| The Research BackgroundSepsis is a life-threatening organ dysfunction caused by the host's maladjusted response to infection.The heart is one of the most vulnerable organs.About 40%?60%of patients with sepsis are accompanied by myocardial injury,with a mortality of 70%?90%,while those without myocardial injury have a mortality of 20%Sepsis myocardial injury brings great burden to medical and health industry and national economy.Save Sepsis Campaign(Surviving Sepsis Campaign,SSC)international Sepsis and septic shock treatment guidelines(2016)denied many previously recommended therapeutic regimen,means the Sepsis myocardial damage diagnosis are still facing many problems,such as the pathophysiological mechanism of complex,extensively drug-resistant bacteria produce and treatment,medical treatment of iatrogenic damage,high,etc.It is of great significance to study the pathogenesis,evolution and effective therapeutic drugs of myocardial injury in sepsis.Ginkgo Biloba is the dried leaf of Ginkgo Biloba.Ginkgo Biloba Extract,Ginkgo Biloba Extract,GBE)mainly contain flavonoids(mainly quercetin,rhizoma kaempferiae phenol,rat Li Su,mignonette,rutin,celery and Yang mei),terpenoids(including Ginkgo lactone and Ginkgo lactone),has long been used in the prevention and treatment of cardiovascular diseases,for the protection of the myocardial cell function involves multiple aspects,such as anti-inflammatory,antioxidant stress.GBE can increase the expression of Hsp72 protein in cardiomyocytes and reduce the oxidative damage of cardiomyocytes under heat stress.Inhibition of myocardial inflammatory response and platelet activation improved cardiac function in rats with myocardial injury caused by ischemia reperfusion,reduced infarcted area,and alleviated endoplasmic reticulum stress and inhibited myocardial cell injury induced by AGEs by antagonizing the expressions of GRP78 and CHOP.In addition,some cell experiments have found that compound ginkgo leaf granules can improve the morphological changes of damaged myocardial cells,reduce LDH and CK leakage,reduce MDA and NO levels in cells,increase SOD activity,reduce ROS production and participate in the protection of damaged myocardial.How to protect the myocardium at or before the occurrence of sepsis has been one of the hot topics in critical care medicine.According to the literature search,there are few studies on the application of ginkgo biloba extract in myocardial injury caused by sepsis.During myocardial infarction,myocardial cells suffer from ischemia and hypoxia for a long time,which can lead to irreversible cell death(or apoptosis)and cardiac dysfunction.Reducing myocardial cell apoptosis can improve cardiac function and cardiac remodeling process after MI.Reducing oxidative stress and direct intervention to inhibit apoptosis pathway can provide potential molecular targets for treatment.MicroRNA(miRs/miRNAs)is a class of non-coding RNA molecules that regulate gene expression,and there is increasing evidence that miRNAs play an important role in heart disease.Mir-370 has long been considered as a tumor suppressor,which can down-regulate endothelin,inhibit cell proliferation and induce apoptosis of endometrioid ovarian cancer cells.In gastric cancer tissues and cells,mir-370 was significantly down-regulated by targeting the regulation of progesterone and fat Q receptor family member 4.Studies have shown that the up-regulation of mir-370 may inhibit vascular inflammation and oxidative stress,and the expression of mir-370 in myocardium is decreased after acute myocardial infarction.Ginkgo biloba extract has long been used in the treatment of cardiovascular disease,we through the in vivo animal experiments found that ginkgo biloba extract can inhibit myocardial injury caused by sepsis,through the cell in vitro experiment found that the active components of ginkgo biloba extract ginkgo double yellow ketone can through the miR-370/FOXO1 suppress the apoptosis induced by oxidative stress,this finding provides a theoretical basis for the clinical applicationIn this paper,we established a model of myocardial injury in sepsis rats,designed a drug prevention group,and studied the protection of ginkgo biloba extract in advance on myocardial injury in sepsis rats and the changes in survival time,proving the preventive value of drugs and the theoretical value of "prevention before disease" in TCM.Tested the troponin I,CK-MB,such as myocardial injury markers,tested the IL-6,TNF-? inflammatory factors,such as myocardial tissue MDA content and SOD activity were measured and analyzed the Bcl-2,BAX,Caspase 3,Caspase-6 protein expression changes,and observed the rat myocardial tissue inflammation and apoptosis,prompt ginkgo biloba extract can relieve the oxidative stress and reduce the myocardial cell apoptosis of myocardial cells to sepsis in the rat myocardial injury play a role of protection,and improving survival time.It has been proved by cell experiments that regulation of mir-370/FOXOl is the mechanism by which ginkgo diflavone inhibits apoptosis of cardiomyocytes induced by oxidative stress,which provides theoretical basis for the prevention and protection of ginkgo leaf extract against myocardial injury in sepsis.Part I protective effect of ginkgo biloba extract on myocardial injury in sepsis ratsObjectiveWhether ginkgo biloba extract can reduce myocardial injury in sepsis and the timing of administration were studied preliminarily.Methods1.Grouping experimental animals:96 male Wistar rats were randomly divided into 4 groups according to the random number table:Sham operation group(S group),sepsis group(C group),Prevention group(injection of ginkgo biloba damol injection 6 hours before CLP modeling,group P),Treatment group(0.5 h after CLP injection of ginkgo dameo injection,T group),There were 24 rats in each group.12 animals were randomly selected from each group and their survival time was observed up to 72h.2.Establishment and intervention of sepsis rat model:(1)The model of myocardial injury in sepsis rats was established by cecal ligation and perforation(CLP).In the pseudo-operation group,the abdominal cavity was opened,the cecum was turned over,the abdominal cavity was put back into the abdominal cavity without puncture treatment,and the abdominal wall incision was sutured layer by layer.