Shuxuening Injection Inhibits Apoptosis and Reduces Myocardial Ischemia-Reperfusion Injury in Rats through PI3K/AKT Pathway

Objective: To investigate the main components and potential mechanism of Shuxuening Injection(SXNI) in the treatment of myocardial ischemia-reperfusion injury(MIRI) through network pharmacology and in vivo research. Methods: The Traditional Chinese Medicine Systems Pharmacology(TCMSP) and Pharm Mapper databases were used to extract and evaluate the effective components of Ginkgo biloba leaves, the main component of SXNI. The Online Mendelian Inheritance in Man(OMIM) and Gene Cards databases were searched for disease targets and obtain the drug target and disease target intersections. The active ingredient-target network was built using Cytoscape 3.9.1 software. The STRING database, Metascape online platform, and R language were used to obtain the key targets and signaling pathways of the anti-MIRI effects of SXNI. In order to verify the therapeutic effect of different concentrations of SXNI on MIRI in rats, 60 rats were first divided into 5 groups according to random number table method: the sham operation group, the model group, SXNI low-dose(3.68 mg/kg), medium-dose(7.35 mg/kg), and high-dose(14.7 mg/kg) groups, with 12 rats in each group. Then, another 60 rats were randomly divided into 5 groups: the sham operation group, the model group, SXNI group(14.7 mg/kg), SXNI+LY294002 group,and LY294002 group, with 12 rats in each group. The drug was then administered intraperitoneally at body weight for 14 days. The main biological processes were validated using in vivo testing. Evans blue/triphenyltetrazolium chloride(TTC) double staining, hematoxylin-eosin(HE) staining, terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) assay, enzyme-linked immunosorbent assay(ELISA), and Western blot analysis were used to investigate the efficacy and mechanism of SXNI in MIRI rats. Results: Eleven core targets and 30 Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways were selected. Among these, the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) pathway was closely related to SXNI treatment of MIRI. In vivo experiments showed that SXNI reduced the myocardial infarction area in the model group, improved rat heart pathological damage, and reduced the cardiomyocyte apoptosis rate(all P<0.01). After SXNI treatment, the p-PI3K/PI3K and p-AKT/AKT ratios as well as B-cell lymphoma-2(Bcl-2) protein expression in cardiomyocytes were increased, while the Bax and cleaved caspase 3 protein expression levels were decreased(all P<0.05). LY294002 partially reversed the protective effect of SXNI on MIRI. Conclusion: SXNI protects against MIRI by activating the PI3K/AKT signaling pathway.

Shenmai Injection Attenuates Myocardial Ischemia/Reperfusion Injury by Targeting Nrf2/GPX4 Signalling-Mediated Ferroptosis

Objective:To examine the effect of Shenmai Injection(SMJ)on ferroptosis during myocardial ischemia reperfusion(I/R)injury in rats and the underlying mechanism.Methods:A total of 120SPF-grade adult male SD rats,weighing 220–250 g were randomly divided into different groups according to a random number table.Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion.SMJ was injected intraperitoneally at the onset of 120 min of reperfusion,and erastin(an agonist of ferroptosis),ferrostatin-1(Fer-1,an inhibitor of ferroptosis)and ML385(an inhibitor of nuclear factor erythroid-2 related factor 2(Nrf2))were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments.Cardiac function before ischemia,after ischemia and after reperfusion was analysed.Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed,and the myocardial infarction area was measured.Additionally,the concentration of Fein heart tissues and the levels of creatine kinase-MB(CK-MB),troponin I(cTnI),malondialdehyde(MDA)and superoxide dismutase(SOD)in serum were measured using assay kits,and the expressions of Nrf2,glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4)were examined by Western blot.Results:Compared with the sham group,I/R significantly injured heart tissues,as evidenced by the disordered,ruptured and oedematous myocardial fibres;the increases in infarct size,serum CK-MB,cTnI and MDA levels,and myocardial Feconcentrations;and the decreases in SOD activity(P<0.05).These results were accompanied by ultrastructural alterations to the mitochondria,increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling(P<0.05).Compared with the I/R group,pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury,Feconcentrations and ACSL4 expression and attenuated mitochondrial impairment,while 14 mg/kg erastin exacerbated myocardial I/R injury(P<0.05).In addition,cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385,as evidenced by the increased myocardial infarct size,CK-MB,cTnI,MDA and Feconcentrations,and the decreased SOD activity(P<0.05).Conclusions:Ferroptosis is involved in myocardial I/R injury.Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis,thereby providing a strategy for the prevention and treatment of ischemic heart diseases.

