| Objective:Cerebral ischemic stroke(CIS)has the characteristics of high prevalence,high recurrence rate,high disability rate and fatality rate.Sodium Tanshinone ⅡA Sulfonate(STS),as water-soluble substance after sulfonation of Tanshinone ⅡA,has a definite effect in the treatment of CIS,but the mechanism of it has not been fully elucidated.In this study,the network Meta-analysis system will be used to evaluate the effectiveness and safety of Danshen traditional Chinese medicine injections in the treatment of acute ischemic stroke.By means of network pharmacology and proteomics technology,we will explore the potential mechanism of Tanshinone ⅡA,and verify the targets in ischemia/reperfusion rat model and glucose deprivation/reoxygenation PC 12 cell model.Methods:(1)We searched the following 6 databases until February 2021:China National Knowledge Infrastructure(CNKI),VIP Database(VIP),Wanfang Database,PubMed and Cochrane Library.Two investigators independently screened the literature,extracted the data,and evaluated the quality of the literature according to the Jadad scale.Stata 16.0 was used for statistical analysis.(2)We employed TCMSP database to search for the active ingredients and targets of Danshen,as well as obtained the targets of CIS in GeneCards,NCBI and OMIM database.Then drug targets and disease targets were overlapped,and put into STRING database to establish a protein interaction network.Moreover,drug-compound-target-disease network was constructed and GO function and KEGG pathway enrichment analysis were carried out in Bioconductor.(3)The modified suture method was used to establish the middle cerebral artery occlusion reperfusion injury(MCAO/R)model of rats,and the SD rats were randomly divided into sham operation group,model group,and STS group.The MCAO/R model and drug efficacy were evaluated according to the neurological function score(Z-Longa),TTC staining and HE staining.(4)We employed DIA quantitative proteomics to analyze the protein expression in the ischemic penumbra of rats in each group,and screened the differential proteins according to the criteria of |log2FC|≥0.58 andp<0.05.R software was used to draw volcano maps and cluster analysis heat map,as well as STRING database to construct differential protein interaction(PPI)network.Then we performed GO enrichment analysis and KEGG signal pathway analysis with the aid of DAVID,ClueGO and other tools,and brought parallel reaction monitoring technology(PRM)method to detect protein expression.(5)We constructed oxygen and glucose deprivation reoxygenation(OGD/R)model in PC 12 cell,and added STS of different concentrations(1μM,5μM,10μM,20μM,40μM)for 3h,6h,12h.Then we detected cell viability by CCK-8 kit to determine the optional drug dose and time,and tested genetic expression by real-time fluorescent quantitative PCR(RT-qPCR)technology.Results:(1)A total of 160 studies were included,with a total of 18079 samples.The measures included 7 Chinese medicine injections and 8 treatment measures,covering Danhong injection combined with conventional treatment(DH+CT),Danshen injection combined with conventional treatment(DS+CT),Danshen ligustrazine injection combined with conventional treatment(DSCXQ+CT),Danshen polyphenolic acid for injection combined with conventional treatment(DSDFS+CT),compound Danshen injection combined with conventional treatment(FFDS+CT),Danshen polyphenolate for injection combined with conventional treatment(SI+CT),Sodium Tanshinone ⅡA Sulfonate injection combined with conventional treatment(STS+CT)and conventional treatment(CT).On the part of total effective rate,results of network meta-analysis showed that the rank of cumulative probability was:DSDFS+CT(93.0%)>DH+CT(80.5%)>STS+CT(66.7%)>DSCXQ+CT(66.4%)>SI+CT(50.0%)>DS+CT(26.7%)>FFDS+CT(16.7%)>CT(0.1%).In terms of NIHSS,the rank of cumulative probability was:STS+CT(95.5%)>DH+CT(80.9%)>DSCXQ+CT(70.1%)>SI+CT(64.7%)>DSDFS+CT(42.0%)>FFDS+CT(24.4%)>DS+CT(20.1%)>CT(2.4%).In the aspect of Barthel index,the rank of cumulative probability was:DH+CT(76.2%)>DSCXQ+CT(74.3%)>STS+CT(64.1%)>DSDFS+CT(62.2%)>FFDS+CT(51.8%)>SI+CT(46.0%)>DS+CT(21.7%)>CT(3.8%).