Effects of astragalus polysaccharide on cell injury and mitochondrial pathway apoptosis in the hypoxia reoxygenation of myocardial cells

Objective: To study the effects of astragalus polysaccharide on cell injury and mitochondrial pathway apoptosis in the hypoxia reoxygenation of myocardial cells. Methods: H9c2 myocardial cell lines were cultured and divided into negative control group (NC group), hypoxia reoxygenation group (H/R group) and astragalus polysaccharide group (APS), H/R group underwent hypoxia reoxygenation treatment, and APS group underwent both hypoxia reoxygenation treatment and astragalus polysaccharides intervention. The cell viability was measured 8 h, 16 h and 24 h after reoxygenation;the expression of mitochondrial apoptosis genes, apoptosis pathway genes as well as the contents of ROS metabolism indexes were determined 24 h after reoxygenation. Results: 8 h, 16 h and 24 h after reoxygenation, the cell viability of H/R group were lower than those of NC group, and the cell viability of APS group were higher than those of H/R group;24 h after reoxygenation, BIM, BAX, Caspase-9, Caspase-3, p-PI3K, p-AKT and p-FoxO3a protein expression as well as ROS, HSP70 and p-p38MAPK contents in H/R group were significantly higher than those in NC group whereas BCL2 protein expression and SOD content were significantly lower than those in NC group;BIM, BAX, Caspase-9, Caspase-3, p-PI3K, p-AKT and p-FoxO3a protein expression as well as ROS, HSP70 and p-p38MAPK contents in APS group were significantly lower than those in H/R group whereas BCL2 protein expression and SOD content were significantly higher than those in H/R group. Conclusion: Astragalus polysaccharide can reduce the cell damage and inhibit the mitochondrial pathway apoptosis in the hypoxia reoxygenation process of myocardial cells.

Sevoflurane pretreatment inhibits the myocardial apoptosis caused by hypoxia reoxygenation through AMPK pathway:An experimental study

Objective:To study whether sevoflurane pretreatment inhibits the myocardial apoptosis caused by hypoxia reoxygenation through AMPK pathway.Methods:H9c2 myocardial cell lines were cultured and divided into control group(C group),hypoxia reoxygenation group(H/R group),sevoflurane pretreatment+hypoxia reoxygenation group(SP group) and sevoflurane combined with Compound C pretreatment+hypoxia reoxygenation group(ComC group),and the cell proliferation activity and apoptosis rate,myocardial enzyme levels in culture medium as well as the expression of apoptosis genes and p-AMPK in cells were determined.Results:p-AMPK expression in cells of H/R group was significantly lower than that of C group,SP group was significantly higher than that of H/R group;cell proliferation activity value and Bcl-2 expression in cells of H/R group were significantly lower than those of C group,SP group were significantly higher than those of H/R group,Com C group were significantly lower than those of SP group;apoptosis rate,LDH,CK and AST levels as well as the Bax and Caspase-3 expression in cells of H/R group were significantly higher than those of C group,SP group were significantly lower than those of H/R group,ComC group were significantly higher than those of SP group.Conclusions:Sevoflurane pretreatment can activate AMPK signaling pathway to inhibit the myocardial apoptosis caused by hypoxia reoxygenation.

Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

Aldehyde dehydrogenase 2 preserves mitochondrial morphology and attenuates hypoxia/reoxygenationinduced cardiomyocyte injury

BACKGROUND:Disturbance of mitochondrial fi ssion and fusion(termed mitochondrial dynamics)is one of the leading causes of ischemia/reperfusion(I/R)-induced myocardial injury.Previous studies showed that mitochondrial aldehyde dehydrogenase 2(ALDH2)conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes.However,whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown.METHODS:In the present study,we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation(H/R)as an in vitro model of myocardial I/R injury.RESULTS:Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation(OGD/R),and ALDH2 activation largely decreased the cardiomyocyte apoptosis.Additionally,we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R.Furthermore,we found that ALDH2 dominantly suppressed dynamin-related protein 1(Drp1)phosphorylation(Ser616)and adenosine monophosphate-activated protein kinase(AMPK)phosphorylation(Thr172)but not interfered with the expression levels of mitochondrial shaping proteins.CONCLUSIONS:We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.

