| Objective: The purpose of this study was to determine the optimum combination of SM and SA based on their effects on MIRI to facilitate the elucidation of their functional mechanisms.All the results achieved would provide solid theoretical evidences for the guidance of TCM clinical application and the development of modern TCM new drugs.Methods: 1.Single factor experiments and response surface methodology were employed to investigate the extraction method of Salvia miltiorrhiza and Sandalwood,the particle size of the medicinal material,the solvent type,the extraction solvent concentration and the ratio of material to liquid,based on the index of extract yield.The optimum preparation process for SM and SA extracts were established according to the gained results.2.The MIRI model was established on rat by left anterior descending artery ligation.70 rats were divided into 7 groups,10 in each group(5male+5female),representing sham operation group,model group,positive control group and dose group 1(high dose of Salvia miltiorrhiza + high dose of sandalwood,DGTG),dose group 2(high dose of Salvia miltiorrhiza + low dose of sandalwood,DGTD),dose group 3(low dose of Salvia miltiorrhiza + sandalwood High-dose group,DDTG),dose group 4(low-dose Salvia miltiorrhiza + low-dose sandalwood group,DDTD).The myocardial infarct size of each group was measured,and the pathological changes of myocardial tissue was observed by HE staining.The activity of CK and LDH in serum as well as SOD,GSH-Px and MDA in myocardial tissue were also measured by biological methods.Further,the relative expression of Bcl-2 and Bax genes in myocardial tissue were determined by PT-PCR.The above indicators were used to either determine the optimum combination of SM and SA or leave scientific clues for the discussion of their synergistic mechanisms on MIRI in vivo.3.The oxygen and glucose deprivation/reoxygenation model(OGD/R)was established on H9C2 cell.The drug-containing serum was isolated from three groups of rats after being feed 10 days by SM extracts,SA extracts,and SM-SA mixed extracts,respectively.The MTT method was used to investigate the cytotoxicity and protective effect on OGD/R H9C2 of the above three groups of drug-containing serum.Cell morphology was observed by Crystal Violet Staining.Cell apoptosis was observed by Hochest 33258 and EB/AO staining.Mitochondrial damage was examined by JC-1 staining.All those experiments above were designed to provide trends and clues for subsequent proteomics studies.4.The iTRAQ quantitative method was used to study the proteomics variation of blank control group(DZ),OGD/R model group(MX),SM drug serum group(FA1),SA drug serum group(FA2)and SM-SA mixed drug serum group(FA3).The proteins with significantly different expression level between different experiment groups(P-value<0.01,FC?1.5)had been subjected to GO annotation of biological process,cell localization and molecular function,according to UniProt-GOA database.The online tool KAAS was used to annotate the function of those differentially expressed proteins(P-value<0.01,FC?1.5),while the signal pathway of the annotation protein was mapped using KEGG mapper.By analyzing the function and signaling pathway of key differential proteins,a network of SM-SA synergistic effects against MIRI was constructed.Then,the protein of key nodes in the network was verified by Western blot so that the synergistic mechanisms of SM-SA on antti-MIRI effects could be elucidated.Results: 1.The optimum extraction processes for SM and SA were as follows: the optimum extraction process for SM(100 mesh of medicinal material,ethanol concentration of about 54%,ratio of material to liquid 1:25)and the optimum extraction process for SA(160 mesh of medicinal material,ethanol concentration of about 81%,ratio of material to liquid 1:23).2.The results of myocardial infarct size measurement showed that the positive control group and the drug-administered group 1,2,3 all significantly reduced the myocardial infarct size in rats(P<0.01)with compare to OGD/R group.Although the drug-administered group 4 appeared to be effective,its myocardial infarct size of the rats was significantly lower than that of the other three groups.The results of HE staining showed that SM contributed more to the amelioration of myocardial tissue injury.The therapeutic effects of all SM high-dose groups(Administered group 1 and 2)were better than those of SMlow-dose group(Administered group 3 and 4);On the other hand,the protective effect of SA on myocardial injury was also dose-dependent,leading to a better performance of the high-dose groups than the low-dose groups.The results of serum enzyme assay showed that both the drug-administered group and the positive control group could significantly reduce the levels of CK and LDH in serum(P<0.01).The effect of the drug-administered group were dose-dependent with the amount of SM and SA extracts in the mixture.In general,the effect of administered group 1 was the best,followed by administered group 2,administered group 3,and administered group 4;The results of cardiac tissue oxidative stress injury indicators showed that the SOD activity of positive control group and all administered groups increased significantly(P <0.01,P <0.05).The GSH-Px activity in positive control group and drug-administered group 1,2,and 3 was significantly increased(P <0.01,P<0.05)during which the content of MDA was significantly decreased(P <0.01);While the administered group 4 had a significant effect on the activity of SOD(P <0.05),its effects on the content of GSH-Px and MDA only showed no significant difference with the model group.The results of RT-PCR showed that the relative expression of Bcl-2 was significantly increased in the drug-administered