脑卒中是我国成年人致死致残的首要病因，分为出血性卒中和缺血性卒中，其中缺血性脑卒中约占全部卒中的80%。目前临床上常用的治疗手段包括溶栓和取栓两类，但由于严格的时间窗限制，许多患者无法得到有效的治疗。因此，深入了解缺血性脑卒中的发病机制及开发具有良好治疗效果的药物具有十分重要的意义。

       丹参葛根作为药对记载于《施今墨对药》中，两者配伍具有活血化瘀、祛瘀生新的功效。根据其现代药理作用，研发出了一些新的中成药制剂如通脉颗粒、脑心通胶囊等，在心脑血管疾病领域有着广泛的应用。现代药理研究表明，丹参葛根药对具有扩张脑血管、抑制炎症反应等作用。课题组前期研究也表明，丹参葛根提取物预给药7天，能够降低脑缺血再灌注大鼠的组织学损伤、改善脑血流并促进神经功能恢复，有效改善大鼠脑缺血再灌注损伤。

       细胞焦亡是近年来新发现的一种细胞死亡方式，与炎症反应密切相关。多项研究显示细胞焦亡上游分子在脑缺血半暗带中高度表达，抑制细胞焦亡能在一定程度上减轻缺血性脑卒中造成的损伤，因此开发能有效调控细胞焦亡的药物，有可能成为缺血性脑卒中药物研发的新方向。脑缺血再灌注发生后，炎症反应会对脑组织带来再次的损害，抑制炎症反应在脑缺血再灌注损伤的治疗中至关重要。丹参葛根药对具有很好的抗炎作用，而炎症反应又与细胞焦亡密切相关，基于以上理论基础，本课题拟从细胞焦亡的角度，探讨丹参葛根药对改善脑缺血再灌注损伤的作用，以及其可能的机制。

第一部分 丹参葛根药对对脑缺血再灌注损伤大鼠细胞焦亡的调节作用

目的：探究丹参葛根提取物对脑缺血再灌注大鼠脑损伤的改善作用，并对其是否通过细胞焦亡途径发挥药效进行探索。方法：选用健康雄性SD大鼠，采用线栓法，建立大脑中动脉阻塞（MCAO）模型以模拟脑缺血再灌注损伤，术后2 h进行再灌注。待大鼠清醒后随机分为对照组、模型组、丹葛提取物100 mg/kg、200 mg/kg、400 mg/kg组及NLRP3抑制剂MCC950 5 mg/kg组，其中丹葛提取物100、200、400 mg/kg剂量组进行灌胃给药，MCC950组腹腔注射给药，对照组和模型组给予等量的生理盐水，给药时间分别为3天和7天。在此期间对大鼠进行体重及改良神经功能缺损评分（mNSS）行为学监测，分别在第3/7天取大鼠脑组织进行TTC染色进行脑梗死率及脑半球肿胀度的计算。同时，检测大鼠血清中LDH的含量及炎症因子IL-18和IL-1β以及TNF-α的变化。此外，利用HE染色及Nissl染色检测大鼠脑组织的神经细胞损伤水平，采用RT-qPCR对脑缺血半暗带细胞焦亡相关的mRNA如NLRP3、caspase-1、GSDMD、ASC和IL-1β的表达水平进行检测，利用免疫荧光caspase-1和TUNEL双染来检测脑缺血半暗带中焦亡的发生水平，并利用免疫组化来检测焦亡相关的关键蛋白IL-18、IL-1β和TNF-α的表达水平。结果：与模型组相比，丹参葛根提取物100、200、400 mg/kg剂量组给药3天均能显著降低MCAO大鼠的脑梗死率（P <0.05，P <0.01），而仅有400 mg/kg剂量组能显著降低其脑半球肿胀度（P <0.01）；200、400 mg/kg剂量组能显著恢复MCAO大鼠的体重和降低改良神经功能缺损评分（P <0.05，P <0.01）；与模型组相比，丹参葛根提取物100、200、400 mg/kg剂量组给药7天均能显著降低MCAO大鼠的脑梗死率（P <0.05，P <0.01），200、400 mg/kg剂量组能显著降低其脑半球肿胀度（P <0.05，P <0.01）；200、400 mg/kg剂量组能显著恢复MCAO大鼠的体重、降低改良神经功能缺损评分和运动功能评分（P <0.05，P <0.01），100、200、400 mg/kg剂量组均能显著降低MCAO大鼠的平衡功能评分（P <0.05，P <0.01）。丹葛提取物及MCC950给药7天均能降低MCAO大鼠脑组织损伤水平，改善神经细胞形态结构。丹参葛根提取物100、200、400 mg/kg剂量组给药7天均能显著降低MCAO大鼠血清中LDH的水平(P <0.05，P <0.01)，200、400 mg/kg剂量组给药降低血清中炎症因子IL-18、IL-1β和TNF-α水平(P <0.05，P <0.01)，丹葛提取物高剂量组和MCC950均能降低NLRP3、caspase-1、ASC、IL-18和IL-1β的mRNA表达水平(P <0.05，P <0.01)，降低IL-18、IL-1β和TNF-α蛋白的表达水平，也降低脑组织中细胞焦亡的水平。结论：丹参葛根提取物能减轻MCAO大鼠的脑组织损伤水平，促进神经功能恢复，降低炎症因子水平，这可能与其能够抑制细胞焦亡相关。