Effects of Crocin on Nox2 Expression and ROS Level of Hypoxia/Reoxygenation-induced Injury of Cardiomyocytes

[Objectives]To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells.[Methods]Newborn male SD rats were selected,left ventricular cardiomyocytes(CMs)were isolated,and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia/reperfusion injury.The cells were randomly divided into four groups:normal cell group(control group),crocin group),hypoxia/reoxygenation group(H/R group),hypoxia/reoxygenation+crocin group(H/R+crocin group).H/R+crocin group selected the concentration of crocin 1,10,and 100μmol/L,and determined the optimal concentration of crocin by detecting the cell proliferation ability.After the cells were pretreated using the optimal concentration of crocin,the levels of superoxide anion,cell proliferation,apoptosis and Nox2 levels in each group of cells were detected.[Results]Compared with the control group,the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced(P<0.05),while cell apoptosis and intracellular superoxide anion levels were significantly increased(P<0.01);the CMs pretreated with crocin can reduce the level of Nox2(P<0.01),increase the cell proliferation ability of CMs,reduce cell apoptosis,and accordingly reduce the level of superoxide anion in the cell(P<0.05).[Conclusions]Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

Background:Administration of propofol,an intravenous anesthetic with antioxidant property,immediately at the onset of post-ischemic reperfusion(propofol postconditioning,P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion(I/R) injury,while the underlying mechanism remains incompletely understood.The forkhead box O(FoxO) transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection,however,the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.Methods:Rat heart-derived H9c2 cells were exposed to high glucose(HG) for 48 h,then subjected to hypoxia/reoxygenation(H/R,composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol(P-PostC) at the onset of reoxygenation.After having identified the optical concentration of propofol,H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.Results:The results showed that HG with or without H/R decreased cell viability,increased lactate dehydrogenase(LDH) leakage and the production of reactive oxygen species(ROS) in H9c2 cells,all of which were significantly reversed by propofol(P-PostC),especially at the concentration of 25 μmol/L(P25)(P<0.05,NC vs.HG;HG vs.HG+HR;HG+HR+P12.5 or HG+HR+P25 or HG+HR+P50 vs.HG+HR).Moreover,we found that propofol(P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression(P<0.05,HG+HR+P25 vs.HG+HR).The protective effects of propofol(P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a(P<0.05,HG+HR+P25 vs.HG+HR+P25+siRNA-1 or HG+HR+P25+siRNA-5).Conclusions:It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

Epac1/Rap1 signaling pathway is involved in the pathogenesis of myocardial ischemia/reperfusion injury in rats

OBJECTIVE In this study we explored the role of Epac1-Rap1 pathway in the acute myocardial ischemia/reperfusion injury(MIRI) in vitro and in vivo.METHODS An acute myocardial ischemia/reperfusion injury model was established by the ligation of left anterior descending coronary.Myocardial architecture,fibers and apoptosis was evaluated by the Masson trichrome staining,Sirius red staining and TUNEL assay.H9c2 cells were subjected to hypoxia for 5 h followed by 1-h reoxygen.ation in vitro.Cell viability was measured by MTT assay and cellular injury was evaluated by measuring the release of lactate dehydrogenase(LDH).Western blot,real-time PCR and immunofluorescence were used to detect the expressions of Epac1 and relative downstream molecules.RESULTS Myocardial IR-induced cardiac apoptosis and accumulation of Epac1 and Rap1 in rat IR injury model.Direct Epac activation by 8-CPT(8-(4-chlorophenylthio)-2′-O-methyl-cAMP) exacerbated cardiomyocyte death and dysfunction following hypoxia-reoxygenation(H/R),selective activation of Epac in response to H/R was evident which enriched for cytosolic/membrane proteins and mRNA.Harmacological inhibitor of Epac(ESI-09) significantly ameliorated myocardial injury with the decline of Epac expression.Epac inhibitor and agonist studies also implicated the effect of Rap1,which is downstream of Epac in this pathway.The expression of Rap1 elevated when activated by Epac agonist and was blocked by Epac inhibitor.The same result was true for myocyte CaMK-II and intracellular calcium ions activation.Moreover,ESI-09 also increased ERK1/2 phosphorylation.CONCLUSION Our study reveal that Epac1/Rap1 signaling pathway is involved in the pathogenesis of myocardial I/R injury in rats,which provides evidence on the development of therapeutic strategies target this pathway for myocardial I/R injury.

