Protective function of tocilizumab in human cardiac myocytes ischemia reperfusion injury

Objective:To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL,3.0 mg/mL,5.0 mg/mL) for 24 h.then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h.The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes,respectively.The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot,respectively.Results:Compared to the negative group,pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury(P<0.05).The expression of Bcl-2 in tocilizumab treated group were higher than NC group(P<0.05).while the Bax expression were lower(P<0.05).Conclusions:Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury.Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.

Exploring the mechanism of icariin in regulat⁃ing cardiac microvascular endothelial cells based on network pharmacology,molecular docking and in vitro experiments

OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro⁃vascular endothelial cells(CMEC)after oxygenglucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemiareperfusion model,and were divided into normal,model,low(10μmol·L^(-1)),medium(20μmol·L^(-1))and high(40μmol·L^(-1))ICA group,and high ICA+inhibitor group(40μmol·L^(-1)+20 nmol·L^(-1)).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col⁃lect ICA-related targets,the GeneCards data⁃base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar⁃get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft⁃ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec⁃tive effect at 10^(-1)60μmol·L^(-1);the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P<0.01,P<0.01);and the results of tube-for⁃mation assay showed that each group of ICA could significantly promote the generation of branches(P<0.01)and the capillary length exten⁃sion(P<0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar⁃gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich⁃ment analysis found 53 results,involving AGERAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu⁃lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra⁃tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha⁃nism of action of ICA against myocardial isch⁃emia-reperfusion injury may be related to the reg⁃ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.

Tauroursodeoxycholic acid and 4-phenyl butyric acid alleviate endoplasmic reticulum stress and improve prognosis of donation after cardiac death liver transplantation in rats

BACKGROUND： Inevitable warm ischemia time before organ procurement aggravates posttransplantation ischemia- reperfusion injury. Endoplasmic reticulum （ER） stress is involved in ischemia-reperfusion injury, but its role in donation after cardiac death （DCD） liver transplantation is not clear and the effect of ER stress inhibitors, tauroursodeoxycholic acid （TUDCA） and 4-phenyl butyric acid （PBA）, on the prognosis of recipient of DCD liver transplantation remains unclear. METHODS： Male Sprague-Dawley rats （8-10 weeks） were randomly divided into control group： liver grafts without warm ischemia were implanted; DCD group： warm ischemia time of the liver grafts was 60 minutes; TUDCA and PBA groups： based on the DCD group, donors were intraperitoneally injected with TUDCA or PBA 30 minutes before the organ procurements. Serum aminotransferase levels, oxidative stress activation and expression of ER stress signal molecules were evaluated. Pathological examinations were performed. The survivals of the recipients in each group were compared for 14 days.RESULTS： Compared with the control group, DCD rats had significantly higher levels of serum aminotransferase at 6 hours, 1 day and 3 days after operation （P〈0.01, 0.01 and 0.05, respectively） and oxidative indices （P〈0.01 for both malondialdehyde and 8-hydroxy deoxyguanosine）, more severe liver damage （P〈0.01） and up-regulated ER stress signal expressions （P〈0.01 for GRP78, phos-eIF2al, CHOP, ATF-4, ATF-6, PERK, XBP-1 and pro-caspase-12）. All recipients died within 3 days after liver transplantation. Administration of TUDCA or PBA significantly decreased aminotransferase levels （P〈0.05）, increased superoxide dismutase activities （P〈0.01）, alleviated liver damage （P〈0.01）, down-regulated ER stress signal expressions （P〈0.01） and improved postoperative survivals （P〈0.01）. CONCLUSIONS： ER stress was involved with DCD liver trans- plantation in rats. Preoperative intraperitoneally injection of TUDCA or PBA protected ER stress and improved prognosis.