第二部分 丹参葛根药对通过细胞焦亡途径改善大鼠脑缺血再灌注损伤的机制探讨

目的：运用网络药理学的研究方法预测丹参葛根药对可能参与调节细胞焦亡发挥抗脑缺血再灌注损伤作用的通路，并利用western blot技术对其进行验证，以探究丹参葛根药对治疗缺血性脑卒中可能的机制。方法：利用GEO数据库对NLRP3炎症小体调节相关的基因靶点进行搜集。在TCMSP数据库中对丹参葛根药对的药效成分和重要靶标进行搜集，并在Uniprot数据库中将其转换成基因名，与NLRP3炎症小体调节相关的靶标取交集，获得丹参葛根药对发挥调节NLRP3炎症小体作用的潜在靶标。将获得的潜在靶点数据导入cytoscape3.8.0软件，绘制药对成分-疾病-靶点网络图。之后将成分-靶点交集数据导入STRING网站预测各基因之间的联系，得到蛋白质-蛋白质互作网络（PPI）图，将获得的结果再次导入cytoscape软件筛选关键靶点。最后对筛选得到的关键靶点进行GO富集分析和KEGG信号通路分析，筛选获得关联性前20的丹参葛根调节NLRP3炎症小体活化的信号通路。利用western blot 方法对预测到的MAPK信号通路进行验证。结果：通过筛选获得了10个与NLRP3炎症小体调节相关的核心靶点，GO分析及KEGG信号通路富集分析得到MAPK信号通路与丹参葛根调节NLRP3炎症小体的关联性较大。Western blot的实验结果验证了网络药理学的预测，相较于I/R组，丹参葛根提取物400 mg/kg组及MCC950组能显著降低脑缺血半暗带细胞焦亡相关蛋白NLRP3、caspase-1、ASC表达水平(P <0.05，P <0.01)，能显著降低MAPK信号通路蛋白JNK及p38 MAPK水平(P <0.05，P <0.01)。结论：丹参葛根提取物通过细胞焦亡途径减轻MCAO大鼠的脑组织损伤水平可能与其调节MAPK信号通路相关。

第三部分 丹酚酸B-葛根素联用对氧糖剥夺/复氧损伤的SH-SY5Y细胞焦亡的作用

目的：基于细胞焦亡探讨丹酚酸B和葛根素合用对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation, OGD/R)损伤的SH-SY5Y神经细胞的保护作用及机制。方法：利用SH-SY5Y细胞构建OGD/R损伤模型，设立正常对照组(control)、模型组(OGD/R)、丹酚酸B组(salvianolic acid B, Sal B)、葛根素组(puerarin, Pue)、丹酚酸B和葛根素合用组(SP)和NLRP3炎症小体抑制剂MCC950组。除正常组外，其余各组细胞在氧糖剥夺6 h后迅速复糖复氧12 h进行OGD/R造模。采用MTT法检测细胞存活率，光学显微镜下观测细胞形态，分光光度计法检测培养上清中乳酸脱氢酶(LDH) 释放率，Hoechst/PI染色检测细胞损伤情况，RT-qPCR检测NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平，免疫荧光检测NLRP3和caspase-1蛋白表达的变化，western blot检测IL-1β、ASC、NLRP3、caspase-1和cleaved caspase-1蛋白表达的变化。结果：与模型组相比，丹酚酸B、葛根素与丹酚酸B和葛根素合用均能显著提高OGD/R损伤后SH-SY5Y细胞的存活率(P <0.01)；与单用组相比，合用组具有更好的效果(P <0.01)。丹酚酸B和葛根素合用及MCC950均能改善细胞形态，降低细胞上清液LDH的渗漏(P <0.01)，减轻细胞损伤水平，降低SH-SY5Y细胞NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平(P <0.05，P <0.01)，降低IL-1β、ASC、NLRP3、caspase-1和cleaved caspase-1蛋白的表达水平(P <0.05，P <0.01)。结论：结果表明，丹酚酸B和葛根素合用能减轻OGD/R对SH-SY5Y细胞造成的损伤，这可能与其能够抑制细胞焦亡相关。

Stroke is the leading cause of death and disability in Chinese adults. It is divided into hemorrhagic stroke and ischemic stroke, of which ischemic stroke accounts for 80%. Currently, there are two main types of clinical treatment, including thrombolysis and embolectomy. However, due to the strict time window, many patients cannot get effective treatment. Therefore, it is of great significance to explore the pathogenesis and develop drugs with good therapeutic effects of ischemic stroke.