Effects of L-THP on Ca^(2+) Overload of Cultured Rat Cardiomyocytes during Hypoxia and Reoxygenation

The effects of L-tetrahydropalmatine (L-THP) on the cultured rat cardiomyocytes during hypoxia and reoxygenation and the mechanism of L-THP treating reperfusion-arrythmias were stud- ied. The concentration of intracellular free calcium ([Ca2+]i) of single cultured ventricular myocyte was determined by using EPC-9 light-electricity measurement system. It was found that L-THP (100μmol/L) could reduce the [Ca2+]i augmentation in single cultured ventricular myocyte during hypoxia and reoxygenation. Verapamil (10 μmol/L ) had the similar effect. It was concluded that L- THP could inhibit the Ca2+ overload of cultured rat cardiomyocytes during hypoxia and reoxygena- tion.

Relation or Influence of RVOTO in the Inflammatory Response to Reoxygenation in Patients with Tetralogy of Fallot

Background:This study evaluated differential inflammatory response to cardiopulmonary bypass reoxygenation in tetralogy of Fallot repair.Methods:We performed a retrospective study at a cardiovascular center from 2012 to 2018,including 500 patients aged 1 week–18 years who received complete repair of tetralogy of Fallot.Patients were grouped according to tertiles of preoperative RVOT gradient on echocardiography into mild,moderate,and severe stenosis.We measured the highest perfusate oxygenation(PpO_(2))during aortic occlusion as independent variable.Primary outcome was systemic inflammatory response syndrome(SIRS)within 7 days postoperatively or the time of death or discharge.Results:Overall,rate of SIRS was 24.2% without significant differences among three groups(P>0.05).Older age,male,and smaller indexed left ventricular end-diastolic volume is independent risk factor of SIRS.There were significant interactions between RVOT stenosis and PpO2 on SIRS(P interaction=0.011):higher PpO_(2) was associated with a greater SIRS risk among combined moderate and severe stenotic children(OR 1.46395%CI[1.080,1.981]per-SD increase,P=0.014)but not among mild stenotic children(OR 0.900[0.608,1.333]per-SD increase;P=0.600),independent of covariates.Conclusion:The association of PpO_(2) with SIRS was modified by RVOT obstruction severity in tetralogy of Fallot repair.

Salvianolic Acid A Protects Neonatal Cardiomyocytes against Hypoxia/Reoxygenation-Induced Injury by Preserving Mitochondrial Function and Activating Akt/GSK-3β Signals

Objective: To investigate the effects of salvianolic acid A(SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation(H/R) injury and to determine whether the Akt signaling pathway might play a role. Methods: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/m L SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate(ATP) and reactive oxygen species(ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleavedcaspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential(△Ψm) and mitochondrial permeability transition pore(m PTP), followed by the phosphorylation of Akt(p-Akt) and GSK-3β(p-GSK-3β), which were measured by Western blotting. Results: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cel s and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of m PTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. Conclusion: SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.

Therapeutic effects of Qishen Yiqi Dropping Pill on myocardial injury induced by chronic hypoxia in rats

The present study was designed to determine the effects of a traditional Chinese medicine, called Qishen Yiqi Dropping Pill on chronic hypoxia-induced myocardial injury. To establish a rat chronic hypoxia model to be used in the evaluation of the therapeutic effects of the Qishen Yiqi Dropping Pill, Sprague-Dawley （SD） rats were randomly divided into three groups： the control, model, and treatment groups （n - 10 per group）. The animals were housed in a plexiglass container. The control animals were under normal oxygen concentration and the model and treatment groups were exposed to air and nitrogen for 5 weeks. The rats in the treatment group were orally administered the Qishen Yiqi Dropping pill （35 mg-kg-1.d-1） for 5 weeks. After the treatment, the cardiac function and morphology were analyzed, and the expression levels of hypoxia-inducible factor la （HIF-1a） were determined using Western blotting. Our results indicated that the cardiac function was impaired, cell apoptosis was enhanced, and HIF-1a expression was up-regulated in the model group, compared to the control group. These changes were ameliorated by the treatment with the Qishen Yiqi Dropping Pill. In conclusion, Qishen Yiqi Dropping pill can ameliorate myocardial injury induced by chronic hypoxia, improve cardiac function, and decrease myocardial cell apoptosis, which may provide a basis for its clinical use for the treatment of chronic cardiovascular diseases

