Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

Salvianolate Reduces Murine Myocardial Ischemia and Reperfusion Injury via ERK1/2 Signaling Pathways in vivo

Objective： To analyze the effects of salvianolate on myocardial infarction in a murine in vivo model of ischemia and reperfusion （I/R） injury. Metheds： Myocardial I/R injury model was constructed in mice by 30 min of coronary occlusion followed by 24 h of reperfusion and pretreated with salvianolate 30 min before I/R （SAL group）. The SAL group was compared with SHAM （no I/R and no salvianolate）, I/R （no salvianolate）, and ischemia preconditioning （IPC） groups. Furthermore, an ERK1/2 inhibitor PD98059 （1 mg/kg）, and a phosphatidylinositol-3-kinase （PI3-K） inhibitor, LY294002 （7.5 mg/kg）, were administered intraperitoneal injection （i.p） for 30 min prior to salvianolate, followed by I/R surgery in LY and PD groups. By using a double staining method, the ratio of the infarct size （IS） to left ventricle （LV） and of risk region （RR） to LV were compared among the groups. Correlations between IS and RR were analyzed. Western-blot was used to detect the extracellular signal-regulated kinase 1/2 （ERK1/2） and protein kinase B （AKT） phosphorylation changes. Results： There were no significant differences between RR to LV ratio among the SHAM, I/R, IPC and SAL groups （P〉0.05）. The SAL and IPC groups had IS of 26.1% ± 1.4% and 22.3% ±2.9% of RR, respectively, both of which were significantly smaller than the I/R group （38.5% ± 2.9% of RR, P〈0.05, P〈0.01, respectively）. Moreover, the phosphorylation of ERK1/2 was increased in SAL group （P〈0.05）, while AKT had no significant change. LY294002 further reduced IS, whereas the protective role of salvianolate could be attenuated by PD98059, which increased the IS. Additionally, the IS was not linearly related to the RR （r=0.23, 0.45, 0.62, 0.17, and 0.52 in the SHAM, I/R, SAL, LY and PD groups, respectively）. Conclusion： Salvianolate could reduce myocardial I/R injury in mice in vivo, which involves an ERK1/2 pathway, but not a PI3-K signaling pathway.

Protective effects of combination of Xuesaitong and aspirin on cerebral ischemia and reperfusion injury in rats

Objective：To investigate the protective effects of the combination of Xuesaitong（XST）and aspirin on cerebral ischemia and reperfusion injury（CIRI）in rats,and further explore the underlying mechanisms.Methods：A total of 150 male Sprague-Dawley（SD）rats were randomly divided into five groups with 30rats in each group：sham group,middle cerebral artery occlusion/reperfusion（MCAO/R）model group,XST group,aspirin group,and XST＋aspirin group.Rats were pretreated with XST,aspirin,or XST＋aspirin for7 d.One hour after the last administration,a model of CIRI was induced by MCAO/R.Neurological deficits were assessed using Longa’s five-point scale.Cerebral edema was detected by the measurement of brain water content.The volume of cerebral infarction was determined by 2,3,5-triphenyltetrazolium chloride（TTC）staining.The activities of superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH-Px）,as well as levels of malonaldehyde（MDA）were detected by commercial kits.Enzyme-linked immunosorbent assay（ELISA）was used to determine the levels of interleukin-1（）,interleukin-4（IL-4）,interleukin-6（IL-6）,interleukin-10（IL-10）,tumor necrosis factor-alphamonocyte chemotactic protein 1（MCP-1）,and kynurenine in serum,cerebral cortex,and hippocampus of MCAO/R rats.The protein expression of nuclear factor erythroid 2-related factor（Nrf2）,heme oxygenase-1（HO-1）,I-kappa B alpha（IκBα）,and nuclear factor kappa B（B）/p65 in the cortex were analyzed by western blotting.Results：Treatment of XST,aspirin,and XST＋aspirin significantly alleviated the neurological deficits,cerebral edema,and cerebral infarct volume induced by MCAO/R.Treatment of XST,aspirin,and XST＋aspirin also reduced MDA,,MCP-1,and kynurenine levels,and increased SOD,CAT,GSH-Px,IL-4,and IL-10 levels in serum,cerebral cortex,and hippocampus of MCAO/R rats.Furthermore,treatment of XST,aspirin,and XST＋aspirin decreased the expression of nuclearB/p65 and increased the expression of IκBα,nuclear Nrf2,and HO-1.Importantly,the combination of XST and aspirin enhanced the protective effects of XST or aspirin treatment alone on CIRI in rats.Conclusion：The combination of XST and aspirin significantly inhibited oxidative stress and inflammation in serum,cerebral cortex,and hippocampus of MCAO/R rats.The combination of XST and aspirin exerted more protective effects than XST or aspirin treatment alone.The combination of XST and aspirin might provide the synergistic therapeutic effects on CIRI,and deserve further clinical investigation.