Mapping Changes of Whole Brain Blood Flow in Rats with Myocardial Ischemia/Reperfusion Injury Assessed by Positron Emission Tomography

l8F-labeled fluorodeoxyglucose positron emission tomography (l8F-FDG PET) is the most sensitive tool for studying brain metabolism in vivo.We investigated the image patterns of 18F-FDG PET during reperfusion injury and correlated changes of whole brain blood flow utilizing a rat myocardial ischemia/reperfiision injury (MIRI) model.The results assessed by echocardiography indicated resultant cardiac dysfunction after ischemia-reperfusion in the rat heart.It was found that the average standardized uptake value (SUVaverage) of the whole brain was significantly decreased in model rats,and the glucose uptake of different brain regions including accumbens core/shell (Acb),left caudate putamen (LCPu),hippocampus (HIP),left hypothalamus (LHYP),olfactory (OLF),superior colliculus (SC),right midbrain (RMID),ventral tegmental area (VTA),inferior colliculus (IC) and left thalamus whole (LTHA) was significantly decreased in MIRI rats whereas no significant difference was found in the SUVaverage of amygdala (AMY),right Cpu,RHYP,right HYP,left MID,right THA,pons and medulla oblongata (MO).These l8F-FDG PET data provide a reliable identification method for brain metabolic changes in rats with MIRI.

Cardio-Protective Effects of Oral Nicorandil in Patients Undergoing Cardiac Valve Surgery

Background: Reduction of myocardial reperfusion injury during cardiopulmonary bypass is an essential requirement for increasing the success rate, decreasing morbidity and mortality of open-heart surgery. Aim: To study the role of pre-operative oral nicorandil in decreasing reperfusion cardiac injury in patients subjected to cardiac valve surgery. Patients and Methods: The study included 62 patients, who were equally randomized into two groups: nicorandil group and control group. Pre-operative, intra-operative and post- operative data were reported and analyzed. Left Ventricle Ejection Fraction (LVEF) was estimated pre-operatively and postoperatively for both groups. Troponin I, creatine kinase-muscle/brain (CK-MB), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured before surgery by 24 hours then 4, 12 and 48 hours after aortic cross clamp removal. Results: Nicorandil considerably decreased TNF-α and IL-6 after 4 and 12 hours following the removal of aortic clamping. It also reduced troponin-I and CKMB at the same time points. However, there were no important changes in IL-6, TNF-α, troponin-I and CK-MB levels in control group in comparison to nicorandil group in the next 48 hours following the removal of aortic clamping. Conclusions: Pre-operative oral nicorandil expressively decreased myocardial reperfusion damage during open heart valve operations, this evidenced by the decrease in the postoperative use of inotropic drugs, considerable reduction of postoperative elevation of cardiac enzymes and inflammatory cytokines with no reported complications.

Cardiac fibroblast heat shock protein 47 aggravates cardiac fibrosis post myocardial ischemia-reperfusion injury by encouraging ubiquitin specific peptidase 10 dependent Smad4 deubiquitination

Despite complications were significantly reduced due to the popularity of percutaneous coronary intervention(PCI) in clinical trials, reperfusion injury and chronic cardiac remodeling significantly contribute to poor prognosis and rehabilitation in AMI patients. We revealed the effects of HSP47 on myocardial ischemia-reperfusion injury(IRI) and shed light on the underlying molecular mechanism.We generated adult mice with lentivirus-mediated or miRNA(mi1/133TS)-aided cardiac fibroblastselective HSP47 overexpression. Myocardial IRI was induced by 45-min occlusion of the left anterior descending(LAD) artery followed by 24 h reperfusion in mice, while ischemia-mediated cardiac remodeling was induced by four weeks of reperfusion. Also, the role of HSP47 in fibrogenesis was evaluated in cardiac fibroblasts following hypoxia-reoxygenation(HR). Extensive HSP47 was observed in murine infarcted hearts, human ischemic hearts, and cardiac fibroblasts and accelerated oxidative stress and apoptosis after myocardial IRI. Cardiac fibroblast-selective HSP47 overexpression exacerbated cardiac dysfunction caused by chronic myocardial IRI and presented deteriorative fibrosis and cell proliferation.HSP47 upregulation in cardiac fibroblasts promoted TGFβ1-Smad4 pathway activation and Smad4 deubiquitination by recruiting ubiquitin-specific peptidase 10(USP10) in fibroblasts. However, cardiac fibroblast specific USP10 deficiency abolished HSP47-mediated fibrogenesis in hearts. Moreover, blockage of HSP47 with Col003 disturbed fibrogenesis in fibroblasts following HR. Altogether, cardiac fibroblast HSP47 aggravates fibrosis post-myocardial IRI by enhancing USP10-dependent Smad4 deubiquitination,which provided a potential strategy for myocardial IRI and cardiac remodeling.