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.

Shexiang Tongxin Dropping Pill（麝香通心滴丸）Protects against Na2S2O4-Induced Hypoxia-Reoxygenation Injury in H9c2 Cells

Objectives: To investigate the protective effects of Shexiang Tongxin Dropping Pill(麝香通心滴丸,STP) on Na2S2O4-induced hypoxia-reoxygenation injury in cardiomyoblast H9c2 cells. Methods: The cell viability and levels of mRNA and protein expression in H9c2 cells were determined following Na2S2O4-induced hypoxia using Hoechst staining, annexin V/propidium iodide(PI) flow cytometry, real-time polymerase chain reaction and Western blot analysis. Results: STP pretreatment signi?cantly increased the viability and inhibited aberrant morphological changes in H9c2 cardiomyoblast cells induced by Na2S2O4 treatment(P<0.05). In addition, STP pretreatment attenuated Na2S2O4-induced hypoxic damage, down-regulated the expression of pro-apoptotic Bax,and up-regulated the expression of anti-apoptotic Bcl-2 in H9c2 cells(P<0.05). Conclusions: STP was strongly cardioprotective in hypoxia-reoxygenation injury by preventing hypoxic damage and inhibiting cellular apoptosis.These results further support the use of STP as an effective drug for the treatment of ischemic heart disease.

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。

瓜蒌皮注射液调控JNK/p38通路对缺氧/复氧诱导的心肌细胞损伤和凋亡的影响

目的探究瓜蒌皮注射液通过调节c-Jun N末端激酶/p38丝裂原活化蛋白激酶(JNK/p38)通路提高缺氧/复氧损伤条件下心肌细胞活力及抑制其凋亡的作用及可能机制。方法使用H9c2心肌细胞构建缺氧/复氧(H/R)模型,随后根据处理情况将细胞分为对照组、H/R组、H/R+瓜蒌皮组和H/R+瓜蒌皮+茴香霉素组。通过细胞计数试剂盒(CCK-8)检测各组细胞活力,试剂盒检测各组细胞中乳酸脱氢酶(LDH)、半胱天冬酶3(Caspase 3)活性;采用末端脱氧核苷酸转移酶介导的dUTP末端标记法(TUNEL)检测各组细胞凋亡并采用免疫印迹(WB)检测各组细胞中凋亡相关蛋白如B细胞淋巴瘤2(BCL-2)、BCL-2相关的X蛋白(BAX)、前半胱天冬蛋白3(Pro-caspase 3)、裂解的半胱天冬酶3(Cleaved-caspase 3)及JNK/p38通路蛋白表达。结果与对照组相比,H/R组的细胞活力显著降低,LDH和Caspase 3活性显著增加,细胞凋亡率升高,促凋亡蛋白BAX、Pro-caspase 3、Cleaved-caspase 3的表达上升,而抗凋亡蛋白Bcl-2的表达下降;同时,JNK/p38通路蛋白(JNK、p-JNK、p38、p-p38)的表达水平也显著升高,差异有统计学意义(P<0.05)。与H/R组相比,不同浓度的瓜蒌皮注射液处理后细胞活力均有所提升,尤其是2%瓜蒌皮注射液组表现出最佳效果(P<0.05)。与H/R组相比,H/R+瓜蒌皮组以及H/R+瓜蒌皮+茴香霉素组的细胞活力均显著升高,且两组的LDH和Caspase 3活性降低,细胞凋亡率及促凋亡蛋白(BAX、Pro-caspase 3、Cleaved-caspase 3)的表达下降,抗凋亡蛋白Bcl-2的表达上升,JNK/p38通路蛋白(JNK、p-JNK、p38、p-p38)的表达受到抑制(P<0.05)。与H/R+瓜蒌皮组相比,H/R+瓜蒌皮+茴香霉素组细胞活力受到抑制,LDH和Caspase 3活性升高;BAX、Pro-caspase 3、Cleavedcaspase 3及JNK、p-JNK、p38、p-p38的表达显著上升,而Bcl-2表达下降(P<0.05)。结论瓜蒌皮注射液能够通过抑制JNK/p38通路的激活增加细胞活力并减少细胞凋亡。

