| BackgroundWith the accelerated pace of life and the surge of work pressure in our country,cardiovascular and cerebrovascular diseases have seriously threatened people’s life and health.Among patients with those diseases,the incidence,disability rate,and mortality of stroke are remarkably high,and the damage caused by cerebral ischemia-reperfusion injury during treatment is extremely serious.However,there are no effective methods to treat cerebral ischemia-reperfusion injury in clinical practice at present.Therefore,searching for safe,effective,and prophylactic drugs for cerebral ischemia-reperfusion injury has become a research focus in recent years.The study of the effects and mechanism of the effective substances in traditional Chinese herbs plays an important role in the research and development of innovative and therapeutic strategies against cerebral ischemia-reperfusion injury.Traditional Chinese medicine has a long history in the research and treatment of ischemic stroke with blood stasis.Qi deficiency is the root cause of the disease and blood stasis is the core of the disease development.The commonly used traditional Chinese herbs for cerebral ischemia-reperfusion injury are Safflower,Salvia and Astragulus since Qi and blood circulation are the key essence of treatment for it.Numerous studies at home and abroad have shown that Hydroxysafflor yellow A(HSYA),Danshensu(DSS)and Astragaloside IV(AST-Ⅳ)as their main active ingredients respectively have curable effects on cerebral ischemia-reperfusion injury.Nowadays the traditional Chinese herbs in the treatment of cerebral ischemiareperfusion injury are mainly Chinese herbal compound formulas and compound preparations in clinical practice and have reaped good results.Both Safflower-Salvia and Safflower-Huangqi are classic couplet herbs,which are commonly used for the prevention and treatment of cardiovascular and cerebrovascular diseases.Although the compatibility of HSYA with DSS and AST-Ⅳ respectively has been effectively applied to the treatment of some cardiovascular and cerebrovascular diseases,there is no report of such treatment for cerebral ischemia-reperfusion injury and the synergy of its multi-components and multi-targets is still unknown.In addition,HSYA is not only the main active ingredient of safflower yellow with the efficacy of promoting blood circulation but also the most effective water-soluble ingredient of medicinal safflower.As a result,it is the main active ingredient used in several Chinese compound injections in clinical practice.However,its mechanism of fighting against cerebral ischemiareperfusion injury has not been fully elucidated until now.Since the pathophysiological mechanism of cerebral ischemia-reperfusion injury is complex and the ingredients of traditional Chinese herbs are numerous,the single pathway and single effect of research strategies in traditional molecular biology cannot elaborate the mechanism of traditional Chinese herbs for cerebral protection.With the rapid development of proteomics technology and comprehensive research in traditional Chinese medicine,we have been provided with a good opportunity to screen and analyze the differentially expressed proteins of HSYA against cerebral ischemia-reperfusion injury by label-free quantitation and bioinformatics techniques.Furthermore,we have investigated the compatibility of HSYA with DSS and AST-Ⅳ respectively to fight against cerebral ischemia-reperfusion injury in rats from a new perspective.Objectives1.To analyze the differentially expressed proteins of HSYA against cerebral ischemia-reperfusion injury by label-free quantitation and bioinformatics techniques for the preliminary determination of the multi-effects of safflower constituents to provide evidence for further study of stroke with blood stasis.2.To clarify the synergistic effects and mechanism of HSYA and DSS in combination to activate blood circulation and dissolve blood stasis on cerebral ischemiareperfusion injury in rats.3.To explore the synergistic effects and mechanism of HSYA and AST-Ⅳ in combination to invigorate Qi and activate blood circulation on cerebral ischemiareperfusion injury in rats.Materials and Methods1.60 SD rats were randomly divided into sham group,model group and HSYA group,with 20 rats in each group.MCAO model was established using the suture method and the rats were evaluated according to Neurological Deficit Scale.After 72 hours of reperfusion,the infarct areas of the rats with TTC staining were calculated by Image Pro Plus 7.0 software.Pathological changes were observed by HE staining and apoptosis was observed by TUNEL staining.Then label-free quantitation was used to screen the differentially expressed proteins of HSYA against cerebral ischemia-reperfusion injury and bioinformatics techniques were employed to analyze their effects and mechanism.Finally three essential proteins in the differentially expressed proteins of HSYA were verified by Western blot.2.40 SD rats were randomly divided into sham group,model group,HSYA group,DSS group,and HSYA+DSS group,with 8 rats in each group.MCAO model was established using the suture method and the