粉防己碱激活Akt/GSK-3b信号通路保护心肌I/R大鼠研究

目的:探究粉防己碱对心肌缺血/再灌注损伤(Ischemia/Reperfusion injury,I/R)大鼠的心肌梗塞面积、心脏功能蛋白激酶B(Protein kinase B,Akt)/糖原合酶激酶-3b(Glycogen synthase kinase-3b,GSK-3b)信号通路的影响。方法:SPF级健康雄性SD大鼠180只,随机分为假手术组、模型组、辛伐他汀组及粉防己碱低、中、高剂量组。辛伐他汀组于造模前14天灌胃给予辛伐他汀2.0 mg/(kg·d);粉防己碱低、中、高剂量组于造模前20 min分别腹腔注射给予粉防己碱1.5 mg/kg、3.0 mg/kg、6.0 mg/kg;假手术组与模型组给予等体积生理盐水。通过结扎冠状动脉左前降支建立I/R大鼠模型。再灌注24 h后,测定大鼠血清肌钙蛋白T(cTnT)含量及肌酸激酶同工酶(CK-MB)、乳酸盐脱氢酶(LDH)活性;氯化硝基四氮唑蓝(NBT)染色法测定心肌梗死面积;超声心动图检测心脏功能;Western blot检测心肌组织Akt、p Akt、GSK-3b、p GSK-3b蛋白表达。结果:与假手术组比较,模型组大鼠血清cTnT含量及CK-MB、LDH活性均显著升高,心肌梗死面积显著增大,心脏左室舒张末期内径(Left ventricular internal diameter at diastole,LVIDd)和左室收缩末期内径(Left ventricular internal diameter at systole,LVIDs)均显著增大,心肌组织pAkt/Akt、p GSK-3b/GSK-3b比值均显著减小,差异均有统计学意义(P<0.0 5);与模型组比较,粉防己碱中、高剂量组及辛伐他汀组大鼠血清cTnT含量及CK-MB、LDH活性均显著降低,心肌梗死面积及心脏LVIDd、LVIDs均显著减小,心肌组织pAkt/Akt、pGSK-3b/GSK-3b比值均显著增加,且呈剂量依赖性,差异均有统计学意义(P<0.0 5)。结论:粉防己碱能明显减轻I/R大鼠的心肌损伤,改善心脏功能,其机制可能与Akt/GSK-3b通路活化有关。

Liver graft preservation methods during cold ischemia phase and normothermic machine perfusion

The growing demand for donor organs requires measures to expand donor pool.Those include extended criteria donors, such as elderly people, steatotic livers,donation after cardiac death, etc. Static cold storage to reduce metabolic requirements developed by Collins in late 1960 s is the mainstay and the golden standard for donated organ protection. Hypothermic machine perfusion provides dynamic organ preservation at 4°C with protracted infusion of metabolic substrates to the graft during the ex vivo period. It has been used instead of static cold storage or after it as short perfusion in transplant center. Normothermic machine perfusion(NMP) delivers oxygen, and nutrition at physiological temperature mimicking regular environment in order to support cellular function. This would minimize effects of ischemia/reperfusion injury.Potentially, NMP may help to estimate graft functionality before implantation into a recipient. Clinical studies demonstrated at least its non-inferiority or better outcomes vs static cold storage. Regular grafts donated after brain death could be safely preserved with convenient static cold storage. Except for prolonged ischemia time where hypothermic machine perfusion started in transplant center could be estimated to provide possible positive reconditioning effect. Use of hypothermic machine perfusion in regular donation instead of static cold storage or in extended criteria donors requires further investigation. Multicenter randomized clinical trial supposed to be completed in December 2021. Extended criteria donors need additional measures for graft storage and assessment until its implantation. NMP is actively evaluating promising method for this purpose.Future studies are necessary for precise estimation and confirmation to issue clinical practice recommendations.

