| Stroke,also known as cerebral apoplexy or cerebrovascular accident,is one of the leading causes of death worldwide,including two common types:ischemic stroke and hemorrhage stroke.Ischemic stroke is a common form of stroke caused by insufficient blood supply to the brain.However,due to the complication of the pathological process of ischemic stroke,so far the safe and effective anticerebral ischemic drugs are very few.Traditional Chinese medicine Xiaoxuming decoction is a classic prescription for treating ischemic stroke.Active components group of Xiaoxuming decoction is the effective pharmacological activity components of Xiaoxuming decoction for the treatment of cerebral ischemia.Our previous study confirmed that the active components group of Xiaoxuming decoction can significantly improve the mitochondrial energy metabolism disorder,improve the mitochondrial membrane potential,and inhibit mitochondrial apoptosis pathway.Recent studies have found that cerebral ischemia-reperfusion injury is closely linked to the dysfunction of mitophagy.Ischemic injury leads to impaired mitochondrial function,while mitophagy can remove damaged mitochondria and maintain their own steady-state balance to achieve mitochondrial quality control.Therefore,we think targeted adjustment of mitophagy may reduce the injury of cerebral ischemia-reperfusion.So,we use focal cerebral ischemia rats induced by MCAO to investigate the effect and mechanism of the active components group of Xiaoxuming decoction on mitophagy in ischemic stroke.We observed the changes of mitochondrial function and mitophagy in the brain to reveal the mechanism of the active components group of Xiaoxuming decoction on ischemic stroke through regulating mitophagy.Part 1 Effect of active components group of Xiaoxuming decoction on mitochondrial function after focal cerebral ischemia/reperfusion injuryObjective To explore the effects of the active components group of Xiaoxuming decoction(XXMD)on mitochondrial function in rats after focal cerebral ischemia/reperfusion injury.Methods The healthy male SD rats were randomly divided into Sham group,the ischemia/reperfusion group,Xiaoxuming decoction active components low,medium,high dose groups and positive drug Ginaton group(extract of ginkgo biloba leaves EGb 761).The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on rats.Rats were orally administrated with active components group of Xiaoxuming decoction and Ginaton.We measured mitochondrial membrane swelling,mitochondrial ATPase enzyme activity(Na+-K+-ATPase,Ca2+-Mg2+-ATPase),mitochondrial superoxide anion(O2-)and calcium ion content to explore the protective effect of XXMD on the mitochondrial function in MCAO rats.Results Compared with the model group,active components group of XXMD could significantly improve the activity of the brain mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase(P<0.05,P<0.01)and significantly decreased the content of mitochondrial calcium ion(P<0.01)and superoxide anion(P<0.05).Conclusion The active components group of XXMD can improve mitochondrial function by alleviating the oxidative stress reaction and calcium overload of mitochondria caused by ischemia-reperfusion,which plays a protective role on cerebral ischemia-reperfusion injury.Part 2 Effect of active components group of Xiaoxuming decoction on cerebral mitophagy after focal cerebral ischemia/reperfusion injuryObjective To explore the effect of the active components group of Xiaoxuming decoction(XXMD)on mitophagy at different time points after cerebral ischemia/reperfusion in MCAO rats.Methods The healthy male SD rats were randomly divided into Sham group,the ischemia/reperfusion group,Xiaoxuming decoction active components medium dose group and rapamycin(RAP)group.The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on rats.Rats were orally administrated with active components group of Xiaoxuming decoction,while rapamycin was given by intraperitoneal injection.The effects of XXMD and rapamycin on behavioral study of focal cerebral ischemia-reperfusion in rats were measured by Zea-Longa’s level standard scoring method and neurological defect Score(mNSS).The volume percentage and hemispheric swelling of rat cerebral infarction were detected by TTC staining.Western blot was used to detect the expression level of autophagy and mitophagy-related protein LC3,P62,Beclin-1 and BNIP3 at different time points after ischemia-reperfusion,and the mitochondrial morphology and ultrastructure of mitophagy in cortical neurons of ischemic penumbra were observed by transmission electron microscopy at 24 h after reperfusion.Results The behavioral results showed that compared with the model group,active components group of Xiaoxuming