脑卒中又称脑中风或脑血管意外，是由于脑部供血障碍或脑血管破裂引起的一种急性脑血管疾病，分为缺血性脑卒中和出血性脑卒中两种，死亡率和致残率极高，严重威胁着人类的生命健康。然而，由于缺血性脑卒中病理进程复杂，迄今为止正式被批准使用的有效治疗药物还为数不多。中药复方小续命汤是治疗缺血性脑卒中的经典方剂，小续命汤有效成分组是依据小续命汤适应症脑缺血的发病机制，通过体外筛选模型优化确定的小续命汤抗脑缺血的有效药理活性组分。我们前期研究证实小续命汤有效成分组能够作用于缺血区神经细胞线粒体，显著改善缺血后神经元线粒体能量代谢障碍，提高线粒体膜电位，抑制线粒体凋亡途径。近年来的研究发现，脑缺血再灌注损伤与线粒体自噬功能紊乱有着密切的联系，缺血性损伤导致线粒体功能受损，而线粒体自噬清除受损线粒体，完成损伤线粒体的降解，维持自身稳态平衡，实现线粒体质量控制，因此靶向调节线粒体自噬或能降低脑缺血再灌注损伤。

基于此，本课题以线粒体自噬为切入点，提出线粒体网络的稳态失衡在缺血性脑卒中发生发展过程中具有重要意义，小续命汤有效成分组可通过调节线粒体自噬，调控线粒体稳态失衡改善神经元功能来治疗缺血性脑卒中。因此，本研究采用局灶性脑缺血再灌注大鼠为观察对象，以小续命汤有效成分组为干预手段，探讨小续命汤有效成分组对模型大鼠脑缺血改善情况，观察其对线粒体功能及线粒体自噬变化的影响，从调节线粒体自噬的角度揭示小续命汤有效成分组对缺血性脑卒中的作用机制。

第一部分 小续命汤有效成分组对局灶性脑缺血再灌注大鼠脑线粒体

功能损伤的影响

目的 考察小续命汤有效成分组对局灶性脑缺血再灌注大鼠脑线粒体功能损伤的影响。方法 选用健康雄性SD大鼠，体重250 g-280 g，随机分为假手术(Sham)组、模型(Model)组、小续命汤有效成分组(XXMD)低、中、高剂量组和金纳多(EGb 761)组，插线法制备大鼠右侧大脑中动脉缺血再灌注(MCAO)模型，缺血2 h后实现再灌注，假手术组大鼠不进行线栓插入操作。待大鼠清醒后给予药物，小续命汤有效成分组和金纳多均采用灌胃给药，每日一次，连续四天。测定给药4天时MCAO大鼠缺血周边区大脑皮层组织线粒体膜肿胀度、ATP酶活性(Na⁺-K⁺-ATPase、Ca²⁺-Mg²⁺-ATPase)以及线粒体超氧阴离子(O₂^-)和钙离子含量，研究小续命汤有效成分组对脑缺血再灌注大鼠脑线粒体功能的影响。结果 与模型组相比，给药4天时小续命汤有效成分组能够显著改善MCAO大鼠的脑线粒体功能损伤，表现为显著改善脑缺血再灌注大鼠脑线粒体Na⁺-K⁺-ATPase(P<0.05，P<0.01)和Ca²⁺-Mg²⁺-ATPase活力(P<0.05，P<0.01)，降低线粒体钙离子含量(P<0.01)和超氧阴离子含量(P<0.05)。结论 小续命汤有效成分组能够减轻脑缺血再灌注导致的线粒体氧化应激反应和钙超载，改善线粒体功能。