Danshen-Gegen as a traditional Chinese medicine pair recorded in the "Shi Jinmo Pair medicine", has the effect of promoting blood circulation, disperse stasis and promoting regeneration. According to its modern pharmacological action, some new Chinese patent medicine preparations have been developed, such as Tongmai Granules and Naoxintong Capsules, which have been widely used in the field of cardiovascular and cerebrovascular diseases. Studies have shown that Danshen and Gegen can dilate cerebral blood vessels and inhibit inflammation. Our previous study also showed that preadministration of water extract from Danshen-Gegen for 7 days could reduce the histological injury, improve the cerebral blood flow and promote the recovery of nerve function, and effectively improve the cerebral ischemia reperfusion injury in rats.

Pyroptosis is a newly discovered cell death mode in recent years, which is closely related to inflammatory responses. Multiple studies have shown that inhibition of pyroptosis can reduce the damage caused by ischemic stroke to some extent, so the development of drugs that can effectively regulate pyroptosis might become a new direction of drug research for ischemic stroke. After the occurrence of cerebral ischemia-reperfusion, inflammation will cause repeated damage to the brain tissue, so the inhibition of inflammation is very important in the treatment of ischemic stroke. Danshen and Gegen have a good effect on anti-inflammation, and inflammation is closely related to pyroptosis. Based on the research theory mentioned above, the study aims to explore the effect of Danshen-Gegen on improving cerebral ischemia reperfusion injury and its possible mechanism from the perspective of pyroptosis.

Part 1 The regulation effect of Salvia miltiorrhiza Bunge and Radix Puerariae herbal pair on pyroptosis of cerebral ischemia reperfusion rats

Objective: To explore the amelioration effect of water extract of Danshen and Gegen on brain injury in rats with cerebral ischemia reperfusion, and whether it plays its role through pyroptosis pathway. Method: The model of MCAO (middle cerebral artery obstruction) was established in healthy male SD rats to simulate cerebral ischemia reperfusion injury, and the control group, I/R group, DG 100 mg/kg group, DG 200 mg/kg group, DG 400 mg/kg group and NLRP3 inhibitor MCC950 group were set up randomly. DG extract 100、200、400 mg/kg groups were administrated by intragastric, and MCC950 group was administrated by intrabitoneal injection. The control group and I/R group were given the same amount of normal saline. During this period, the body weight and modified neurological deficit score(mNSS) of rats were monitored. TTC staining was performed on the brain tissue of rats on day 3/7 to calculate the cerebral infarction rate and cerebral hemisphere swelling degree separately. At the same time, the levels of LDH and the change of inflammatory cytokines IL-18, IL-1β and TNF-α in serum were then determined. What’s more, HE staining and Nissl staining were used to detect the level of nerve cell damage in rat cerebral ischemic penumbra. RT-qPCR was used to detect the expression levels of NLRP3, caspase-1, GSDMD, ASC and IL-1β related to pyroptosis in cerebral ischemic penumbral zone cells. Immunohistochemistry was used to detect the expression of IL-18 and IL-1β, and immunofluorescence of caspase-1 and TUNEL double staining were used to detect the occurrence level of pyroptosis in ischemic penumbra. Results: Compared with the I/R group, 100、200 and 400 mg/kg groups of DG for 3 days could significantly reduce the cerebral infarction rate of MCAO rats (P <0.05, P <0.01), but only 400 mg/kg dose group could apparenttly lower the cerebral hemisphere swelling degree (P <0.01). 200 and 400 mg/kg dose groups could markedly restore the weight of MCAO rats and reduce the score of mNSS (P <0.05, P <0.01); Compared with the I/R group, the 100、200 and 400 mg/kg dose groups of DG for 7 days could significantly reduce the cerebral infarction rate of MCAO rats (P <0.05, P <0.01), and the 200 and 400 mg/kg dose groups could apparently bring down the cerebral hemisphere swelling degree (P <0.05, P <0.01). 200 and 400 mg/kg dose groups could markedly restore the weight of MCAO rats and reduce the score of mNSS (P <0.05, P <0.01). DG and MCC950 can lower the level of brain tissue injury and improve the morphological structure of nerve cells in MCAO rats for 7 days. After 7 days of administration of DG in 100、200 and 400 mg/kg dose groups, the serum LDH level of MCAO rats was significantly decreased (P <0.05, P <0.01), and the serum inflammatory cytokines IL-18, IL-1β and TNF-α levels in 200 and 400 mg/kg dose groups were decreased (P <0.05, P <0.01). In 400 mg/kg DG group and MCC950 group, the expression levels of IL-18 and IL-1β protein, mRNA expression levels of NLRP3, caspase-1, ASC, IL-18 and IL-1β were cut down (P <0.05, P <0.01), and the pyroptosis level of cells in brain tissue was decreased. Conclusion: The water extract of Danshen and Gegen can reduce the level of brain tissue injury in MCAO rats, promote neurological recovery, lower the level of inflammatory cytokines, which may be related to the inhibition of pyroptosis.