Effect of electroacupuncture pretreatment on adenine nucleotides in myocardial tissues of rats with myocardial ischemia-reperfusion injury detected by high performance liquid chromatography(HPLC)

Objective:To observe the effect of electroacupuncture(EA)pretreatment on adenine nucleotides in the myocardial tissues of the myocardial ischemia-reperfusion injury(MIRI)rats,and to explore the mechanism of EA pretreatment on myocardial prevention and protection in MIRI rats.Methods:Forty SPF male Sprague-Dawley(SD)rats were randomly divided into 5 groups:a blank group,a sham operation group,a model group,an EA at Neiguan(PC 6)group and an EA at Hegu(LI 4)group,with 8 rats in each group.Rats in the blank group only received binding to the rat plate,30 min/time,once a day for 7 d;on the 7th day,rats in the sham operation group were subjected to threading for 40 min at the left anterior descending coronary artery without ligation,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day,rats in the model group were subjected to threading at the left anterior descending coronary artery with ligation,for 40 min before the blood flow was restored,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day of pretreatment with EA at Neiguan(PC 6)or Hegu(LI 4)for 30 min per day(once a day for 7 d),rats in the EA at Neiguan(PC 6)group and EA at Hegu(LI 4)group were subjected to modeling and sample collection same as in the model group.The left ventricular myocardium of the lower left anterior descending coronary artery was collected from rats in all 5 groups.Hematoxylin-eosin(HE)staining and transmission electron microscope(TEM)were used to observe the changes in myocardial pathological morphology.The change in the adenine nucleotide level of myocardial tissue was measured by high performance liquid chromatography(HPLC).Results:The HE staining and ultrastructure showed that the myocardial injury was severer in the model group compared with the sham operation group.Compared with the model group,the myocardial injury in the EA at Neiguan(PC 6)and the EA at Hegu(LI 4)groups was mild or hardly any.The adenine nucleotide levels in the sham operation group and the model group were all decreased compared with the blank group(all P<0.05);compared with the sham operation group,the adenine nucleotide level of the model group was also decreased,but the difference was not statistically significant(P>0.05);compared with the model group,the adenine nucleotide level in the EA at Neiguan(PC 6)group was increased(P<0.05),and the adenine nucleotide level in the EA at Hegu(LI 4)group was significantly increased(P<0.01).The adenine nucleotide level in the EA at Hegu(LI 4)group was higher than that in the EA at Neiguan(PC 6)group,but the difference was not statistically significant(P>0.05).Compared with the EA at Neiguan(PC 6)group,the levels of adenosine triphosphate(ATP),adenosine diphosphate(ADP)and adenosine monophosphate(AMP)in the EA at Hegu(LI 4)group were significantly increased(all P<0.01).Conclusion:Both EA at Neiguan(PC 6)and Hegu(LI 4)can alleviate the pathological damage to myocardium in MIRI rats,and increase the adenine nucleotide level in myocardial tissues,and thus protect MIRI rats.EA at Hegu(LI 4)has a better protective effect than Neiguan(PC 6).

N-acetylcysteine attenuates ischemia/reperfusion-induced cardiocyte apoptosis in diabetic rats

Objective：To study the effects of N-acetylcysteine （NAC） on ischemia/ reperfusion （I/R）-induced myocyte apoptosis in diabetic rats. Methods：The I/R heart model was made by ligation of the left anterior descending coronary artery （LAD） close to its origin. The LAD was occluded for 30 min followed by removal of ligation to allow subsequent reperfusion for 3 h. 72 rats were randomly divided into two groups , non-diabetic group （C, n = 36） and diabetic group ,（D, n = 36）. The animals in C group were randomly reassigned into sham-operated group （CS, n = 12） , I/R group （C I/R, n = 12） and treated with NAC group （CN, n = 12）. The rats in D group were also reassigned to sham-operated group （DS, n = 12） , I/R group （DI/R, n = 12） and treated with NAC group （DN, n = 12）. Malondialdehyde （MDA） and creatine kinase isoenzyme-MB （CK-MB） were measured. Infarct size（IS/AAR%）, the apoptosis index（AI） by TUNEL staining, the number of the cells positive for Caspase-3 and positive expression index （PEI） were calculated. Results：After I/R, the IS/AAR%, CK-MB, MDA, AI and Caspase-3 PEI were higher in diabetic group than those in non-diabetic group. Treatment with NAC decreased the above parameters in both non-diabetic and diabetic rats, but the parameters in diabetic rats were higher than those in non-diabetic rats. Conclusion：Diabetic rat hearts are more susceptible to I/R-induced myocardial necrosis and myocyte apoptosis. NAC can decrease the infarct size and attenuate cardiomyocyte apoptosis in both non-diabetic and diabetic rats, but the therapeutic effects are less effective in diabetic rats than those in non-diabetic rats.

