract can inhibit the oxidative stress of HEK293-APPswe transgenic cells,reduce the aggregation of A?1-42.However,there are few studies on the protective effect of FLDK on cerebral ischemia-reperfusion injury.In vitro oxygen glucose deprivation/reperfusion(OGD/R)model can simulate the in vivo brain ischemia reperfusion,which can eliminate the interference of many factors in the body,and can be more direct and accurate to understand the changes of cell function and regulation mechanism after ischemia-reperfusion.Therefore,in this study,the OGD/R damage model of HT22 cells was established to observe the neuroprotective effect of FLDK and to explore its possible molecular mechanism.Objective1.Observation the protective effect of FLDK on oxygen glucose deprivation/reperfusion(OGD/R)induced HT22 cell injury.2.To investigate whether the anti oxidative stress and anti apoptotic effects of FLDK mediate by activating the PI3K/Akt/GSK-3? signaling pathway.Methods1.Determine the appropriate time of oxygen deprivation HT22 cells were randomly divided into 4 groups:normal control group(Control group),OGD(1h)/R(24 h)group,OGD(3h)/R(24 h)group,OGD(6h)/R(24 h)group.C.CK-8 assay was used to detect cell activity to determine the appropriate time of OGD,2.Determine the safe concentration range of FLDK and screen the appropriate effective concentrationDifferent concentrations of FLDK(0,1,5,10,20,50,100,200 ?g/mL)added to the normal HT22 cells were incubated for 24 h,CCK-8 assay was used to detect cell activity to determine the safe concentration range of drugs.Then established the OGD/R damage model,adding the drug within the safe concentration range,using CCK-8 assay combined with lactate dehydrogenase(LDH)assay to screen the effective concentration suitable for subsequent test.3.Effect of FLDK on oxidative stress in HT22 cells induced by OGD/R injuryHT22 cells were randomly divided into normal control group(Control group),OGD/R group,and OGD/R+FLDK group.Analysis Kit were used to detect the malondialdehyde(MAD)content and superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activity,2',7T-two chlorofluorescein diacetate(DCFH-DA)fluorescence probe was used to detect intracellular reactive oxygen species(ROS)level.4.Effect of FLDK on the apoptosis in HT22 cells induced by OGD/R injuryHT22 cells were randomly divided into normal control group(Control group),OGD/R group,and OGD/R+FLDK group.Flow cytometry analysis was used to determain the cell apoptosis,rhodamine 123(Rh123)and tetramethylrhodamine methyl ester(TMRM)staining method were used to detect the mitochondrial membrane potential(MMP)change,and Western-bloting was used to detect the Bax,Bcl-2,cleaved,Caspase-3,Caspase-8,cleaved caspase-9,cleaved PARP and cytochrome c(cyt-c)proteins expression.5.Exploring the possible neuroprotective molecular mechanism of FLDKHT22 cells were randomly divided into normal control group(Control group),OGD/R group,OGD/R+FLDK group and LY294002(PI3K specific inhibitor)group(OGD/R+FLDK+LY group).Western-blotting was used to detect the Akt,p-Akt,GSK-3? and p-GSK-3? protein expression.Then HT22 cells were divided into normal control group(Control group),OGD/R group,OGD/R+FLDK group,TDZD-8(GSK-3pspecific inhibitor)group(OGD/R+FLDK+TDZD-8 group).According to the above method,respectively,related indexes of oxidative stress in each group:the MAD content,SOD and GSH-Px activity,the ROS level,and Nrf2 and HO-1 proteins expression;apoptosis related indexes:cleaved caspase-3,caspase-8,cleaved caspase-9 protein expression and apoptosis rate.Results1 Determine the appropriate time of oxygen deprivationCompared with control group,the cell activity was not significantly decreased when OGD 1 h,and the difference was not statistically significant(P>0.05).When OGD 3 h and 6 h,the cell activity decreased to(45.4 ± 12.9)%and(37.1 ± 9.4)%of control group respectively,and the difference was statistically significant(P<0.05).In this experiment,OGD 3 h was used for follow-up test.2 Determine the safety concentration and appropriate effective concentration of FLDKDifferent concentrations of FLDK(1?200 ? g/mL)was added to normal HT22 cells for 24 h.When FLDK concentration was 0?50 ??g/mL,cell viability was not affected.When FLDK>100?g/mL,cell