INCREASED VULNERABILITY OF HYPERTROPHIED MYOCARDIUM TO ISCHEMIA AND REPERFUSION INJURY RELATION TO CARDIAC RENIN-ANGIOTENSIN SYSTEM

Hearts of pressure-overload hypertrophy show an increased activation of intracardiac renin-angiotensin system which may contribute to ischemia and reperfusion injury. The purpose of this study is to evaluate whether the hypertrophied myocardium is more vulnerable to ischemia and reperfusion injury and to find out its relation to the cardiac renin-angiotensin system. Hypertrophied rat hearts induced by abdominal aortic banding for 6 weeks were subjected to 2 hours of hypothermic ischemic arrest followed by 30 minutes of reperfusion, and their cardiac function recovery was compared with that of sham-operated normal control hearts. The cardiac renin activity and angiotensin II content before ischemia and after reperfusion were determined. It was found that both the pre-ischemic renin activity and angiotensin II level were higher in hypertrophied myocardium than those in the control: ischemia and reperfusion injury increased both renin activity and angiotensin II content in the two groups, but the renin activity and angiotensin II level were further elevated after reperfusion in the hypertrophied hearts than those in the control hearts. Meanwhile, the cardiac function recovery after 30 minutes reperfusion in the hypertrophied hearts was poorer than that in the control. Correlation analysis revealed that there was a negative correlation between the cardiac output recovery and the myocardial angiotensin II content (r=-0.841), P<0.001), It is concluded that ischemia and reperfusion injury can activate cardiac renin-angiotensin system in isolated rat heart, which may be responsible for the increased susceptibility of the hypertrophied myocardium to ischemia and reperfusion injury.

A New Method of Superoxide Free Radical Determination in Ischemia and Reperfusion Injury of Rat Liver

The present study was designed to utilize the chemiluminescence(CL)method for O2- analysis in ischemia and reperfusion of the whole rat liver. The results indicated that the O2- level was lower after 30 and 60 min ischemia. After 40 min reperfusion, the level of O2- increased obviously,reaching about 8. 3-fold (30min ischemia) and 9. 0-fold (60 min ischemia) (P<0.001,P<0.01) respectively.The results also show that Chinese Traditional Medicines ligustrazine and salvia compound can scavenge O2- effectively.

MicroRNA-670 aggravates cerebral ischemia/reperfusion injury via the Yap pathway

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury.MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury.However,whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood.In this study,we established mouse models of transient middle artery occlusion and Neuro 2a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo.Our results showed that after ischemia/reperfusion injury,miR-670 expression was obviously increased.After miR-670 expression was inhibited with an miR-670 antagomir,cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced.When miR-670 overexpression was induced by an miR-670 agomir,neuronal apoptosis was increased.In addition,we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits.Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury.These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway,which may be a potential target for treatment of cerebral ischemia/reperfusion injury.The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27,2017(IRB No.2017PS035K).

PROTECTIVE EFFECTS OF ERYTHROPOIETIN ON MYO-CARDIUM AGAINST ISCHEMIA-REPERFUSION INJURY

Objective To explore the protective effects of erythropoietin （EPO） on myocardium against ischemia-reperfusion injury （ IRI）. Methods The Langendorff model of isolated rat heart was set up and a 3-stage protocol was performed： 20 min stabilization, 30 min global ischemia, and 120 min reperfusion. Sixty SD rats were randomly divided into sham group, ischemia-reperfusion group （I/R group ） and EPO treated group （ EPO group）. Heart rate （ HR）, left ventricular developed pressure （ LVDP）, the first derivative （ △dp/dt max） and coronary flow （CF） were recorded at the 20th minute of stabilization and the 120th minute of reperfusion. Lactate dehydrogenase （LDH） and creatine phosphokinase （CK） in the coronary effluent at the 60th minute of reperfusion , the levels of myocardial nuclear factor-kappa B （NF-KB） and the myocardial content of tumor necrosis factor-α （TNF-α） ,interleukin-lβ （1L-lβ） were measured at the end of reperfusion. Results No statistically significant differences were observed on the aspect of hemodynamic parameters among the groups at the 20th minute of stabilization, but at the 120th minute of reperfusion, the recovery ratio of EPO group was higher than I/R group （P 〈0. 05）. LDH and CK in the coronary effluent, the levels of myocardial NF-KB and TNF-α,IL-lβ expression in EPO group were significantly lower than those in I/ R group, but higher than sham group （ P 〈 0. 05 ）. Conclusion EPO has protective effects on myocardium against 1RI possibly through the mechanism of relieving the myocardial inflanmtatory reaction by regulating the activation of NF-KB and then decreasing the expression of proinflammatory factors TNF-α and IL-lβ.

