| Object ive:1.To assess the function and dose-response relationship of salvianolic acid B preprocessing of against myocardial ischemia-repursion injury in rats.2.To assess the effect of salvianolic acid B on the inflammatory response,apoptotic protein of myocardial ischemia-repursion injury in rats.3.To explore that in myocardial protection of myocardialischemia-repursion injury in rats,salvianolic acid B expression to myocardial SIRT1,and the effect of related protein expression mediated by SIRT1.4.Under the model of oxygen glucose deprivation/reperfusion,OGDR,to explore the relationships between myocardial protection of salvianolic acid B and SIRT1 protein expression,inflammatory factors.Methods:A.Animal experiment section:Models of myocardial ischemia-repursion injury in SD rats were adopted in the study,SD rats were randomlly into the group,and the groups were respectively:SHAMgroup,ischemia-repurfusion model group(I/R),Sa1B LD group(LD:30mg/kg),Sa1B HDgroup(HD:60mg/kg),Sa1B HD+ EX527 group(EX527:2mg/kg);N=16 per group.After administration for four days,besides group SHAM was conducted SHAM-operation,the other groups were all conducted ligation LAD for 0.5h,and were conducted repurfusion for 24h,then the next step was conducted:1.cardiac ultrasonography was underwent in all rats to detect their cardiac function.2.Blood was collected at posterior abdominal aorta,and after centrifugation,supernatant was removed at-20 C and frozen for Elisa use.3.Cardiac tissues were removed,heart paryformaldehyde of three rats was removed randomly from each group and fixed,then paraffin section was made for TUNEL/IHC use.Another three rats were underwent TTC staining.After the remaining rat s hearts were removed,2mm cardiac tissues along infarction border zone were clipped and protein was extracted for western blot use.Specific observation indicators were as follows:1.Cardiac function and size:cardiac ultrasonography ?left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular end systolic diameter(LVESD),left ventricular end diastolic diameter(LVEDD),left atrial diameter(LAD),etc.2.Marker of myocardial injury and inflammation marker:LDH/CK-MB/TNF-?/IL-1?/IL-18/HMGB1 was measured by Elisa;3.Area detection of cardiac infarction:TTC staining;4.Pathological observation of cardiac tissues of myocardial infarction ischemia-repurfusiion:TUNEL staining;5.Immuno-histochemistry of tissue protein:SIRT1/ac-HMGB1/TLR4.6.Detection of signaling pathway targets in myocardial infarction ischemia-repurfusion injury:western bolt:SIRT1,ac-HMGB1,TLR4,NF-KB,Bcl-2,Bax;B.Cell experiment section:Appling H9C2 cell to explore the establishment of cell model for cell OGDR,the minimum effective concentration of sirtl inhibitor EX527 and the testing of SalB cytotoxicity,and to determine Sa1B intervention concentration;they were divided into:controlgroup,SalBgroup,Modelgroup,Sal B LDgroup,SalB HD+EX527group;the experimental steps were as follows:The first stage:cardiomyocytes with density of 2*104/ml were planted in the 96 orifice plate to hatch for 24h.ODGR group was respectively cultivated with sugar-free culture-medium in a low oxygen(1%O2)invironment,then changed.into normal culture-medium.Cultivation with conventional incubator was to explore the establishment of ODGR model;after 24h incubation in 96 orifice plate,not any treatment was done in group control;the minimum effective concentration was tested in EX527 group with three respective concentrations 50 ?m,25 ?m,12.5 ?m;toxicity test in SalBgroup:MTT method,the SalBmaximal safe concentration was tested respectively with 200 ?m,100 ?m,50 ?m,0 ?m.The second stage:the control group,SalB group,group Model,Sal B LD group,Sal B HD group,and SalB HD+EX527 group were respectively administrated medication,after cultivated for 24h,OGDR model was established,then the following indicators were detected:1.Detection of marker of cell injury and inflammation marker:LDH/CK-MB/TNF-?