(2)Experimental rat administration1)false operation group:Saline 4 ml kg was injected intraperitoneally at 6 h before operation and 0.5 h after operation.2)Sepsis group:Saline 4 ml kg was injected intraperitoneally at 6 h before operation and 0.5 h after operation.3)Prevention group:Ginkgo biloba injection 4 ml·kg?(-1)was injected intravenously 6 hours before operation,and saline 4 ml·kg?(-1)was injected intraperitoneally 0.5 h after CLP.4)Treatment group:Normal saline 4 ml·kg?(-1)was injected intraabdominal 6 hours before operation and Ginkgo biloba injection(4 ml·kg?(-1)was injected intravenously 0.5 h after operation.3.Specimen collection and detection of related indicators(1)Blood sample extraction from experimental ratsThe rats were randomly sacrificed at 6h and 12h,and the blood samples were taken from the abdominal aorta.After centrifugation,the supernatant was placed in EP tube and stored in low temperature refrigerator at-80?.(2)Troponin I,CKMB,IL 6 and TNF-were detected by ELISA.(3)HE staining and light microscopic examination were performed onpathological sections of heart.4.Statistical treatmentSPSS22.0 statistical software was used for statistical analysis,and all experimental results were expressed as mean±standard error(x±s).For multi-group comparison,if the data met normal distribution and homogeneity of variance,anova was used for inter-group comparison;if the difference between groups was statistically significant,Bonferroni method,LSD method and dunnett-t method were further used for pairwise comparison.If normal distribution was not followed,kruskal-wallis rank sum test was used for inter-group comparison and kaplan-meier analysis was used for survival analysis.P<0.05 was considered as statistically significant difference.Results1.The model of myocardial injury in sepsis rats was successfully established.Compared with the sham operation group,ck-mb and cTnl levels in sepsis group,prevention group and treatment group were significantly higher than those in the sham operation group(p<0.05),indicating that myocardial injury occurred in rats after CLP modeling.2.After CLP modeling,the survival rate of rats showed a decreasing trend,with the highest survival rate in the prevention group,followed by the treatment group,and the lowest survival rate in the CLP group.Compared with the treatment group,the prevention group was more significant for the long-term prognosis of sepsis rats.3.Compared with the sepsis group,the levels of ck-mb,cTnI,il-6 and tnf-alpha in the prevention group and the treatment group after the intervention of ginkgo biloba extract were significantly decreased(p<0.05).The levels in the prevention group before the model administration were more significantly decreased than those in the treatment group after the model administration(p<0.05).4.In the sham group,the myocardial striation was clear and the cells were neatly arranged.There was no obvious nuclear swelling or inflammatory cell infiltration,and no obvious congestion,edema or inflammatory cell infiltration in the interstitium.In the sepsis group,the myocardial edema was denaturated and the cytoplasm was slight,with hyaline degeneration of cardiac muscle fibers,edema and nuclear membrane rupture,obvious infiltration of inflammatory cells,interstitial edema,congestion,and inflammatory cell infiltration.The edema and volume of cardiomyocytes in the treatment group and the prevention group were reduced,and the interstitial inflammatory response was significantly improved.Compared with the treatment group,the cardiomyocyte edema under microscope was less in the prevention group,and the interstitial inflammatory response was not significantly different.ConclusionGinkgo biloba extract has a protective effect on myocardial injury in septic rats.The application of ginkgo biloba extract before sepsis can significantly improve the long-term survival prognosis of septic rats.Part ii study on the protective mechanism of ginkgo biloba extract against myocardial injury in septic ratsObjectiveTo investigate the possible mechanism of protection of ginkgo biloba extract against myocardial injury in septic rats.Methods1.Grouping of experimental animals:96 male Wistar rats were randomly divided into 4 groups according to the random number table:Sham operation group(S group),Sepsis group(group C),Prevention group(injection of ginkgo biloba damol injection 6 hours before CLP modeling,group P),Treatment group(0.5 h after CLP injection of ginkgo dameo injection,T group),Each group had 24 rats.2.Establishment and intervention of sepsis rat modelSame as the first part.3.Specimen collection and related indicators detection(1)the antioxidant capacity of myocardial tissue was determined by colorimetryThe left ventricular myocardial tissue of each rat was taken 0.2g,and the tissue homogenate was prepared with pre-cooled normal saline.MDA content and SOD activity in the myocardial tissue were measured according to the kit instructions.