三叶青黄酮通过TLR4/NF-κB通路调控NLRP3小体激活改善大鼠心肌细胞缺氧再复氧损伤的作用及机制

目的探究三叶青黄酮(RTHF)对大鼠心肌细胞缺氧再复氧(H/R)损伤的作用及其可能机制。方法以大鼠心肌细胞H9C2为研究对象,分为对照组、H/R组、H/R+低浓度RTHF(L-RTHF)组(25μg/mL)、H/R+中浓度RTHF(M-RTHF)组(50μg/mL)和H/R+高浓度RTHF(H-RTHF)组(100μg/mL)。通过CCK-8法检测各组细胞活力;试剂盒检测各组细胞上清液中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)含量;DCFH-DA探针法检测细胞内活性氧(ROS)水平;实时荧光定量聚合酶链反应(qRT-PCR)和Western blot检测各组细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸蛋白酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)、消皮素D的N端切割产物(GSDMD-N)、Toll样受体4(TLR4)、磷酸化核因子κB p65(p-NF-κB p65)、核因子κB p65(NF-κB p65)的表达水平。结果与对照组比较,H/R组H9C2细胞活力[(71.33±1.70)%比(100.00±2.94)%,P<0.01]、SOD水平[(15.54±0.36)U/mg prot比(34.54±0.87)U/mg prot,P<0.01]降低,ROS相对DCF强度[(3.72±0.12)比(1.00±0.10),P<0.01]和LDH水平[(387.57±9.37)U/L比(182.80±11.53)U/L,P<0.01]升高,NLRP3[(2.75±0.04)比(1.00±0.02)、(4.71±0.18)比(1.00±0.11),P<0.01]、Caspase-1[(3.02±0.11)比(1.00±0.06)、(3.33±0.09)比(1.00±0.12),P<0.01]、ASC[(3.32±0.12)比(1.00±0.09)、(5.30±0.15)比(1.00±0.10),P<0.01]、GSDMD-N[(3.69±0.14)比(1.00±0.13)、(3.23±0.08)比(1.00±0.06),P<0.01]、TLR4[(4.00±0.12)比(1.00±0.09)、(5.68±0.20)比(1.00±0.10),P<0.01]、p-NF-κB p65[(6.81±0.16)比(1.00±0.10)、(3.25±0.07)比(1.00±0.05),P<0.01]相对mRNA和蛋白水平升高;与H/R组比较,H/R+M-RTHF组和H/R+H-RTHF组的细胞活力[(77.00±2.16)%、(82.00±2.16)%比(71.33±1.70)%,P<0.05或P<0.01]、SOD水平[(23.43±1.50)U/mg prot、(28.66±1.22)U/mg prot比(15.54±0.36)U/mg prot,P<0.05或P<0.01]升高,ROS[(3.24±0.05)、(2.57±0.04)比(3.72±0.12),P<0.05或P<0.01]、LDH[(332.77±5.76)U/L、(253.36±9.43)U/L比(387.57±9.37)U/L,P<0.05或P<0.01]水平降低,NLRP3[(1.86±0.06)、(1.56±0.09)比(2.75±0.04),(2.97±0.11)、(1.86±0.06)比(4.71±0.18),P<0.05或P<0.01]、Caspase-1[(2.06±0.13)、(1.27±0.06)比(3.02±0.11),(2.12±0.15)、(1.42±0.09)比(3.33±0.09),P<0.05或P<0.01]、ASC[(2.40±0.25)、(2.19±0.14)比(3.32±0.12),(2.46±0.08)、(1.28±0.05)比(5.30±0.15),P<0.05或P<0.01]、GSDMD-N[(2.43±0.07)、(2.19±0.13)比(3.69±0.14),(1.91±0.07)、(1.46±0.07)比(3.23±0.08),P<0.05或P<0.01]、TLR4[(3.52±0.09)、(1.88±0.11)比(4.00±0.12),(4.04±0.32)、(2.07±0.07)比(5.68±0.20),P<0.05或P<0.01]、p-NF-κB p65[(4.09±0.12)、(3.03±0.20)比(6.81±0.16),(1.93±0.31)、(1.39±0.12)比(3.25±0.07),P<0.05或P<0.01]相对mRNA和蛋白水平降低,且这种变化呈现RTHF剂量依赖性。结论在H/R诱导的大鼠心肌细胞中,RTHF可能通过抑制ROS和TLR4/NF-κB通路调控NLRP3小体激活,进而抑制细胞焦亡。