rats were evaluated according to Neurological Deficit Scale.After 48 hours of reperfusion,the infarct areas of the rats with TTC staining were calculated by Image Pro Plus 7.0 software.Pathological changes were observed by HE staining and apoptosis was observed by TUNEL staining.The anti-oxidation of SOD,GSH-Px and MDA was measured by ELISA Kit.The anti-inflammation of TNF-α,IL-1β and IL-6 was detected by ELISA Kit.TLR4/NF-κB and Nrf2/HO-1 were measured by Western blot.The OGD-induced apoptosis of LDH was detected by MTT.3.100 SD rats were randomly divided into sham group,model group,HSYA group,AST-Ⅳ group,and HSYA+AST-Ⅳ group,with 20 in each group.MCAO model was established using the suture method and the rats were evaluated according to Neurological Deficit Scale.After 72 hours of reperfusion,the infarct areas of the rats with TTC staining were calculated by Image Pro Plus 7.0 software.Pathological changes were observed by HE staining.The expression of CD31 was observed by SABC method.The anti-oxidation of SOD and GSH-Px was measured by ELISA Kit.The anti-inflammation of TNF-α and IL-1β was detected by ELISA Kit.Results1.The findings of the differentially expressed proteins of HSYA against cerebral ischemia-reperfusion injury by label-free quantitation and bioinformatics techniques indicated that the anti-cerebral ischemia-reperfusion injury of HSYA was mainly represented by three essential proteins.Compared with the rats in model group,the infarct areas of the rats in HSYA group were reduced,their scales of neurological deficit evaluation before and after treatment were significantly different,and there were less pathological changes in the cortex.1982 differentially expressed proteins associated with HSYA were screened from 9107 proteins by label-free quantitation and 13 of them showed high expression.Western blot analysis confirmed that mTOR,Rab11 and Ppp2r5 e were the essential proteins of HSYA against cerebral ischemia-reperfusion injury.The up-regulation of mTOR and Rab11 and the down-regulation of Ppp2r5 e demonstrated the curable effects of HSYA on cerebral ischemia-reperfusion injury and their mechanism may be related to anti-inflammation,anti-oxidation,anti-apoptosis and angiogenesis.2.The findings of HSYA+DSS group against cerebral ischemia-reperfusion injury showed that the synergistic effects of HSYA and DSS in combination significantly enhanced the efficacy of anti-cerebral ischemia-reperfusion injury in rats.Although both HSYA and DSS improved the cerebral ischemia-reperfusion injury,their compatibility reaped better results in decreasing neurological deficit and reducing cerebral infarct size,cerebral cortex damage and TUNEL-positive cells(P<0.05).Compared with HSYA group and DSS group,SOD and GSH-Px in HSYA+DSS group were increased to 11.8,16.8 and 3.9,12.9,respectively while TNF-α,IL-1β,IL-6 and MDA in HSYA+DSS group were decreased to 39.7,41.9;10.6,6.3;49.5,31.9 and 5.38,16.82;respectively(P<0.05).Furthermore,the levels of TLR4/NF-κB were tremendously down-regulated while the levels of Nrf2/HO-1 were significantly up-regulated,with the remarkably increased level of LDH in OGD-induced neuronal cells in HSYA+DSS group.The mechanism of the enhanced curable effects of HSYA and DSS in combination may be related to inflammatory reaction and oxidative stress.3.The findings of HSYA+AST-Ⅳ group against cerebral ischemia-reperfusion injury demonstrated that the synergistic effects of HSYA and AST-Ⅳ in combination significantly enhanced the efficacy of anti-cerebral ischemia-reperfusion injury in rats.Although both HSYA and AST-Ⅳ improved the cerebral ischemic reperfusion injury,their combination reaped better results in decreasing neurological deficit,reducing cerebral infarct size and increasing CD31 cells(P<0.05).Compared with HSYA group and AST-Ⅳ group,SOD and GSH-Px in HSYA+AST-Ⅳ group were increased to 3.9,11.5 and 8.9,16.9,respectively while TNF-α and IL-1β in HSYA+AST-Ⅳ group were decreased to 3.7,5.9 and 12.9,7.3,respectively(P<0.05).The mechanism of the enhanced curable effects of HSYA and AST-Ⅳ in combination may be related to angiogenesis,anti-oxidation and anti-inflammation.ConclusionmTOR,Rab11 and Ppp2r5 e have been discovered and verified to be the essential proteins of HSYA against cerebral ischemia-reperfusion injury by label-free quantitation,bioinformatics techniques and Western blot analysis for the first time in this study.HSYA has displayed its curable effects on cerebral ischemia-reperfusion injury through the up-regulation of mTOR and Rab11 and the down-regulation of Ppp2r5 e.Their effects may be related to anti-inflammation,anti-oxidation,anti-apoptosis and angiogenesis,which has provided experimental evidence for further study of stroke with blood stasis.In addition,the compatibility mechanism of multi-components and multi-targets of such Chinese herbs as HSYA,DSS and AST-Ⅳ has been elucidated through the synergistic effects of HSYA with DSS/AST-Ⅳ against cerebral ischemia-reperfusion in rats,which has provided a new perspective for the treatment of ischemic stroke in clinical practice. |
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