Part 2 The mechanism of Salvia miltiorrhiza Bunge and Radix Puerariae herbal pair to improve cerebral ischemia reperfusion injury by pyroptosis pathway

Objective: Using the research method of network pharmacology, to predict the pathway that DG may participate in the regulation of pyroptosis and play the role of anti-cerebral ischemia reperfusion injury, and to verify it by western blot. Methods: GEO database was used to collect gene targets related to NLRP3 inflammasome regulation. Pharmacodynamic components and important targets of Danshen and Gegen were collected in the TCMSP database and converted into gene names in the Uniprot database. The intersection of targets related to the regulation of NLRP3 inflammasome was selected to obtain potential targets for Danshen and Gegen to play the role of regulating NLRP3 inflammasome. The potential target data obtained was imported into cytoscape3.8.0 software to map the drug pair component-disease-target network. Cytoscape software will import the cytoscape intersection data into the STRING website to predict the association between genes and obtain the protein-protein interaction network (PPI) map. The obtained results will be imported into Cytoscape software to screen key targets. Finally, GO enrichment analysis and KEGG signaling pathway analysis were performed on the selected key targets, and the top 20 signaling pathways regulating the activation of NLRP3 inflammasome were screened. Western blot was used to verify the predicted MAPK signal pathway. Results: Ten core targets related to NLRP3 inflammasome regulation were obtained through screening. GO analysis and KEGG signal pathway enrichment analysis showed that MAPK signaling pathway had the greatest correlation with Danshen and Gegen in regulating NLRP3 inflammasome regulation. Western blot verified the prediction of network pharmacology. Compared with I/R group, Danshen-Gegen water extract 400 mg/kg group and MCC950 group could significantly reduce the level of NLRP3、caspase-1、ASC、JNK and p38 MAPK in ischemic penumbra (P <0.05, P <0.01). Conclusion: The reduction of brain tissue injury in MCAO rats by the water extract of Danshen and Gegen through pyroptosis pathway may be related to the regulation of MAPK signaling pathway.

Part 3 The effect of salvianolic acid B-puerarin combination on pyroptosis of SH-SY5Y cells after OGD/R injury

Objective: To investigate the effect and mechanism of salvianolic acid B and puerarin on SH-SY5Y cells damaged by oxygen glucose deprivation and reoxygenation (OGD/R) based on pyroptosis. Method：SH-SY5Y cells were used to construct the model of OGD/R damage, and the control group, OGD/R group, salvianolic acid B group (Sal B), puerarin group (Pue), salvianolic acid B and puerarin group (SP) and MCC950 group were set up. Cell survival rate was detected by MTT method, cell morphology was observed by optical microscope, lactic dehydrogenase (LDH) content in culture supernatant was detected, cell damage was detected by Hoechst/PI staining, mRNA expression levels of NLRP3, caspase-1, GSDMD, ASC and IL-1β were determined by RT-qPCR.Caspase-1 and NLRP3 protein activation were detected by immunofluorescence. Protein expression levels of IL-1β、ASC、NLRP3、caspase-1 and cleaved caspase-1 were observed by western blot. Result: Salvianolic acid B, puerarin and salvianolic acid B combined with puerarin could improve cell survival rate (P <0.01), and the combined treatment group had better effect (P <0.01). Salvianolic acid B combined with puerarin and MCC950 can both improve cell morphology, reduce the leakage of LDH (P <0.01), reduce the level of cell damage, reduce the mRNA expression levels of NLRP3, caspase-1, GSDMD, ASC and IL-1β (P <0.05, P <0.01), and also reduce the activation of IL-1β、ASC、NLRP3、caspase-1 and cleaved caspase-1 protein. (P <0.05, P <0.01) Conclusion: It’s indicated that salvianolic acid B combined with puerarin can alleviate the damage of SH-SY5Y cells caused by OGD/R, which may be related to the inhibition of pyroptosis.