groups 1,2,3 and the positive control group(P <0.01,P<0.05).All the drug-administered groups and the positive control group were observed to significantly reduce the relative expression of Bax(P <0.01,P <0.05),indicating that the above experimental groups can inhibit cardiomyocyte apoptosis to a certain extent.Relatively speaking,administered group 1 and 2 had extremely significant expression regulation of Bcl-2 and Bax,and were proved to be the best two groups on protecting cardiomyocytes among all administered groups.3.The results of MTT assay showed that the protective effect of SM-SA mixture group was the most significant at 5% concentration(P <0.01),followed by 5% concentration of SM extract group(P <0.05)and 5% concentration of SA extract group.The morphology of cell was confirmed by crystal violet staining.It was further confirmed that the combination of SM and SA was better than the single usage after dissection;Hochest 33258 staining showed that SM-SA mixed group and SM group can significantly reduce the number ofapoptotic cells(P <0.01),while the SA group has little effect on the proportion of apoptotic cells;The results of EB/AO staining showed that the proportion of apoptotic cells in SM+SA mixed group was significantly decreased(P <0.01),chromatin morphology was also normal,proving that it had better cardioprotection effect.Compared with the model group,the proportion of apoptotic cells of SM group and SA group also decreased significantly(P<0.05).However the proportion of early apoptotic cells(green spots)in SA group was significantly higher than that of SM group.The effect of JC-1 staining showed that the SM-SA mixed group significantly reduced the proportion of green fluorescence(P <0.01),which was better than the other two groups(P <0.05).4.The results of GO function annotation indicate different degrees of influence on the expression of extracellular space and matrix-related proteins,cell growth regulation,lipid metabolism,cholesterol metabolism,triglyceride metabolism,and clearance of various microparticles in myocardial cell,after pre-treatment with SM drug-containing serum.Nevertheless,SA-containing serum has different effects on ATP synthesis,oxygen binding and oxygen carrying capacity in addition to the expression of extracellular space and matrix related proteins.Combination of the two ingredients would produce a wider range of influences on external spatial environment response,emergency stress response,hypoxic stress response,oxidative stress response,mechanical stimulation stress response,mitochondrial ATP synthesis,proton transport,lipid metabolism and transport,protein synthesis,calcium regulation,protein binding ability and so on.KEGG functional annotation and signal pathway analysis showed that after administration of SM drug-containing serum,collagen adhesion protein was highly expressed,and the downstream PI3K-Akt signaling pathway was activated to produce an inhibitory effect on apoptosis.Also,the down-regulation on lysosome expression could ameliorate the damages for organelles,leading to a certain therapeutic effects on MIRI.The SA drug-containing serum group shared similar pathways with SM group in focal adhesion and PI3K/Akt.On the other hand,the SA group was discovered to have certain effects on cell adhesion factors(CAMs),which can activate the corresponding apoptotic pathway andoften appear as a target for anti-MIRI.After the combination of the two,their targets expanded to a larger range of proteins and pathways,for instances,the classical apoptotic pathways such as MAPK,PI3K/Akt and VEGF,as well as the mitochondria-related pathways such as oxidative phosphorylation and fatty acid metabolism.In general,the combination has a positive effect on inhibiting apoptosis,preventing mitochondrial oxidative damage and enhancing energy metabolism.The results of Western blot showed that the high-dose combination of SM and SA could significantly up-regulate(P <0.01)the expression of cell survival and apoptosis inhibition proteins,like Vtn,PI3 K and XIAP,as well as either significantly up-regulate(P<0.01)the phosphorylation level of Akt1 or significantly down-regulated(P <0.01)the phosphorylation level of pro-apoptotic genes NF-?B,p38 and MAPKAPK2.All the results above proved the synergistic effects of SM-SA combination on protecting cardiomyocytes from MIRI.Conclusion: The optimal dosage for SM and SA were 15g/d and 6g/d,which could synergistically exert anti-MIRI effects in vivo.Their mechanisms related to the inhibition of cardiomyocyte apoptosis and the regulation on oxidative stress environment of cardiac tissue.The experiments on OGD/R model proved that the combination of SM and SA had better cardioprotective effects as compared to single dosage of SM or SA.To be specific,single dosage of SM can reduce the number of dead cells by inhibiting apoptosis,while single dosage of SA can only reduce the number of cells in the cellular circle of late apoptosis with an insufficient ability on the early apoptosis inhibition.In addition,SM and SA extract could also effectively regulate the variation of mitochondrial membrane potential,indicating that the mechanism of its protection on OGD/R injury was directly related to the amelioration of mitochondria damage.Through proteomics research,it is further clarified that the combination of SM and SA could inhibit cell apoptosis,ameliorate mitochondrial damage,reduce ATP depletion and alleviate calcium overload,so as to exert the anti-MIRI effects.Their central function mechanisms were determined by western blot to be either increasing cell proliferation and viability through the Vtn/PI3K/Akt/NF-?B pathway orinhibiting apoptosis through the p38/MAPK pathway. |
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