Cardioprotection of Shenfu Injection(参附注射液) against Myocardial Ischemia/Reperfusion Injury in Open Heart Surgery

Objective： To investigate the protective effect of Shenfu Injection （参附注射液, SFI） against myocardium ischemia/reperfusion injury （IRI） in mitral valve replacement （MVR） with cardiopulmonary bypass （CPB）. Metheds： Forty patients undergoing selective MVR were randomly assigned to the control group and trial Groups Ⅰ, Ⅱ,Ⅲ, and Ⅳ according to the different administrations of SFI, 8 patients in each group. The changes of systolic blood pressure （SBP）, mean blood pressure （MBP）, diastolic blood pressure （DBP） in each group were monitored, respectively. The recovering percentage of spontaneous heart beat, the heart rate （HR） and cardiac rhythm as well as the abnormal duration of ECG-ST segment were recorded after the restoration of heart beat. The serum concentration of cardiac troponin Ⅰ （cTnl）, malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） were determined as well. Results： （1） The SBP, MBP and DBP values, the recovering rate of spontaneous heart beat, HR, ECG-ST, atrioventricular block and ventricular arrhythmia were significantly improved in group Ⅳ compared with any other groups. （2） Compared with the control group, the postoperative serum contents of cTnl and MDA were significantly decreased, but the activity of SOD was significantly increased in group Ⅳ. Cenclusiens： SFI had a certain protective effect against myocardium IRI. Moreover, better efficacy was seen with the administration of 1.5 mL/kg SFI into CPB priming fluid and pumping 1.5 mL/kg SFI via CPB as soon as the clamped aorta was unclamped.

Single intravenous injection of CoQ₁₀reduces infarct size in a rat model of ischemia and reperfusion injury

Maintenance of mitochondrial activity and antioxidant features of coenzyme Q10 (CoQ10) could be an effective background for treatment of acute myocardial ischemia. Dietary uptake of CoQ10 is limited to only a few percent. In urgent cases, parenteral administration of CoQ10 could provide fast increase of its plasma and myocardial levels. The aim was to evaluate whether a single intravenous (i.v.) injection of solubilized CoQ10 before ischemia/reperfusion (IR) could lead to replenishment of its myocardial levels and limits subsequent myocardial IR injury. Methods: 30 min prior to coronary artery occlusion rats received i.v. solubilized CoQ10 (30 mg/kg) or saline (1 ml/kg). After 30 min of ischemia and 120 min of reperfusion, infarct zone of left ventricle (LV) and quantity of CoQ10 in LV were determined. Cardiac rhythm was monitored through the whole experiment. Results: At the beginning of reperfusion, arrhythmias were recorded in 8 (from 9) in saline and 2 (from 9) in CoQ10-treated rats. Arrhythmias in CoQ10-treated rats arose later (40 ± 8 sec) and had less duration (26 ± 14 sec);14 ± 13 sec and 52 ± 17 sec in saline treated rats respectively. At the end of reperfusion CoQ10 treated rats revealed: 2 fold higher CoQ10 content in LV (p 10 were accompanied by less infarct size (r = ﹣0.77, p i.v. injection of CoQ10 effectively increased its myocardial levels and protected heart against IR injury by diminishing the size of the irreversibly damaged myocardium, decreasing frequency and duration of arrhythmias. The infarct zone inversely correlated with the quantity of CoQ10 in LV.

Effects of glucagon-like peptide 1 analogs in combination with insulin on myocardial infarct size in rats with type 2 diabetes mellitus

AIM To evaluate the effects of glucagon-like peptide-1 analogs(GLP-1 a) combined with insulin on myocardial ischemiareperfusion injury in diabetic rats.METHODS Type 2 diabetes mellitus(T2 DM) was induced in maleWistar rats with streptozotocin(65 mg/kg) and verified using an oral glucose tolerance test. After anesthesia, the left coronary artery was occluded for 40 min followed by 80 min reperfusion. Blood glucose level was measured during surgery. Rats were randomized into six groups as follows:(1) control rats;(2) insulin(0.1 U/kg) treated rats prior to ischemia;(3) insulin(0.1 U/kg) treated rats at reperfusion;(4) GLP-1 a(140 mg/kg) treated rats prior to ischemia;(5) GLP-1 a(140 mg/kg) treated rats at reperfusion; and(6) rats treated with GLP-1 a(140 mg/kg) prior to ischemia plus insulin(0.1 U/kg) at reperfusion. Myocardial area at risk and infarct size was measured planimetrically using Evans blue and triphenyltetrazolium chloride staining, respectively.RESULTS There was no significant difference in the myocardial area at risk among groups. Insulin treatment before ischemia resulted in a significant increase in infarct size(34.7% ± 3.4% vs 18.6% ± 3.1% in the control rats, P < 0.05). Post-ischemic administration of insulin or GLP-1 a had no effect on infarct size. However, pre-ischemic administration of GLP-1 a reduced infarct size to 12% ± 2.2%(P < 0.05). The maximal infarct size reduction was observed in the group treated with GLP-1 a prior to ischemia and insulin at reperfusion(8% ± 1.6%, P < 0.05 vs the control and GLP-1 a alone treated groups).CONCLUSION GLP-1 a pre-administration results in myocardial infarct size reduction in rats with T2 DM. These effects are maximal in rats treated with GLP-1 a pre-ischemia plus insulin at reperfusion.