(2)expression of apoptosis-related proteins BAX,BCL-2,Caspase-3 and Caspase-6 were detected by Western blot.(3)TUNEL method:to observe the apoptosis of cardiomyocytes in sepsis rats by ginkgo biloba extract.(4)After homogenization of myocardial tissue,the expression of mir-370 was detected by qPCR and FOXO1 was detected by Western Blot.4.Statistical processingSPSS 24.0 statistical software was used,the measurement data were tested by t,and Graphpad Prism 6.0 software was used for statistical processing and mapping.The results were expressed as mean ± standard deviation(±S).Univariate analysis of variance was used for comparison between groups,and P<0.05 was considered statistically significant.The protein expression levels of each group were analyzed by Image J 2.0 software.Results1.Compared with the sham operation group,the concentration of MDA in the myocardial tissue of the rats was significantly increased,and these changes were significantly decreased after treatment with GBE.SOD activity(2-2)of group C was significantly lower than that of control group,and SOD concentration of group P and group T was significantly increased compared with that of group C.2.Compared with the control group,the proportion of apoptotic protein BAX and anti-apoptotic protein BCL-2 in the prevention group and the treatment group was significantly increased,and the activity of cleaved Caspase-3 and Caspase-6 was also significantly increased.GBE normalized the ratio of BAX to BCL-2 in sepsis rats and reduced the levels of Caspase-3 and Caspase-6.3.After TUNEL staining,the apoptotic cells in the S group were very small,compared with the apoptotic cells in the C group,and the number of apoptotic cells in the rat cardiomyocytes was significantly decreased after GBE treatment.4.The expression of mir-370 in the CLP group was significantly down-regulated,and the expression of mir-370 in the P group and T group after GBE intervention was up-regulated compared with that in the C group.FOXO1 expression in P group and T group was down-regulated compared with that in C group.ConclusionGBE inhibits apoptosis by reducing inflammatory response and oxidative stress in rat cardiomyocytes.Regulation of mir-370/FOXO1 is a possible mechanism by which GBE inhibits apoptosis in cardiomyocytes induced by oxidative stress.Part iii study on regulating mir-370/FOXO1 to inhibit oxidative stress and apoptosis of cardiomyocytesObjectiveTo study the effect of ginkgo diflavone on mir-370/FOXO1 on h2o2-induced myocardial cell injury in oxidative stress and apoptosis.Methods1.H9C2 cells were cultured in vitro,cultured and subcultured according to the instructions,and cells of logarithmic growth stage were taken for experimental study.2.Grouping of cells The Normal group Cells were normally cultured in DMEM medium containing FBS without any The Normal group treatment H2O2 group The cells were treated with a final concentration of 100 mol/1 H2O2 solution H202 group in DMEM medium containing FBS for 24h H2O2+Ginkgetin group The cells were treated for 24h in DMEM medium containing FBS with a final H2O2+Ginkgetin group concentration of 100 mol/1 H2O2 solution and a final concentration of 15 mol/l ginkgo diflavone solution H2O2+Ginkgetin+miR-370 inhibitor group The cells were treated with 100 mol/1 H2O2 solution and 15 mol/l ginkgo H2O2+Ginkgetin+miR-370.diflavone solution at the same time in DMEM medium containing FBS for inhibitor group 24h,and mir-370 was down-regulated3.The transfection level was detected after cell transfection.4.Western Blot experiments were performed to extract total protein from cells and determine the protein concentration.Then acrylamide(sds-page)gel electrophoresis was performed,and the expression intensity of the target protein was calculated after membrane transfer,sealing,primary antibody incubation,secondary antibody binding and development.5.RT-qPCR,Cells of the control group and the experimental group were collected,total RNA was extracted,purity and integrity were detected,genomic DNA was removed,and real-time PCR reaction was performed after RNA reverse transcription.6.Statistical methods:SPSS 24.0 software was used for statistical analysis of the data.All experiments were repeated three times,and the data were expressed as mean±standard deviation(±S).T test was used to compare the mean of two samples,and anova was used to compare the mean of multiple samples.P<0.05 was considered to be statistically significant.The ggplot2 package of R 3.5.3 was used for data visualization.Results1.After H202 treatment of H9C2 cells,the expressions of Caspase-3(p<0.01)and Caspase-6(p<0.01)were up-regulated,and the ratio of BAX to BCL-2 was increased(p<0.01).At the same time,the expression of mir-370 was down-regulated,while the expression of FOXO1 was up-regulated.2.When h2o2-treated H9C2 cells were treated with ginkgo diflavone,the expressions of Caspase-3(p<0.01)and Caspase-6(p<0.01)were down-regulated,and the ratio of BAX to BCL-2 was decreased(p<0.01).The expression of mir-370 was up-regulated by qPCR and down-regulated by Western BlotConclusionGinkgo diflavone inhibits apoptosis by upregulating mir-370,and regulating mir-370/FOXO1 is the mechanism of inhibiting apoptosis of cardiomyocytes induced by oxidative stress. |
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