缺氧后处理通过piRNA-005854调控衰老心肌细胞自噬发挥保护心肌作用

背景:缺血后处理是减轻缺血再灌注损伤的有效方式之一,近年来被越来越广泛地应用于临床实践,但其具体分子机制还有待研究。目的:探讨piRNA-005854在衰老心肌细胞缺氧后处理中的作用及机制。方法:体外给予心肌细胞8 mg/mL D-半乳糖9 d诱导其衰老,β-半乳糖苷酶染色观察心肌细胞的衰老情况;衰老后细胞给予缺氧/复氧处理和缺氧后处理,ELISA检测心肌损伤标志物肌酸激酶同工酶MB以及乳酸脱氢酶水平;Western blot检测衰老心肌细胞中自噬相关蛋白LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达;qRT-PCR检测piRNA-005854的表达水平;进一步用piRNA-005854 inhibitor及piRNA-005854 mimics转染衰老心肌细胞并进行缺氧后处理,Western blot检测LC3Ⅱ、p62和ULK1及其磷酸化ULK1的表达。结果与结论:①D-半乳糖诱导9 d后心肌细胞出现明显衰老;②与正常氧组比较,缺氧/复氧组肌酸激酶同工酶MB以及乳酸脱氢酶水平增加(P<0.01);LC3Ⅱ/Ⅰ表达升高、p62表达降低、ULK1磷酸化水平升高、piRNA-005854表达升高(P<0.01);③与缺氧/复氧组比较,缺氧后处理组肌酸激酶同工酶MB以及乳酸脱氢酶水平明显减少(P<0.01);LC3Ⅱ/Ⅰ表达明显降低(P<0.05)、p62表达升高(P<0.01)、ULK1磷酸化水平降低(P<0.05)、piRNA-005854表达降低(P<0.01);④转染piRNA-005854 inhibitor后,LC3Ⅱ/Ⅰ表达降低(P<0.01),p62表达明显升高(P<0.05),ULK1磷酸化水平明显降低(P<0.01);转染piRNA-005854 mimics后,LC3Ⅱ/Ⅰ表达显著升高,p62表达降低,ULK1磷酸化水平明显增加(P<0.01);⑤结果表明,piRNA-005854介导的ULK1依赖性自噬水平降低是衰老心肌细胞缺氧后处理发挥保护作用的可能机制。