Effects of Acupuncture Pretreatment on Ischemic Cardiac Muscle Cell Apoptosis and Gene Expression in Ischemia-reperfusion Rats

Objective： To investigate the protective effects of acuptmcture pretreatment on ischemic myocardium, the protective mechanism of acupuncture pretreatment on ischemic myocardium was explored by observing the cardiac muscle cell apoptosis and the expression of HSP70 mRNA of ischemia-reperfusion injury rats treated with acupuncture pretreatment. Methods： Sixty-four Wistar rats were randomly divided into eight groups： control group, sham surgery group, ischemia-reperfusion group, ischemia pretreatment group, manual acupuncture pretreatment group （once a day）, electroacupuncture pretreatment group （once a day）, manual acupuncture pretreatment group （twice a day）, and electroacupuncture pretreatment group （twice a day）. The reperfusion model of rat myocardial ischemia was made. Expression of HSP70 mRNA was assayed by in situ hybridization, and cell apoptosis by TUNEL. Results： Compared with those in the control group and the sham surgery group, the apoptosis and the expression of HSP70 mRNA were increased in the ischemia-reperfusion group. Compared with those in the ischemia-reperfusion group, the cardiac muscle cell apoptosis was decreased and the HSP70 mRNA was increased in the rats treated with acupuncture pretreatment; meanwhile, acupuncture pretreatment twice a day had stronger effects than acupuncture pretreatment once a day and ischemia pretreatrnent. Conclusion： Acupuncture pretreatment can inhibit the cardiac muscle cell apoptosis, and up-regulate the expression of HSP70 mRNA in ischemia-reperfusion rats. Acupuncture pretreatment twice a day has stronger effects than pretreatment once a day.

Progresses in the research of Danhong Injection against the cardial ischemia-referfusion injury

Researches in the field of the myocardial ischemia-reperfusion injury are attracting the attentions of clinicians for the treatments that protect cardiac muscle cells from being injured can not only help the patients get recovery but also keep them in health. By clearing the free radicals and reducing calcium overload of myocardial cell, treatments with Danhong Injection will help myocardial cells survive from inflammatory reactions which are triggered by ischemia reperfusion so as that endothelial function will be improved and myocardial cell apoptosis will be inhibited. In all, Danhong Injection is an ideal medicine for protecting myocardial cell against ischemia reperfusion injury.

上调缺氧诱导因子对老龄小鼠心肌缺血再灌注损伤保护作用的实验研究

目的:分析上调缺氧诱导因子(HIF)对老龄大鼠心肌缺血再灌注(I/R)损伤的保护机制。方法:选择40只SPF级健康老年雄性C57小鼠为研究对象,适应性饲养1周后将其随机分为A、B、C、D四组,每组各10只。A组不做任何处理,B、C、D组均构建小鼠心肌I/R损伤模型,C组于缺血前2 h腹腔注射100 mg/kg HIF-1α激活剂[二甲基乙二酰基甘氨酸(DMOG)],D组于缺血前2 h腹腔注射15 mg/kg HIF-1α抑制剂[2-甲氧基雌二醇(2-ME2)]。比较四组小鼠给药前、给药1、2 h的心率、心肌收缩张力、左室内压最大上升/下降速率、心肌梗死面积情况,以免疫荧光技术、Western blot检测心肌细胞HIF-1α及α-微管蛋白表达。结果:给药后1、2 h,B组、C组和D组小鼠的心率、心肌收缩张力、左室内压最大上升/下降速率较给药前均降低,且D组低于B组、C组,B组低于C组,差异有统计学意义(均P<0.05)。给药后2 h,B组、D组小鼠心肌梗死面积的危险区/左心室、梗死区/左心室、梗死区/危险区水平均低于C组,且D组低于B组(均P<0.05);B组、C组、D组小鼠的HIF-1α蛋白表达水平均高于A组,且D组低于B组、C组,B组低于C组;C组小鼠的α-微管蛋白表达水平高于A组、B组、D组(均P<0.05),但A组、B组、D组小鼠的α-微管蛋白表达水平比较差异无统计学意义(均P>0.05)。结论:上调HIF可改善心肌I/R损伤小鼠的心脏功能,减少心肌梗死面积,改善HIF-1α及α-微管蛋白表达。