第二部分 小续命汤有效成分组对局灶性脑缺血再灌注大鼠脑线粒体自噬

的影响及作用机制研究

目的 考察小续命汤有效成分组对局灶性脑缺血再灌注大鼠皮层神经元不同时间点线粒体自噬的影响及相关机制。方法 选用健康雄性SD大鼠，体重250 g-280 g，随机分为假手术(Sham)组、模型(Model)组、小续命汤有效成分组(XXMD)中剂量组和自噬激活剂雷帕霉素(RAP)组，插线法制备大鼠右侧大脑中动脉缺血再灌注(MCAO)模型，缺血2 h后实现再灌注，假手术组大鼠不进行线栓插入操作。待大鼠清醒后给予药物，小续命汤有效成分组采用灌胃给药，雷帕霉素采用腹腔注射给药，每日一次，连续四天。采用Zea-Longa’s级标准评分法和神经功能缺损评分(mNSS)实验来测评小续命汤有效成分组和雷帕霉素给药对局灶性脑缺血再灌注大鼠行为学的影响，TTC染色法检测大鼠脑梗死体积百分比和半球肿胀。Western blot法检测给药后24h、48h、72h和96h时自噬及线粒体自噬相关蛋白LC3、P62、Beclin-1及BNIP3的表达水平变化，透射电镜观察再灌注24 h时缺血半暗带皮层神经元细胞中线粒体形态及线粒体自噬超微结构。结果 与模型组相比，给药4天时小续命汤有效成分组和雷帕霉素能够显著改善MCAO大鼠神经功能缺损，表现为降低MCAO大鼠mNSS评分(P<0.05, P<0.01)，延长MCAO大鼠在平衡木上的停留时间(P<0.05, P<0.01)。TTC结果显示，小续命汤有效成分组和雷帕霉素能够显著减少给药4天时MCAO大鼠脑组织梗死体积百分比(P<0.05, P<0.01)，并可显著缩小其半球肿胀(P<0.01)。Western blot检测结果显示，脑缺血再灌注24 h时，缺血周边区脑组织中自噬及线粒体自噬均被显著激活，表现为细胞总蛋白和纯化线粒体蛋白中LC3-I向 LC3-II的转化显著增多(P<0.05, P<0.01)，细胞总蛋白中P62、Beclin-1及BNIP3的表达水平发生下降(P<0.05)，而纯化线粒体蛋白中P62 及BNIP3的表达水平则显著上升(P<0.05, P<0.01)。缺血再灌注48 h时线粒体自噬被抑制，且随着再灌注时间的延长，线粒体自噬出现持续下降现象。与模型组相比，小续命汤有效成分组给药24 h时，能够通过降低P62、Beclin-1、BNIP3的表达水平以及LC3-II/LC3-I的比值(P<0.05, P<0.01)抑制脑缺血再灌注后24 h时线粒体自噬的过度激活。其对线粒体自噬的抑制作用延续至再灌注48 h，随着时间进一步延长，小续命汤有效成分组对线粒体自噬的抑制作用消失。缺血再灌注时间延长至96 h时，小续命汤有效成分组对P62、Beclin-1及BNIP3的表达出现明显激活作用(P<0.05, P<0.01)，并能增加LC3-II/LC3-I比值(P<0.05)，从而激活线粒体自噬，促进受损线粒体的自噬性清除。电镜检测结果显示，脑缺血再灌注24 h时，模型组大鼠脑组织神经元细胞中有大量空泡结构的自噬泡及大量线粒体自噬结构。与模型组相比，XXMD给药组大鼠脑神经元核膜较为完整，细胞器较模型组明显增多，自噬泡及线粒体自噬结构明显减少。结论 脑缺血再灌注后线粒体自噬随病程进展呈现先激活后抑制现象，过高和过低的线粒体自噬均不利于神经细胞的存活。小续命汤有效成分组能够调节缺血后不同时间点自噬及线粒体自噬相关蛋白的表达水平，抑制缺血再灌注后24 h时线粒体自噬的过度激活，促进缺血再灌注后期线粒体自噬。

第三部分 应用线粒体自噬抑制剂后观察小续命汤有效成分组对脑缺血

再灌注损伤的影响

目的 考察应用线粒体自噬抑制剂mdivi-1后对小续命汤有效成分组脑缺血再灌注损伤保护作用的影响及机制。方法 选用健康雄性SD大鼠，体重250 g-280 g，随机分为假手术(Sham)组、模型(Model)组、小续命汤有效成分组(XXMD)中剂量组、小续命汤有效成分组联合线粒体自噬抑制剂mdivi-1作用(XXMD+MDI)组及线粒体自噬抑制剂mdivi-1组，插线法制备大鼠右侧大脑中动脉缺血再灌注(MCAO)模型，缺血2 h后实现再灌注，假手术组大鼠不进行线栓插入操作。待大鼠清醒后给予药物，小续命汤有效成分组采用灌胃给药，mdivi-1采用腹腔注射给药，每日一次，连续四天。采用Zea-Longa’s级标准评分法和神经功能缺损评分(mNSS)实验来观察小续命汤有效成分组以及小续命汤有效成分组联合mdivi-1给药对局灶性脑缺血再灌注大鼠行为学的影响，TTC染色法检测大鼠脑梗死体积百分比和半球肿胀，Western blot法检测给药4天时，MCAO大鼠脑线粒体中自噬及线粒体自噬相关蛋白LC3、P62、Beclin-1及BNIP3的表达水平。结果 与XXMD组相比，应用线粒体自噬抑制剂mdivi-1后，小续命汤有效成分组对脑缺血再灌注损伤的神经保护作用显著减弱，表现为MCAO大鼠mNSS评分升高(P<0.05)，在平衡木上的停留时间减少(P<0.05)，脑组织梗死体积百分比增多(P<0.01)，并可增加其半球肿胀(P<0.05)。Western blot检测结果显示，应用线粒体自噬抑制剂mdivi-1后，小续命汤有效成分组大鼠脑线粒体P62和Beclin-1 的表达降低(P<0.05, P<0.01)，LC3-I向 LC3-II的转化减少(P<0.05)，说明线粒体自噬减弱。结论 线粒体自噬抑制剂mdivi-1能够抑制线粒体自噬，并能进一步加重脑缺血再灌注损伤。应用线粒体自噬抑制剂mdivi-1后，小续命汤有效成分组对MCAO大鼠的神经功能保护作用显著降低，说明小续命汤有效成分组可能通过调节线粒体自噬发挥神经保护作用。

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 injury

Objective 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 injury

Objective 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 inhibitor

Objective 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.