The Effect And Mechanism Of Salvia Miltiorrhiza Bunge And Radix Puerariae Herbal Pair On Cerebral Ischemia-reperfusion Injury By Pyroptosis Pathway

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 ratsObjective: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-1βrelated 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 pathwayObjective: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 p3 8 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 injuryObjective: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.