基于转录组测序探讨加味桃红四物汤缓解大鼠心肌缺血再灌注损伤的作用机制

目的 基于转录组测序研究加味桃红四物汤对大鼠心肌缺血再灌注损伤的影响及作用机制。方法 将15只SPF级大鼠随机分为假手术组、模型组和加味桃红四物汤组,每组5只,加味桃红四物汤组予加味桃红四物汤预灌胃5 d,假手术组和模型组灌胃等体积蒸馏水,建立心肌缺血再灌注损伤大鼠模型。心脏超声检测大鼠心功能,试剂盒检测血清氧化应激和炎症因子含量,TUNEL染色检测心肌细胞凋亡,取心肌组织进行转录组测序,对差异基因进行韦恩图及热图分析,对共同差异基因进行GO功能分析和KEGG通路富集分析。RT-qPCR验证差异基因PTX3和EGR2表达。结果 与假手术组比较,模型组大鼠射血分数(EF)和短轴缩短率(FS)明显降低(P<0.01),心肌组织细胞凋亡率及血清乳酸脱氢酶(LDH)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6含量明显升高(P<0.01),SOD活性和IL-10含量明显降低(P<0.01);与模型组比较,加味桃红四物汤组大鼠EF和FS明显升高(P<0.05),心肌组织细胞凋亡率及血清肌酸激酶同工酶、LDH、TNF-α、IL-1β、IL-6含量明显降低(P<0.01),SOD活性和IL-10含量明显升高(P<0.01)。转录组测序得到假手术组与模型组差异基因4 227个(2 259个上调、1 968个下调),假手术组与加味桃红四物汤组差异基因1 933个(1301个上调、632个下调),模型组与加味桃红四物汤组差异基因94个(46个上调、48个下调),3组共同差异基因35个,差异基因主要富集到流体剪切力和动脉粥样硬化、泛素介导的蛋白质降解、C型凝集素受体信号通路、鞘脂类信号通路、细胞周期、趋化因子信号通路、脂质和动脉粥样硬化等信号通路。RT-qPCR验证结果显示,模型组心肌组织PTX3和EGR2基因表达较假手术组明显升高(P<0.01),加味桃红四物汤组PTX3和EGR2基因表达较模型组明显降低(P<0.01)。结论 加味桃红四物汤对心肌缺血再灌注损伤具有缓解作用,表现为改善模型大鼠心功能、减少细胞凋亡和炎症因子释放及减轻氧化应激水平,其机制可能与调控PTX3和EGR2基因表达有关。

清营生脉汤激活Akt信号通路保护H9c2心肌细胞缺氧复氧损伤的作用研究

目的研究清营生脉汤含药血清对缺氧复氧诱导H9c2心肌细胞损伤的保护机制。方法本研究采用H9c2心肌细胞缺氧/复氧(H/R)方法模拟心肌缺血再灌注损伤(MIRI)细胞模型,细胞实验分组为:空白血清对照组、模型组、含药血清低剂量组、含药血清中剂量组、含药血清高剂量组。应用CCK-8法测定心肌细胞活性,流式细胞术检测细胞凋亡率,ELISA法检测超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、丙二醛(MDA)、肌酸激酶同工酶(CK-MB)水平,Western blotting法检测细胞内磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)及p-Akt的蛋白表达。结果与对照组相比,模型组细胞存活率显著降低、凋亡率显著升高、细胞内SOD水平显著降低,LDH、MDA、CK-MB水平显著升高(P<0.01);与模型组相比,含药血清低、中、高剂量组细胞的存活率显著升高、凋亡率显著降低、细胞内SOD水平显著升高,LDH、MDA、CK-MB水平显著降低(P<0.01);与对照组相比,模型组细胞中p-Akt蛋白表达水平显著降低(P<0.01),p-Akt/Akt比值降低(P<0.05);与模型组相比,含药血清高剂量组细胞内p-Akt蛋白表达量显著升高(P<0.01),p-Akt/Akt比值明显升高(P<0.05)。结论清营生脉汤含药血清能够提高缺氧复氧心肌细胞的活性,降低细胞凋亡率,升高SOD水平,降低LDH、MDA、CK-MB水平,提高细胞抗氧化能力,且可能通过促进Akt磷酸化降低缺氧复氧诱导的H9c2心肌细胞损伤程度。

黄葵素通过核因子κB信号改善老年糖尿病大鼠心肌缺血再灌注的研究

目的:探讨黄葵素对糖尿病心肌缺血再灌注老年大鼠的影响及其作用机制。方法:将老年大鼠按照简单随机法分为假手术组(只穿线不结扎)、模型组(构建糖尿病心肌缺血再灌注模型)、黄葵素低剂量组(75 mg/kg的黄葵素)、黄葵素高剂量组(150 mg/kg的黄葵素)、抑制剂组(10 mg/kg的核因子κB抑制剂BAY11-7082),高剂量+抑制剂组(150 mg/kg的黄葵素+10 mg/kg BAY11-7082)。观察比较大鼠心功能指标、心肌梗死面积、血清指标含量、心肌细胞凋亡率、心肌组织相关蛋白表达。结果:与模型组比较,黄葵素低剂量、黄葵素高剂量组、抑制剂组、高剂量+抑制剂组大鼠心肌梗死面积、心肌肌钙蛋白T(cTnT)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、丙二醛(MDA)、原位末端转移酶标记法(Tunel)阳性细胞率、磷酸化核因子κB(p-NF-κB p65)、裂解胱天蛋白酶-3(Cl-caspase-3)、Bcl-2相关X蛋白(Bax)水平均显著降低(均P<0.05),左室射血分数(LVEF)、核因子κB抑制蛋白(IκBα)、B淋巴细胞瘤-2基因(Bcl-2)水平均显著升高(均P<0.05);与高剂量组比较,高剂量+抑制剂组大鼠心肌梗死面积、cTnT、CK-MB、LDH、MDA、Tunel阳性细胞率、p-NF-κB p65、Cl-caspase-3、Bax水平均显著降低(均P<0.05),LVEF、IκBα、Bcl-2水平均显著升高(均P<0.05)。结论:黄葵素可能通过调节核因子κB信号抑制心肌细胞凋亡、氧化应激改善糖尿病心肌缺血再灌注老年大鼠心功能。