虾青素通过调控氧化应激和炎症反应修复压力性损伤

目的探究虾青素对小鼠压力性损伤创面的修复作用。方法体外实验:用不同浓度虾青素处理成纤维细胞,通过CCK-8实验检测细胞的增殖活性。随后通过缺氧/复氧诱导成纤维细胞损伤,给予最适浓度虾青素,用DHE荧光探针检测细胞内活性氧水平,RT-qPCR检测细胞内TNF-α、IL-1β、IL-6、IL-10、TGF-β的mRNA表达。体内实验:将两块圆形磁铁对称吸附于小鼠皮肤两侧,5 h后移除磁铁,以构建压力性损伤模型。随后分组灌胃等量的生理盐水、低剂量虾青素(10 mg/kg)与高剂量虾青素(20 mg/kg),定期采集创面图像。治疗7 d后,统计创面愈合率,并采集创面组织进行组织病理学染色。结果体外:应用虾青素后,缺氧/复氧损伤成纤维细胞内DHE荧光强度显著降低,TNF-α、IL-1β、IL-6 mRNA表达水平明显下调,TGF-β、IL-10 mRNA表达显著上调(P<0.05)。体内:高剂量虾青素组创面愈合率显著提高,DHE荧光强度显著降低,创面组织TNF-α、IL-6明显减少,TGF-β、IL-10明显增多(P<0.05)。结论虾青素可显著改善氧化应激,减轻炎症反应,对压力性损伤创面具有保护作用。

虫草素调控受体相互作用蛋白激酶1介导的凋亡改善缺氧/复氧诱导的人肾小管上皮细胞损伤

目的基于基因表达综合(gene expression omnibus,GEO)数据库中的基因芯片、网络药理学、分子对接和体外实验探讨虫草素治疗急性肾损伤(acute kidney injury,AKI)的作用机制。方法从GEO(GSE87025)、GeneCards 2个数据库获得AKI的疾病靶点。从TargetNet、BATMAN和GeneCards 3个数据库获得虫草素相关靶点。对AKI和虫草素相关靶点取并集,筛选出共同靶基因。进行基因本体论(gene ontology,GO)注释和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析后对虫草素及关键靶点进行分子对接。细胞实验设计对照组、缺氧/复氧组、缺氧/复氧+虫草素组对相关靶点进行实验验证。结果AKI和虫草素共同靶基因共12个,GO功能注释结果表明主要参与凋亡信号通路的负调控、坏死性凋亡过程等生物学过程,KEGG富集分析结果表明主要参与Toll样受体信号通路、坏死性凋亡、核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain,NOD)样受体信号通路、核因子κB(nuclear factor-κB,NF-κB)信号通路、肿瘤坏死因子(tumor necrosis factor,TNF)信号通路和细胞凋亡等常见信号通路。虫草素和受体相互作用蛋白激酶1(receptor-interacting protein kinase 1,RIPK1)分子对接的最优结合能为-7.1,表明两者有较强的结合活性。蛋白免疫印迹结果表明虫草素可下调RIPK1、Bcl-2相关X蛋白、凋亡指标胱天蛋白酶表达、上调B细胞淋巴瘤/白血病-2的表达,细胞免疫荧光结果表明虫草素可下调RIPK1表达。结论虫草素可通过调控RIPK1介导的凋亡缓解缺氧/复氧诱导的人肾小管上皮细胞损伤,以达到治疗AKI的效果。

交趾黄檀新黄酮类成分及其抗H9c2心肌细胞缺氧/复氧损伤活性研究

目的研究交趾黄檀Dalbergia cochinchinensis Pierre ex Laness的新黄酮类成分及其抗H9c2心肌细胞缺氧/复氧损伤活性。方法交趾黄檀70%乙醇提取物采用硅胶、Sephadex LH-20、反相制备HPLC进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。采用CCK-8法检测其对H9c2心肌细胞的活性及对H9c2细胞缺氧/复氧损伤的保护作用,并分析其构效关系。结果从中分离得到12个化合物,分别鉴定为阔叶黄檀酚(1)、5-O-methyllatifolin(2)、mimosifoliol(3)、5-O-methydalbergiphenol(4)、dalbergiphenol(5)、cearoin(6)、2,4-dihydroxy-5-methoxy-benzophenone(7)、2-hydroxy-4,5-dimethoxybenzophenone(8)、melannoin(9)、2,2′,5-trihydroxy-4-methoxybenzophenone(10)、黄檀素(11)、4-甲氧基黄檀醌(12)。黄檀酚及黄檀内酯类化合物对H9c2细胞毒性较小,黄檀酚类化合物抗H9c2心肌细胞缺氧/复氧损伤活性较强。结论化合物8为新天然产物,化合物4、9为首次从该植物中分离得到。黄檀酚类化合物可能是抗H9c2细胞缺氧/复氧损伤的主要新黄酮类成分。