/HMGB1 was measured by Elisa;2.Detection of intracellular injury signaling pathway-related proteins:SIRT1,ac-HMGB1,TLR-4,NF-?B,Bcl-2,Bax were detected by western bolt.Results:A.Animal expeeriment:1.Cardiac function evaluation:The rats EF,FS,SV in I/R group were significantly lower than that of normal group(P<0.05);compared with I/Rgroup,EF in Sal B LDgroup,Sal B HDgroup had significance difference,and it was higher than that of I/Rgroup(P<0.05);the significance difference in Sal B LD group was higher than that of Sal B HD group(P<0.05);the EF,FS,SV in Sal B HD+EX527 group was significant lower than that of Sal B HDgroup(P<0.05).The cardiac rate in I/R group was significant higher than that of normal group(P<0.05),and that had no difference between Sal B LDgroup,Sal B HDgroup,Sal B HD+EX527 group and I/Rgroup(P<0.05).2.Evaluation of marker of myocardial injuryThe rat LDH,CK-MB levels in I/R group were significantly higher than that of normal group(P<0.05);compared with I/Rgroup,LDH,CK-MB levels in Sal B LDgroup,Sal B HD group were significantly higher than that of I/Rgroup(P<0.05);LDH,CK-MB level in Sal B HD was significantly lower than that of Sal B LDgroup,and had significance difference(P<0.05);compared with Sal B LDgroup,LDH,CK-MB level in Sal B HD+EX527 groupwas significantly increased(P<0.05).3.Anatomic evaluation:The myocardial infarction area was significantly lower than that of normal group(P<0.05);compared with I/Rgroup,the percentage of infarct area to left ventricular area in Sal B LDgroup,Sal B HD group was significantly higher than that of I/Rgroup(P<0.05);the myocardial infarction area in Sal B HD group was significantly higher than that of Sal B LDgroup(P<0.05);compared with Sal B HDgroup,infarct area in Sal B HD+EX527 group was significantly decreased(P<0.05).4.Pathological evaluation:Tunel staining:the rat apoptotic rate in I/R group was significantly higher than that of normal group(P<0.05);compared with I/Rgroup,apoptotic rate in both Sal B LD group and Sal B HD group was significantly lower than that of I/Rgroup(P<0.05);the apoptotic rate in Sal B HD group was significantly lower than that of Sal B LDgroup;compared with Sal B HDgroup,apoptotic rate in Sal B HD+EX527 groupwas significantly increased(P<0.05).5.Evaluation of tissue protein expression;Western blot:the expressions of SIRT1,Bc1-2,N-HMGB1 in I/R group were significantly lower than that of normal group(P<0.05);compared with I/Rgroup,the expressions of SIRT1,Bcl-2,N-HMGB1 in both Sal B LD group and Sal B HD group were significantly higher than that of I/Rgroup(P<0.05);the expressions of SIRT1,Bcl-2,N-HMGB1 in Sal B HD group were significantly higher than that of Sal B LDgroup,and had significant.difference(P<0.05);compared with Sal B HDgroup,the expression of SIRT1,Bcl-2,N-HMGB1 in Sal B HD+EX527 group was significantly decreased(P<0.05).In constrast,the SD rat C-HMGB1,ac-HMGB1,Bax,TLR4,NF-KB expressions in I/R group were significantly higher than that of normal group(P<0.05);the C-HMGB1,ac-HMGB1,TLR4,NF-KB,Bax expressions in Sal B HD group were lower than that of Sal B LDgroup(P<0.05),and had significant difference;compared with Sal B HD group(P<0.05),the C-HMGB1,ac-HMGB1,TLR4,NF-KB,Bax expressions in Sal B HD+EX527 group were significantly increased(P<0.05).Immunohistochemistry:the rat SIRTlexpression in group I/R was significantly lower than that of normal group(P<0.05);compared with I/Rgroup,SIRT1 in Sal B LD group and Sal B HD group was significantly higher than that of I/Rgroup(P<0.05);the expression of SIRT1 in Sal B HD group was significantly higher than that of Sal B LDgroup,and had significant difference(P<0.05);compared withSal B HD group,SIRT1 inSal B HD+EX527 group was significantly decreased(P<0.05).In constrast,SD rat ac-HMGB1 and TLR4 expressions in I/R groupwas significantly higher than that of normal group;compared with I/Rgroup(P<0.05),ac-HMGBl,TLR4 