注射用丹参多酚酸对大鼠脑缺血再灌注损伤的保护作用及机制研究

目的探讨注射用丹参多酚酸(SAFI)对大鼠脑缺血再灌注损伤的保护作用及机制。方法将100只SD大鼠随机分为假手术组、模型组及丹参多酚酸低、中、高剂量组(5、10、20 mg·kg^(-1)),每组20只。采用改良线栓法复制大脑中动脉闭塞再灌注(MCAO/R)大鼠模型。于MCAO/R手术前按照上述剂量腹腔注射给药,每日1次,连续给药3 d;末次给药1 h后,进行MCAO/R模型复制。采用Longa五级评分方法进行神经功能缺失评分;采用TTC染色后测量脑梗塞体积;ELISA法检测血清中氧化应激指标NADPH氧化酶(NOX)、4-羟基壬烯醛(4-HNE)、8-羟基脱氧鸟苷(8-OHd G)及炎症反应指标单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-18、IL-6、细胞间黏附分子1(ICAM-1)的水平;HE及尼氏染色法观察脑组织病理损伤情况;TUNEL染色法检测脑组织神经元细胞凋亡情况;免疫荧光法测定大鼠脑组织中胶质纤维酸性蛋白(GFAP)的表达情况;Western Blot法检测脑组织中NLRP3、Caspase1蛋白表达情况。结果与假手术组比较,模型组大鼠神经功能缺损评分显著升高(P<0.01),脑梗死体积显著增加(P<0.01),脑组织病理损伤明显,神经元密度显著降低(P<0.01),皮层细胞凋亡率显著升高(P<0.01);血清NOX、4-HNE、8-OHd G、MCP-1、TNF-α、IL-1β、IL-18、IL-6、ICAM-1水平均显著升高(P<0.05,P<0.01);脑组织中GFAP、NLRP3、Caspase1蛋白表达显著上调(P<0.01)。与模型组比较,SAFI中、高剂量组大鼠的神经功能缺失评分显著降低(P<0.01),脑梗死体积显著缩小(P<0.01),神经元损伤有不同程度好转,神经元密度显著增加(P<0.05,P<0.01),皮层细胞凋亡率显著降低(P<0.01);血清NOX、4-HNE、8-OHd G、MCP-1、TNF-α、IL-1β、IL-18、IL-6、ICAM-1水平明显降低(P<0.05,P<0.01);脑组织中GFAP、NLRP3、Caspase1蛋白表达显著下调(P<0.01)。结论SAFI预防性给药可明显降低脑缺血再灌注损伤大鼠的炎症反应和氧化应激水平,改善脑组织病理损伤,发挥神经保护作用,其机制可能与抑制星型胶质细胞活化及NLRP3/Caspase-1通路激活有关。

川芎多糖调节p38 MAPK/NF-κB信号通路对大鼠心肌缺血再灌注损伤的影响

目的:探讨川芎多糖(LCPS)调节p38丝裂原活化蛋白激酶(p38 MAPK)/核因子-κB(NF-κB)信号通路对大鼠心肌缺血再灌注损伤(MIRI)的影响。方法:将144只大鼠数字编码后按照随机数字表法分为假手术组、模型组、盐酸维拉帕米(VH)组(20 mg/kg)和LCPS低剂量组(50 mg/kg)、LCPS中剂量组(100 mg/kg)、LCPS高剂量组(200 mg/kg),每组24只大鼠。给药预处理14 d后,采用阻断冠状动脉左前降支30 min制备MIRI大鼠模型。再灌注2 h后,通过2,3,5-三苯基氯化四氮唑(TTC)染色法、心脏超声法、苏木精-伊红(HE)染色法检测大鼠心肌梗死面积、心功能指标[左室舒张末期压力(LVEDP)、左室收缩末期压力(LVESP)、左室内压最大上升速率(+dp/dt_(max))、左室内压最大下降速率(-dp/dt_(max))]和心肌组织病理变化;酶联免疫吸附法(ELISA)检测血清心肌酶和心肌组织炎性因子水平;蛋白免疫印迹法(Western Blot)检测心肌组织p38 MAPK/NF-κB信号通路相关蛋白表达。结果:与假手术组比较,模型组大鼠心肌梗死面积、LVEDP、病理评分、心肌酶[乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)]水平、炎性因子[白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α]水平、p-p38 MAPK、p-NF-κB p65蛋白相对表达量及p-p38 MAPK/p38 MAPK、p-NF-κB p65/NF-κB p65比值升高,LVESP、+dp/dt_(max)、-dp/dt_(max)降低,差异均有统计学意义(P<0.05)。与模型组比较,VH组、LCPS中剂量组和LCPS高剂量组心肌梗死面积、LVEDP、病理评分、心肌酶水平、炎性因子水平、p-p38 MAPK、p-NF-κB p65蛋白相对表达量及p-p38 MAPK/p38 MAPK、p-NF-κB p65/NF-κB p65比值降低,LVESP、+dp/dt_(max)、-dp/dt_(max)升高,差异均有统计学意义(P<0.05),且LCPS上述效应呈一定的剂量依赖性。除+dp/dt_(max)、-dp/dt_(max)、IL-1β外,LCPS高剂量组对其他指标的影响优于VH组(P<0.05)。结论:LCPS对大鼠MIRI和心功能有保护作用,其机制可能与抑制p38 MAPK/NF-κB信号通路及其介导的炎症反应有关。

