| Stroke, which is known as cerebrovascular accident, is a sudden onset disease of cerebral blood circulation disorder. Its main clinical manifestations are sudden fainting, facial asymmetry, hemiplegia, abnormal speech and intellectual disability. The epidemiological studies showed that ischemic stroke accounts for 80% of all stroke, increased by 8.7% through the incidence of hemorrhagic stroke decreased by 1.7% per year in China. With the increase of the aging population, the incidence of ischemic stroke is increasing year by year. However, due to the complexity of the pathological process of ischemic stroke, we almost have no drugs to treat the disease. Many studies have confirmed that mitochondria play an important role in the pathophysiology of ischemic stroke through a variety of mechanisms, which is an important subcellular target of ischemic injury. XXMD is a traditional Chinese medicine prescription for treating ischemic stroke from external wind. The active components group of Xiaoxuming decoction contains all the active ingredients in the decoction for the treatment of cerebral ischemia stroke. Therefore, we use the cerebral ischemic rats induced by MCAO as the observation object to explore the protective effect of the active components group of Xiaoxuming decoction on ischemic stroke and to investigate the role in the development of ischemic stroke by regulating mitochondrial homeostasis imbalance.Part 1 Neuroprotective effect of active components group of Xiaoxuming decoction on focal cerebral ischemia/reperfusion injury in rats during early recovery periodAim To explore the general protective effect of active components group of Xiaoxuming decoction (XXMD) on focal cerebral ischemia/reperfusion injury in rats during early recovery period. Methods The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on healthy male SD rats. The models of MCAO were evaluated by Zea-Longa’s standard score. The model rats were randomly divided into sham operation group, the ischemia/reperfusion model group, the active components group of Xiaoxuming decoction and the positive group (extract of ginkgo biloba leaves EGb 761). Rats were orally administrated with different drugs 24 h after operation for up to 14 days, once a day. The effect of active components group of Xiaoxuming decoction on behavior changes of MCAO rats in different recovery period was evaluated by a series of behavioral assessment methods such as modified neurological severity scores and corner test. The infarct volume and hemispheric swelling was observed by TTC staining. Results Compared with the I/R model group, the active components group of XXMD could significantly alleviate the neurological deficit scores with prolonged administration. The motion function tended to be normal, stayed longer on the balance beam, sensory function gradually restored sensitivity, reduce the radio of turning to the right. Among them, compared with model group, the active components group of XXMD could effectively improve the neurological dysfunction after five days of administration (P<0.05, P<0.01). Meanwhile, the active components group of Xiaoxuming decoction could significantly reduce the infarct volume percentage and hemispheric swelling in the cerebral tissue on post operation day 5 and 14 (P<0.01). Conclusion The active components group of XXMD can generate neuroprotective effect in early stage of recovery.Part 2 Effect of active components group of Xiaoxuming decoction on focal cerebral ischemia/reperfusion injury by recovering the redox level in rats during early recovery periodAim To explore the protective effect of active components group of Xiaoxuming decoction (XXMD) on focal cerebral ischemia/reperfusion injury by restoring the redox level in rats during early recovery period. Methods The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on healthy male SD rats. The models of MCAO were evaluated by Zea-Longa’s standard score. The model rats were randomly divided into sham operation group, the ischemia/reperfusion model group, the active components group of Xiaoxuming decoction and the positive group (extract of ginkgo biloba leaves EGb 761). Rats were orally administrated with different drugs 24 h after operation for up to 5 days, once a day. We measured the content of ROS、protein carbonyl and MDA in the penumbra and core area to evaluate the effect of XXMD on oxidative stress injury in MCAO rats. Meanwhile, we studied the activity of endogenous antioxidant enzymes such as SOD, CAT, GR, GSH-Px and NOS, and the level of antioxidants such as GSH and GSSG to evaluate the protective effect of XXMD in endogenous antioxidant system on MCAO rats. Results Compared with the I/R model group, the active components group of XXMD could significantly reduce the level MDA and protein carbonyl, and significantly reduce the activity of NOS (P<0.05) to relieve the oxidative stress injury on MCAO rats (P<0.05, P<0.01). At the same time, the active components group of XXMD could significantly improve the activity of CAT, SOD, GSH-Px and GR on MCAO rats (P<0.05, P<0.01), but the content of GSH and GSSG had no obvious change. Conclusion The active components group of XXMD can regulate the balance of oxidation and antioxidation in early stage of recovery to play a protective role in MCAO rats.Part 3 Effect of active components group of Xiaoxuming decoction on focal cerebral ischemia/reperfusion injury by protecting mitochondrial function in rats during early recovery periodAim To explore the protective effect of active components group of Xiaoxuming decoction (XXMD) on focal cerebral ischemia/reperfusion injury by protecting mitochondrial function in rats during early recovery period. Methods The ischemia and reperfusion of middle cerebral artery model rats were established by nylon wire with 2 hours ischemic time on healthy male SD rats. The models of MCAO were evaluated by Zea-Longa’s standard score. The model rats were randomly divided into sham operation group, the ischemia/reperfusion model group, the active components group of Xiaoxuming decoction and the positive group (extract of ginkgo biloba leaves EGb 761). Rats were orally administrated with different drugs 24 h after operation for up to 5 days, once a day. We measured the mitochondrial function of oxidative phosphorylation, mitochondrial respiratory enzyme activity (SDH and COX)、membrane potential, the opening degree of MPTP and the content of ATP to explore the protective effect of XXMD on the function of mitochondria on MCAO rats. We measured the expression of mitochondrial apoptosis related protein including Caspase-3, Caspase-9, Bcl-2 and Bax and so on to explore the effect of XXMD on the expression of mitochondrial apoptosis related protein on MCAO rats. Results Compared with the I/R model group, the active components group of XXMD could significantly improve mitochondrial respiratory activity (P<0.05, P<0.01), increase the activity of SDH (P<0.05), reduce the level of ROS (P<0.05, P<0.01)and obviously promote the synthesis of ATP in brain tissue (P<0.05). However, the active components group of XXMD has no obvious effect on mitochondrial membrane potential、swelling degree and the activity of COX. Compared with the I/R model group, the active components group of XXMD could significantly inhibit the expression of Bax and upregulate Bcl-2. Meanwhile, the active components group of XXMD could obviously upregulate Caspase-3 and Caspase-9, at the same time, downregulate cleaved caspase-3 and cleaved caspase-9 (P<0.05, P<0.01). Conclusion The active components group of XXMD can improve mitochondrial function and reduce the expression of the mitochondrial related apoptosis protein in early stage of recovery to play a protective role in MCAO rats. |
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