expressions in Sal B LD group and Sal B HD group were significant lower than that of I/' Rgroup(P<0.05);the ac-HMGBl,TLR4 expressions in Sal B HD group was significantly lower than that of Sal B LD group(P<0.05),and had significant difference;compared with Sal B HD group,the ac-HMGB1,TLR4 expressions in Sal B HD+EX527 group were significantly increased(P<0.05).6.Evaluation of inflammation related factorsThe rat TNF-?,HMGB1 levels in I/R groupwas significantly higher than that of normal group(P<0.05);compared with I/Rgroup;the TNF-a,HMGB1 levels in Sal B LD group and Sal B HD group were significantly lower than that of I/' Rgroup(P<0.05);the TNF-?,HMGB1 levels in Sal B HD group were significantly lower than that of Sal B LD group,and had significant difference(P<0.05);compared with Sal B HDgroup,the LDH,CK-MB levels in Sal B HD+EX527 groupwere significantly increased(P<0.05).B.Cell experiment1.Establishment of OGDR cell model:When it is hypoxia for 24h and reaeration for 24h,the cell viability reached 63.8%,and reaeration for 4h,the cell viability was up to 80.1%.Hereby,the establishment time of OGDR model was confirmed to be:hypoxia for 24h and reaeration for 24h.2.Toxicity evaluation of Sal B to H9C2 cardiomyocytes:The result showed that the 100 ?M,200 ?M cell viabilities reduced to 76.7%and 47.1%;no significant difference was found in 50 ?M,and there was no statistic difference with 0 ?m.Therefore,the maximal administrated concentration of Sal B was 50 ?M,and 25 P M was also set to be a low-dose group.3.In OGDR model,the evaluation of Sal B preprocessing to cell viability:Under OGDR condition,the cell viabilities of Sal B 20 ?M(LD)and 50 ?M were respectively increased to 75.2%and 86.6%,and there was significant difference between Sal B 25 ?M and Sal B 50 ?M(P<0.05),the improved cell viabilities appeared concentration dependence;the function of Sal B was reversed by EX527.4.Evaluation of protein expression:The expression of SIRT1 in Model group was significantly lower than that in control group(P<0.05).Compared with model group,the expressions of SIRT1 and Bcl-2 in Sal B LD group and Sal B HD group were significantly increased(P<0.05),while ac-HMGB1.The expressions of TLR4,NF-?B and Bax were significantly decreased(P<0.05).The expression of SIRT1 and Bcl-2 in Sal B HD group was higher than that in Sal B LD group,and ac-HMGB1,TLR4,NF-?B and Bax were lower than Sal B LD group.There were significant differences(P<0.05).Compared with Sal B HD group,the expression of SIRT1 and Bcl-2 in Sal B HD+EX527 group was significantly decreased(P<0.05),while ac-HMGB1,TLR4,NF-?B and Bax were significantly increased(P<0.05).5.Evaluation of inflammation related factorsThe levels of TNF-? and HMGB1 in I/R group were significantly higher than those in normal group(P<0.05).The levels of TNF-? and HMGB1 in Sal B LD group,Sal B HD group and I/R group were significantly lower than I/R.The R group(P<0.05);the level of TNF-?.and HMGB1 in the Sal B HD group was lower than that in the Sal B LD group(P<0.05);compared with the Sal B HD group,the Sal B HD+EX527 group was compared with the Sal B HD group.-? and HMGB1 levels increased significantly(P<0.05).Conclusion:1.Salvianolic acid B can effectively promote myocardial ischemia-repursion injury,presenting dose dependence;2.The mechanism for salvianolic acid reducing myocardial ischemia-repursion injury maybe:through up-regulating the expression of SIRT1 thus inhibiting the HMGB1 nuclear translocation and secretion;through the SIRT1/HMGB1/TLR4/NF-KB pathway,improving the rat' s myocardial inflammatory level and apoptosis rate.3.We build on the above conclusions,and deduce that "heat toxin" maybe a essence of TCM syndrome of myocardial ischemia-repursion injury;the anti-inflammatory signaling pathway mediated by SIRT1 maybe an effect target of cooling blood and treating boils of salvia. |
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