decoction and rapamycin could significantly alleviate the neurological deficit scores at 4 days after ischemia-reperfusion,decreased the MNSS score(P<0.05)of the MCAO rats,and prolonged the retention time of MCAO rats on the balance beam(P<0.05).TTC results showed that XXMD and rapamycin could significantly reduce the percentage of infarct volume and hemispheric swelling(P<0.01)of MCAO rats at 4 days after ischemia-reperfusion.Western blot results showed that both autophagy and mitophagy were activated at 24 h after cerebral ischemia-reperfusion.The conversion of LC3-I to LC3-II increased both in total brain proteins and isolated mitochondrial proteins(P<0.05,P<0.01),the expression level of P62,Beclin-1 and BNIP3 decreased markedly in total brain proteins(P<0.05),the expression level of P62 and BNIP3 increased markedly in isolated mitochondrial proteins(P<0.05,P<0.01),but the level of Beclin-1 did not increase.Mitophagy was inhibited at 48 h after reperfusion,and continued to decrease with the prolongation of reperfusion time.Compared with the model group,the active components group of XXMD could significantly inhibit the expression of P62,Beclin-1 and BNIP3 and decrease LC3-II/LC3-I ratio(P<0.05,P<0.01)in isolated mitochondrial proteins at 24 h after cerebral ischemia-reperfusion.The inhibitory effect of XXMD on mitochondrial autophagy continued until 48 hours after reperfusion then disappeared.Both the expression of P62,Beclin-1,BNIP3(P<0.05,P<0.01)and the ratio of LC3-II/LC3-I in purified mitochondrial proteins can be significantly elevated by XXMD at 96 h after cerebral ischemia-reperfusion.The electron microscope results showed that there were a large number of vacuoles and mitophagy structures in ischemic penumbra cortical neurons at 24 h after cerebral ischemia-reperfusion.Compared with the model group,the rat brain neuron nucleus membrane was complete,and the cellular device was significantly increased with the model group,and the XXMD of autophagy and mitochondria decreased markedly.Conclusion Autophagy and mitophagy were activated at 24 h after cerebral ischemia-reperfusion,and then it was inhibited.Too high and too low mitophagy are not benefit to the survival of nerve cells.XXMD can protect mitochondrial structures,regulate the level of mitophagy at different points after cerebral ischemia-reperfusion,inhibit the excessive activation of mitophagy in 24 h after ischemia-reperfusion,and improve the level of mitophagy in the later stage of ischemia-reperfusion.Part 3 Effect of active components group of Xiaoxuming decoction on cerebral ischemia reperfusion injury after using mitophagy inhibitorObjective To explore the effect of active components group of Xiaoxuming decoction on cerebral ischemia reperfusion injury after using mitophagy inhibitor mdivi-1.Methods The healthy male SD rats were randomly divided into Sham group,the ischemia/reperfusion group,Xiaoxuming decoction active components medium dose group,XXMD+ mdivi-1(XXMD+MDI)group and mitophagy inhibitor mdivi-1 group.The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on rats.Rats were orally administrated with active components group of Xiaoxuming decoction,while mdivi-1 was given by intraperitoneal injection.The effects of XXMD and XXMD+MDI on behavioral study of focal cerebral ischemia-reperfusion in rats were measured by Zea-Longa’s level standard scoring method and neurological defect Score(mNSS).The volume percentage and hemispheric swelling of rat cerebral infarction were detected by TTC staining.Western blot was used to detect the expression level of autophagy and mitophagy-related protein LC3,P62,Beclin-1 and BNIP3 at 96 h after cerebral ischemia-reperfusion.Results Compared with the XXMD group,the neuroprotective effect of XXMD was significantly reduced in the XXMD+MDI group,such as increasing the MNSS score(P<0.05),shortening the stay time of the MCAO rat on the balance beam(P<0.05),increasing the percentage of infarct volume(P<0.01)as well as the hemispheric swelling(P<0.05)at 4 days after ischemia-reperfusion.Western blot results showed that,compared with the XXMD group,the expressions of P62 and Beclin-1(P<0.05,P<0.01)and the conversion of LC3-I to LC3-II(P<0.05)were markedly inhibited in purified brain mitochondrial proteins of rats in XXMD+MDI group at 96 h after cerebral ischemia-reperfusion.Conclusion Mitophagy inhibitor mdivi-1 can inhibit mitophagy and further aggravate cerebral ischemia-reperfusion injury.After using mitophagy inhibitor mdivi-1,the protective effect of XXMD on the neurological function of MCAO rats was decreased,indicating that XXMD could play the neuroprotective effect by regulating mitophagy. |
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