Role of Polyamines in Ozone Exposed Ischemic-Reperfused Hearts

The effect of chronic ozone exposure to ischemia reperfusion (I/R) injury in isolated perfused rat hearts was previously demonstrated. The present study tested our hypothesis that chronic ozone exposure led to attenuation of polyamines in the heart, which may limit sensitivity to I/R. Sprague Dawley rats were continuously exposed for 8 hrs/day for 28 days to filtered air or 0.8 ppm ozone. Isolated hearts were previously subjected to 0.5 hour of global ischemia followed by 1 hour of reperfusion after which global polyamine content was examined between the two groups. Spermidine production was significantly increased in the experimental group compared to control group (of I/R hearts). These results suggest that ozone-induced sensitivity to chronic I/R injury activates myocardial polyamine stress response characterized by increased enzymatic activities and accumulation of spermidine. Collectively, these results suggest that I/R possibly disturbs polyamine metabolism, and increased oxidative stress and concomitant reduced myocardial cell viability.

运动性和高血压性心肌肥大对再灌注损伤耐受性的研究

为探讨运动性心肌肥大和高血压心肌肥大对缺血再灌注损伤的差异，本文在大鼠游泳训练心肌肥大和主动脉缩窄性高血压心肌肥大离体心脏灌流模型上，观察它们对缺血再灌注损伤的差异。结果表明，反映组织损伤程度的指标如乳酸脱氢酶漏出，心肌脂质过氧化终产物丙二醛（ＭＤＡ）和心肌钙在高血压心肌肥大的心肌较对照组和运动心肌肥大组皆明显升高，而运动组和对照组则无差异，高血压心肌肥大的程度和运动性心肌肥大的程度基本一致。本研究提示运动性心肌肥大的心肌抗缺血再灌注损伤的能力较高血压心肌肥大者增强。

丹参饮预处理对心肌缺血再灌注大鼠血流动力学和线粒体能量代谢的影响

目的 观察丹参饮预处理对心肌缺血再灌注(I/R)大鼠心脏血流动力学、心肌能量代谢及心肌细胞凋亡的影响。方法 将60只Wistar大鼠随机分为假手术组、模型组及丹参饮高、中、低剂量组,每组12只,给药1周后,采用大鼠冠状动脉前降支结扎30 min再复流24 h的方法建立I/R模型。PowerLab/4SP型生物信号处理和分析系统检测大鼠血流动力学指标,HE染色观察心肌形态学改变,TUNEL法观察分析心肌细胞的凋亡水平,HPLC检测心肌组织中ATP、ADP、AMP水平,ELISA法检测心肌组织细胞色素C(Cyt-C)水平,Western blot法检测心肌组织cleaved caspase-3、Bcl-2、Bax蛋白表达。结果 与假手术组比较,模型组大鼠LVSP、LV+dp/dt_(max)、ATP、ADP水平、Bcl-2/Bax蛋白表达比值均降低(P<0.05,P<0.01),LVEDP、心肌细胞凋亡率、心肌组织Cyt-C水平、cleaved caspase-3蛋白表达均升高(P<0.05,P<0.01);与模型组比较,丹参饮预处理后,以上指标均有改善(P<0.05,P<0.01)。结论 丹参饮预处理可以改善缺血再灌注大鼠的心功能异常,提高心肌线粒体能量代谢水平,减少凋亡蛋白表达,降低心肌细胞凋亡率,具有对心肌缺血/再灌注损伤的心脏保护作用。