(2)A total of 65 active ingredients and 108 targets were selected out from Danshen,and 2558 targets were collected related to CIS.After intersection,87 targets were obtained,among which the degree value of 6 targets was greater than 50,including AKT1,IL6,FOS,VEGFA,MAPK1,EGFR,as the core targets of this study.A total of 124 GO entries and 134 signal pathways were identified,with the PI3K/AKT and HIF-1 signal pathway occupying the largest number of targets.The drug-compound-target-disease network showed that active compounds such as tanshinone IIA played a key role in the entire network.(3)Compared with the sham-operated group,MCAO/R group had higher neurological function scores,larger infarct range in TTC staining,and obvious tissue degeneration in cerebral cortex and striatum in HE staining.In contrast,STS can reduce neurological function scores,lessen infarct size,and decrease vacuolar changes and nuclear shrinkage.(4)Proteomics has identified a total of 5880 proteins in rat brain tissues,among which there were 423 different proteins between the model group and sham-operated group.After intervention with STS,there were a total of 285 differential proteins compared with the model group.Then we analyzed the three groups integrally and systematacially,and acquired 127 differential proteins of expression trends.The GO enrichment was mainly involved in biological processes such as hypoxia response,neuronal apoptosis process,and calcium ion transport;KEGG pathway was mainly enriched In PI3K/AKT signal pathway,HIF-1 signal pathway and other pathways.The PPI network contained core differential proteins such as Alb,mTOR,Dynaclhl,Stxbp1,Cltc,and Sptanl.On account of mTOR,PI3K/AKT signaling pathway,and HIF-1 signaling pathway all the upstream signals that regulate autophagy,we detected the key targets of the above pathways and autophagy-related proteins by PRM.The results showed that the expression of mTOR and p62 decreased significantly in model group compared with sham-operated group(p<0.05),while the protein expression of HIF-1α,LC3-II/LC3-I,PI3K and AKT1 were not statistically significant(p>0.05).After treatment with STS,the expression of HIF1α and LC3-II/LC3-I protein decreased significantly(p<0.05),and the expression of p62 protein increased significantly(p<0.05).There was no statistical difference in the expression of mTOR,PI3K and AKT1(p>0.05).(5)The results of CCK-8 showed that compared with OGD/R model group,the concentration of STS 1μM and 5μM at 3h,6h,12h and other different administration times had no significant effect on cell viability.However,10μM,20μM and 40μM STS showed a protective effect on PC12 cells at each time(p<0.05),depending on the time of administration and the concentration of the drug.RT-qPCR showed that compared with the control group,the relative expression of HIF1α,Beclinl,and LC3 in OGD/R model group increased significantly(p<0.01),while the relative expression of mTOR mRNA decreased significantly(p<0.01).STS reversed the expression of the genes above mentioned(p<0.01).Conclusion:According to the results of network Meta-analysis,STS has advantages in improving the NIHSS score.Tanshinone IIA may be the main active compounds of Danshen by means of network pharmacology,and predicted by proteomics that its sulfonation may act on mTOR and other core targets,participate in regulating PI3K/AKT pathway,HIF-1 pathway and other signaling pathways,so as to inhibit cell apoptosis,fight inflammation,and regulate autophagy;STS may act on targets such as mTOR through the HIF-1 signaling pathway,so as to inhibit cell autophagy,remedy neurological deficits of MCAO/R model rats,shrink the area of cerebral infarction,and reduce brain tissue damage in vivo.STS may up-regulate the expression of mTOR through the HIF-1 pathway to inhibit autophagy and exert a protective effect on nerve cells in vitro. |
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