viability decreased significantly(P<0.05),so FLDK(0?50 ?g/mL)was the safe concentration.Drugs in a safe concentration range were added to HT22 cells induced by OGD/R injury.The results about cell activity and LDH release show that when the concentration of FLDK = 20 ?g/mL has the strongest protective effect.Therefore,choosing the concentration of FLDK<20?g/mL for subsequent tests.3 Effects of FLDK on oxidative stress in HT22 cells induced by OGD/R injuryCompared with the control group,the levels of ROS and MDA increased and the activity of SOD and GSH-Px decreased in OGD/R group(P<0.05);compared with the OGD/R group,the levels of ROS and MDA decreased and the activity of SOD and GSH-Px increased in the FLDK treatment group(P<0.05),which indicated that FLDK could inhibit the oxidative stress of HT22 cells mediated by OGD/R injury.4 Effects of FLDK on apoptosis in HT22 cells induced by OGD/R injuryCompared with the control group,the rate of apoptosis and the expression of apoptosis related proteins(Bax,cleaved Caspase-3,Caspase-8,cleaved caspase-9,cleaved PARP and Cyt-C)increased,and the expression of anti apoptotic protein Bcl-2 decreased in OGD/R group(P<0.05);compared with the OGD/R group,the apoptosis rate decreased,the expression of Pro apoptotic protein decreased,and the expression of anti apoptotic protein increased in the FLDK treatment group(P<0.05),which indicated that FLDK could inhibit the apoptosis of HT22 cells mediated by OGD/R injury.5 Effect of FLDK on PI3K/Akt/GSK-3? signaling pathwayOGD/R damage can reduce the phosphorylation levels of Akt and GSK-3p inHT22 cells(P<0.05),compared with the OGD/R group,FLDK treatment can increase the phosphorylation levels of Akt and GSK-3?(P<0.05).In order to verify whether the effect of FLDK on GSK-3 beta phosphorylation level is achieved by activating PI3K/Akt,HT22 cells were retreatmented with LY294002(a PI3K specific inhibitors)before FLDK treatment,and the results showed that LY294002 blocked the promoting effect of FLDK on phosphorylation of Akt and GSK-3?(P<0.05).This suggests that PI3K is a key upstream enzyme that activates the Akt/GSK-3 beta pathway,and FLDK activates the PI3K/Akt/GSK-3 beta signaling pathway of HT22 cells after OGD/R damage.6 Effect of FLDK on oxidative stress may mediated by activating the PI3K/Akt/GSK-3 ? signaling pathwayResults 3 had showed that FLDK could inhibit cellular oxidative stress mediated by OGD/R damage.To verify whether the antioxidant stress effect of FLDK is mediated by activation of PI3K/Akt/GSK-3p,HT22 cells were retreatmented with TDZD-8(a GSK-3 ? specific inhibitor)before FLDK treatment,and the results showed that TDZD-8 could partially block the inhibitory effect of FLDK on the increase of ROS and MDA and the decrease of SOD and GSH-Px activity,and reduce the promoting effect of FLDK on the translocation of Nrf2 and the expression of HO-1 protein(P<0.05).This suggests that FLDK may be mainly by activating PI3K/Akt/GSK-3? signaling pathway to increase Nrf2 translocation and HO-1 protein expression,and ultimately inhibit the oxidative stress of HT22 cells mediated by OGD/R damage.7 Effect of FLDK on oxidative stress may mediated by activating the PI3K/Akt/GSK-3 ? signaling pathwayResults 4 had showed that FLDK could inhibit cellular apoptosiss mediated by OGD/R damage.To verify whether the anti-apoptosiss effect of FLDK is mediated by activation of PI3K/Akt/GSK-3?,HT22 cells were retreatmented with TDZD-8(a GSK-3 p specific inhibitor)before FLDK treatment,and the results showed that TDZD-8 could partially block the inhibitory effect of FLDK on the apoptosis rate and the expression of apoptotic protein(P<0.05),suggesting that FLDK may inhibit the apoptosis of HT22 cells mediated by OGD/R damage mainly by activating the PI3K/Akt/GSK-3 beta signaling pathway.ConclusionsFLDK could inhibit OGD/R injury induced oxidative stress and apoptosis,and play a neuroprotective role in HT22 cells.The possible mechanism of molecular biology is to increase Nrf2 nuclear translocation and downstream antioxidant protein HO-1 expression and inhibit Pro apoptotic protein expression by activating the PI3K/Akt/GSK-3 beta signaling pathway,ultimately exerts the neuroprotective effect of antioxidant stress and anti apoptosis.