NLRP3炎症小体与心脏骤停复苏后心肌缺血再灌注损伤的研究进展

心脏骤停已成为世界范围内的重大公共卫生问题,及时有效的心肺复苏可以挽救生命,改善心脏骤停患者的预后。尽管心肺复苏技术取得了进步,但与心脏骤停相关的死亡率仍然很高。NLRP3炎症小体是细胞内多种蛋白质构成的复合物,在先天免疫中起着重要作用。组织损伤后,NLRP3炎症小体激活产生大量细胞因子如白细胞介素(IL)-1β和IL-18,最终导致炎症性细胞死亡(细胞焦亡)。虽然适度的炎症反应有利于损伤组织愈合,但过度的NLRP3炎症小体激活会产生不利影响。NLRP3炎症小体在大量心血管疾病(cardiovascular diseases,CVDs)中发挥关键作用。心脏骤停和复苏后缺血再灌注损伤(I/R损伤)可以通过各种信号通路激活NLRP3炎症小体。抑制NLRP3炎症小体活性可以改善心脏骤停和复苏后缺血再灌注损伤。该文将讨论NLRP3炎症小体在心脏骤停和复苏过程中对细胞损伤的作用,同时针对抑制NLRP3炎症小体激活,改善心脏骤停和复苏后缺血再灌注损伤的治疗方法进行系统阐述。

心肌缺血再灌注损伤中心肌免疫细胞单细胞图谱绘制及具有心肌保护效应的Ym-1hi中性粒细胞的鉴定

Myocardial ischemia–reperfusion injury(MIRI)is a major hindrance to the success of cardiac reperfusion therapy.Although increased neutrophil infiltration is a hallmark of MIRI,the subtypes and alterations of neutrophils in this process remain unclear.Here,we performed single-cell sequencing of cardiac CD45^(+)cells isolated from the murine myocardium subjected to MIRI at six-time points.We identified diverse types of infiltrating immune cells and their dynamic changes during MIRI.Cardiac neutrophils showed the most immediate response and largest changes and featured with functionally heterogeneous subpopulations,including Ccl3^(hi)Neu and Ym-1^(hi)Neu,which were increased at 6 h and 1 d after reperfusion,respectively.Ym-1^(hi)Neu selectively expressed genes with protective effects and was,therefore,identified as a novel specific type of cardiac cell in the injured heart.Further analysis indicated that neutrophils and their subtypes orchestrated subsequent immune responses in the cardiac tissues,especially instructing the response of macrophages.The abundance of Ym-1^(hi)Neu was closely correlated with the therapeutic efficacy of MIRI when neutrophils were specifically targeted by anti-Lymphocyte antigen 6 complex locus G6D(Ly6G)or anti-Intercellular cell adhesion molecule-1(ICAM-1)neutralizing antibodies.In addition,a neutrophil subtype with the same phenotype as Ym-1^(hi)Neu was detected in clinical samples and correlated with prognosis.Ym-1 inhibition exacerbated myocardial injury,whereas Ym-1 supplementation significantly ameliorated injury in MIRI mice,which was attributed to the tilt of Ym-1 on the polarization of macrophages toward the repair phenotype in myocardial tissue.Overall,our findings reveal the antiinflammatory phenotype of Ym-1^(hi)Neu and highlight its critical role in myocardial protection during the early stages of MIRI.

NLRP3炎性体与心血管疾病的研究进展

炎性体是胞内多蛋白复合体,NLRP3炎性体是其重要成员,可感知病原入侵或细胞应激,通过胞外三磷酸腺苷（ATP） /K＋外流、溶酶体释放组织蛋白酶等机制活化半胱天冬酶-1（caspase-1）使IL-1β及IL-18前体成熟参与固有免疫应答.越来越多心血管疾病被证实与免疫功能紊乱相关,因而NLRP3炎性体与心血管疾病之间的关系越发受到关注.