| Objective:Cerebral ischemia reperfusion injury(CIRI)is an important pathological mechanism of ischemic stroke,and neuronal apoptosis is one of the main modes of death after ischemia and reperfusion injury.Total flavonoids from Rosa rugosa(TFR)is the Total flavonoids extracted from rose petals,which has a protective effect on cardiovascular and cerebrovascular diseases.SD rats were used as experimental subjects to observe the protective effect of TFR on CIRI rats,and to explore whether the protective effect is related to the regulation of PI3K/AKT signaling pathway to inhibit er stress and thus reduce nerve cell apoptosis.Methods:In vivo experiment: The Middle cerebral artery/ Reperfusion occlusion(MCAO/R)model was established by suture occlusion.Normal SD rats were divided into sham group(sham group),model group(MCAO/R group),model group +TFR low-dose group(MCAO/R+50 mg/kg.group),model group +TFR medium-dose group(MCAO/R+100 mg/kg group),model group +TFR high-dose group(MCAO/R+200mg/kg group).MCAO/R+TFR+ LY294002(MCAO/R+TFR+ LY).In vitro experiments: The primary neurons of cerebral cortex were extracted and cultured.The cells were divided into the six groups: Control group,OGD/R group,OGD/R+TFR group,OGD/R+TFR+LY group,TFR group and LY group.Behavioral function was measured by open field test and Longa score.TTC staining was used to observe the infarct volume of brain tissue.The degree of brain edema was detected by wet and dry weight method.SOD and MDA contents in brain tissue were detected by ELISA kit.Nissl and HE staining were used to detect pathological changes in cerebral cortex.TUNEL staining was used to detect the apoptosis of nerve cells in ischemic region.Western Blot was used to detect the protein expressions of Bcl-2,Bax,Cleaved caspase-3,GRP78,CHOP,and Caspase 12,and the apoptosis of neurons was detected by Hoechst33342 combined with flow cytometry.Results:In vivo experiment:(1)In MCAO/R group,Longa score was significantly increased,total exercise distance was significantly decreased,cerebral infarction volume was significantly increased,and cerebral edema was aggravated.Longa score(all P<0.001),total distance of exercise,cerebral infarction volume and brain tissue water content were decreased after TFR medium and high dose pretreatment(P<0.05,P<0.01).(2)SOD content in MCAO/R group decreased and MDA content increased(all P<0.001).Compared with MCAO/R group,SOD content increased and MDA content decreased after medium and high dose TFR pretreatment(all P<0.01).(3)The results of Nissl staining showed that a large number of Nissl bodies were dissolved in MCAO/R group,and HE staining showed that the nuclear membrane of neurons was broken,nucleolar shrinkage and a large number of vacuoles.After the intervention of TFR with medium and high dose,the lysis and degeneration of Nisseni body were relieved,and the morphological damage of neurons was relieved.(4)TUNEL staining showed the apoptosis of a large number of nerve cells in MCAO/R group,and the positive apoptotic cells were significantly decreased after TFR intervention(P<0.05,P<0.01).(5)The expression of apoptosis-related proteins was detected by Western blot,and the results showed that:Compared with MCAO/R group,apoptosis related protein Bax and Cleaved caspase3(P<0.05,P<0.01)levels were decreased in medium and high dose TFR groups,and bcl-2 protein expression was increased(P<0.05).(6)ER stress-related proteins were detected by Western blot.The expression levels of ER stressrelated proteins GRP78,CHOP,and Caspase 12 showed an increased trend in MCAO/R group(all P<0.001).ER stress-related protein expression decreased after TFR pretreatment(all P<0.01),and the effect of TFR on ENDOplasmic reticulum stress-related protein was reversed after PI3K/AKT signaling pathway inhibitor(all P<0.05).In vitro experiments :(7)Compared with OGD/R group,after TFR intervention,the number of floating neurons in the medium was significantly reduced,and the morphological integrity of neurons was improved.After PI3K/AKT signaling pathway inhibitor was given,the protective effect of TFR was inhibited.(8)Hoechst33342 staining combined with flow cytometry showed that the apoptosis rate of neurons in TFR intervention group was lower than that in OGD/R group(P<0.05),and PI3K/AKT signaling pathway inhibitor could reverse TFR inhibition of neuronal apoptosis(P<0.01).(9)Western Blot results showed that compared with OGD/R group,after TFR pretreatment,apoptosis related protein bcl-2 expression was increased(P<0.01),Bax and Cleaved caspase-3 expression was decreased(P<0.05,P<0.01).The expressions of er stress-related proteins GRP78,CHOP,and Caspase 12 were decreased(all P<0.01).The effects of TFR on apoptosis and er stress-related proteins were inhibited by PI3K/AKT signaling pathway inhibitors(all P<0.05).Conclusion:TFR can alleviate cerebral ischemia-reperfusion injury in rats,and the mechanism may be related to the inhibition of er stress response by activating PI3K/AKT signaling pathway,thus reducing neuronal apoptosis. |
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