黄杞总黄酮对心力衰竭大鼠心肌缺血再灌注损伤的保护作用研究

目的:探讨黄杞总黄酮对心力衰竭(HF)大鼠心肌缺血再灌注损伤(IRI)的保护作用。方法:60只雄性SD大鼠建模成功后按随机数表法分为5组:假手术组、IRI模型组、低剂量黄杞总黄酮(TFER)组、中剂量TFER组、高剂量TFER组,每组12只,连续灌胃7d。使用心电图和超声心动图检测大鼠S-T段波幅、左室短轴缩短率(LVFS)、左室射血分数(LVEF)、左室内压最大上升速率(+dp/dt_(max))、左室内压最大下降速率(-dp/dt_(max)),使用苏木精-伊红(HE)染色法检测大鼠心肌梗死面积百分比,使用Masson染色法检测大鼠心肌胶原附着面积百分比,使用比色法检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA),使用蛋白免疫印迹(Western blot)法检测核因子E2相关因子2(Nrf2)、Kelch样ECH关联蛋白1(Keap1)表达水平。结果:与IRI模型组相比,使用TFER灌胃的IRI大鼠心电图S-T段得到显著修复,心肌梗死面积百分比、胶原附着面积百分比、MDA、Keap1表达显著降低,LVFS、LVEF、+dp/dt_(max)比值、-dp/dt_(max)比值、SOD、GSH-Px、Nrf2表达显著提高,差异均有统计学意义(P<0.05),其中高剂量TFER组上述指标差异最显著(P<0.05)。结论:黄杞总黄酮对HF大鼠IRI有保护作用,其机制可能与减少心肌梗死面积、改善心功能、缓解氧化应激反应有关。

加味瓜蒌薤白汤对大鼠心肌缺血再灌注损伤的保护作用

目的:观察加味瓜蒌薤白汤对心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)大鼠心功能指标和心肌组织病理形态的影响。方法:将64只Wistar大鼠随机分为对照组11只、模型组11只、加味瓜蒌薤白汤低剂量组(1.134 g·kg^(-1))11只、中剂量组(2.268 g·kg^(-1))11只、高剂量组(4.536 g·kg^(-1))10只及复方丹参滴丸组(86 mg·kg^(-1))10只。除对照组外,其余大鼠采用结扎冠状动脉的方式建立MIRI模型。造模成功后,各药物组以相应药物灌胃,对照组和模型组给予生理盐水灌胃,每日1次,共7 d。采用ELISA法检测大鼠血清肌钙蛋白T(cardiac troponin T,CTNT)、内皮素-1(endothelin-1,ET-1)、血栓素B2(thromboxane B2,TXB2)含量;采用超声心动图检测大鼠左室舒张末期内径(left ventricular internal dimension diameter,LVIDd)、左室射血分数(left ventricular ejection fraction,LVEF)、左室收缩末期内径(left ventricular internal systolic diameter,LVIDs)、心输出量(cardiac output,CO)、左室舒张末期容积(left ventricular internal diastolic volume,LVEDV)、每搏输出量(stroke volume,SV)、左室收缩末期容积(left ventricular internal systolic volume,LVESV);采用HE染色观察大鼠心肌组织病理形态。结果:与对照组比较,模型组大鼠血清CTNT、ET-1和TXB2含量升高(P<0.05);与模型组比较,加味瓜蒌薤白汤中剂量组和复方丹参滴丸组大鼠血清CTNT、ET-1和TXB2含量降低(P<0.05)。与对照组比较,模型组大鼠LVIDd、LVEDV增加(P<0.01),且LVIDs、LVESV、SV、CO、LVEF减少(P<0.05);与模型组比较,加味瓜蒌薤白汤中剂量组和复方丹参滴丸组大鼠LVIDd、LVEDV减少(P<0.05),且LVIDs、LVESV、SV、CO、LVEF增加(P<0.05)。HE染色结果显示,对照组大鼠心肌细胞排列整齐,无炎症细胞浸润;模型组大鼠大量心肌纤维呈不规则排列,心肌间质水肿和坏死;加味瓜蒌薤白汤高剂量组大鼠心肌细胞轻度肿胀,肌原纤维排列较规则;复方丹参滴丸组大鼠心包膜下心肌细胞坏死,中性粒细胞浸润,间质充血水肿。结论:加味瓜蒌薤白汤对大鼠MIRI具有显著的保护作用。

醛固酮受体拮抗剂螺内酯减轻肢体缺血再灌注致心肌损伤的信号传导机制

目的:分析醛固酮受体拮抗剂螺内酯减轻肢体缺血再灌注致心肌损伤的信号传导机制。方法:将30只健康Wistar雄性大鼠随机分为对照组(10只,正常饲养)、再灌注组(10只,建立肢体缺血再灌注大鼠模型)、螺内酯组(10只,建立模型前给予螺内酯)。检测3组大鼠心肌损伤指标、炎症反应及氧化应激反应变化。结果:对照组大鼠心肌细胞凋亡率为(5.28±1.05)%,再灌注组为(185.47±35.85)%,螺内酯组为(75.25±24.19)%,3组比较差异有统计学意义(P<0.05)。再灌注组、螺内酯组血清肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)表达较对照组增加;螺内酯组血清cTnI、CK、CK-MB表达低于再灌注组(P<0.05)。再灌注组、螺内酯组心肌组织内丙二醛(MDA)表达较对照组升高,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)含量较对照组降低;螺内酯组心肌组织内MDA表达低于再灌注组,SOD、GSH-PX含量高于再灌注组(P<0.05)。再灌注组、螺内酯组心肌与血浆白细胞介素(IL)-6、高迁移率族蛋白B1(HMGB1)表达均高于对照组;螺内酯组心肌与血浆IL-6、HMGB1表达均低于再灌注组(P<0.05)。再灌注组、螺内酯组心肌HMGB1 mRNA、Toll样受体4(TLR4)mRNA表达高于对照组;螺内酯组心肌HMGB1 mRNA、TLR4 mRNA表达低于再灌注组(P<0.05)。结论:肢体缺血再灌注后常导致心肌损伤,应用螺内酯治疗可减轻心肌损伤程度,可能是通过抑制氧化反应、抗炎症反应减轻心肌损伤程度,其作用信号传导机制主要是抑制HMGB1-TLR4信号通路。

芍药苷预处理对大鼠心肌缺血-再灌注损伤氧化应激的保护作用及对Nrf-2/TXNIP/NLRP-3信号通路的影响

目的探讨芍药苷(PF)对心肌缺血-再灌注损伤(MIRI)大鼠氧化应激的影响及其可能作用机制。方法选取65只SD大鼠采用结扎冠状动脉(冠脉)左前降支建立MIRI模型,造模成功后随机分为模型组、PF低、高剂量(60 mg/kg、120 mg/kg)组和西药(1 mg/kg盐酸地尔硫卓)组各15只,另设对照组。检测大鼠天冬氨酸氨基转移酶(AST)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)水平;HE染色观察心肌组织病理学变化;RT-PCR和Western blot检测心肌组织核因子E2相关因子2(Nrf2)、硫氧还蛋白互作蛋白(TXNIP)、NOD样受体蛋白3(NLRP-3)mRNA和蛋白表达情况。结果与模型组比较,各实验组AST、CK、CK-MB、LDH、MDA水平、TXNIP、NLRP-3 mRNA和蛋白水平降低,GSH-Px、SOD、CAT水平、Nrf2 mRNA和蛋白水平升高(P<0.05)。与PF低剂量组比较,PF高剂量组以上指标效果更佳(P<0.05)。结论PF可减轻MIRI大鼠氧化应激,改善心肌损伤,可能与Nrf2/TXNIP/NLRP-3信号通路有关。

老鹳草素对大鼠心肌缺血再灌注损伤的作用及其机制

目的探讨老鹳草素对心肌缺血再灌注损伤的作用及其机制。方法将75只SPF级雄性小鼠用随机数字表分为老鹳草素低剂量组(5μmol/L)、中剂量组(10μmol/L)、高剂量组(20μmol/L)、假手术组和模型组,各15只。观察小鼠心脏功能,检测心肌损伤标志物,观察各组大鼠心肌组织病理变化,检测心肌组织中核转录因子E2相关因子(nuclear transcriPtion factor E2-related factor,Nrf2)信号通路相关蛋白水平。体外培养H9C2细胞,检测H9C2细胞中自噬相关蛋白及Nrf2信号通路相关蛋白水平。结果与假手术组相比,模型组心功能指标均下降;与模型组相比,老鹳草素低、中、高剂量组LVEF、FS、LVWT、HR、LVSP水平均升高(P<0.05);模型组肌红蛋白(myoglobin,Mb)、肌酸激酶同工酶(creatine kinase isoenzyme,CK-Mb)、肌钙蛋白I(cardiac tro Ponin I,c Tn I)水平高于假手术组(P<0.05);与假手术组相比,模型组LC3-II/LC3-I比值、Beclin-1升高,P62、Nrf2、游离血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白水平降低(P<0.05);与模型组相比,老鹳草素低、中、高剂量组P62、Nrf2、HO-1蛋白水平升高(P<0.05);与对照组相比,H9C2细胞模拟缺血/再灌注(SI/R)损伤后LC3-II/LC3-I比值、Beclin-1升高,P62、Nrf2、HO-1蛋白水平降低(P<0.05)。结论老鹳草素可能通过激活Nrf2信号通路,抑制心肌细胞过度自噬,对心肌缺血再灌注损伤起到明显保护作用。