Argon: a novel therapeutic option to treat neuronal ischemia and reperfusion injuries?

Neuronal injury and neuroprotection：Ischemia and reperfusion injuries in neuronal cells such as acute ischemic stroke-represent the third leading cause of death in the world.Current therapeutic concepts mainly aim to re-establish cerebral blood flow within a time window of less than 3 hours with the goal of limiting secondary brain injury.

Protective Mechanisms of Suxiao Jiuxin Pills(速效救心丸) on Myocardial Ischemia-Reperfusion Injury in vivo and in vitro

Objective:To study the protective mechanism of Chinese medicine Suxiao Jiuxin Pills(速效救心丸,SXJ)on myocardial ischemia and reperfusion(I/R)injury.Methods:Mouse myocardial I/R injury model was created by 30-min coronary artery occlusion followed by 24-h reperfusion,the mice were then divided into the sham group(n=7),the I/R group(n=13),the tirofiban group(TIR,positive drug treatment,n=9),and the SXJ group(n=11).Infarct size(IS),risk region(RR),and left ventricle(LV)were analyzed with double staining methods.In addition,H9C2 rat cardiomyocytes were cultured with Na2S2O4 to simulate I/R in vitro.The phosphorylation of extracellular regulated protein kinases1/2(ERK1/2),protein kinase B(AKT),glycogen synthase kinase-3β(GSK3β),and protein expression of GATA4 in nucleus were detected with Western blot assay.Results:The ratio of IS/RR in SXJ and TIR groups were lower than that in I/R group(SXJ,22.4%±6.6%;TIR,20.8%±3.3%;vs.I/R,35.4%±3.7%,P<0.05,respectively).In vitro experiments showed that SXJ increased the Na2S2O4-enhanced phosphorylation of AKT/GSK3βand nuclear expression of GATA4.Conclusion:SXJ prevents myocardial I/R injury in mice by activating AKT/GSK3βand GATA4 signaling pathways.

Therapeutic effect of methane and its mechanism in disease treatment

Methane is the simplest hydrocarbon,consisting of one carbon atom and four hydrogen atoms.It is abundant in marsh gas,livestock rumination,and combustible ice.Little is known about the use of methane in human disease treatment.Current research indicates that methane is useful for treating several diseases including ischemia and reperfusion injury,and inflammatory diseases.The mechanisms underlying the protective effects of methane appear primarily to involve anti-oxidation,anti-inflammation,and anti-apoptosis.In this review,we describe the beneficial effects of methane on different diseases,summarize possible mechanisms by which methane may act in these conditions,and discuss the purpose of methane production in hypoxic conditions.Then we propose several promising directions for the future research.

Study on the inflammatory cytokines after heterotopic transplantation of isolated mouse heart preserved in a high-pressured mixed gas chamber

Background The maintenance of heart viability is important for heart transplantation. Currently, heart preservation is limited to 6 hours of cold ischemic storage. This study explored a new heart preservation method under a high-pressured mixed gas chamber. Methods C57BL/6 male mice were used to establish the model of mice cervical heterotopic heart transplantation. Adult donor mice were randomly divided into three groups subjected to naive operation （Group A）, standard control （Group B） and experimental control （Group C）. The recipient mice were randomly divided into two groups subjected to standard control and experimental control. Group A： hearts were isolated; Group B： hearts were isolated and preserved in HTK solution at 4 ℃ for 8 h and transplanted; Group C： hearts were isolated and preserved in high pressured gas （PO2：3200 hPa ＋ PCO： 800 hPa = 4000 hPa） at 4 ℃ for 8h and transplanted. After transplantation, the state of re-beating and cardiac function were observed for Group B and C. At 24 h after transplantation, samples were collected for HE staining, cardiac cell apoptosis detection by Tunnel staining and analysis of tumor necrosis factor α （TNF-α）, interleukin-1β （IL-1β）, interleukin-6 （IL-6） and interleukin-10 （IL-10） by reverse transcriotion-polymerase chain reaction （RT-PCR）. Results In group C, 15 transplanted hearts were re-beat, while only 6 in Group B. The re-beating rate in Group C was significantly higher than Group B [75.0%（15/20） vs. 30.0%（6/20） ,P = 0.01]. The time of re-beating was significantly different between Group B, and C [（352.35 ± 61.07）s vs. （207.85 ± 71.24） s, P 〈 0.011. HE staining showed that pathologic changes such as ceil edema and inflammatory cell infiltration were more obvious in Group B and C than in Group A, but less obvious in Group C compared with Group B. Tunnel staining showed that Group B had more obvious apoptosis than Group A and C. RT-PCR results showed significant increase of TNF-α, IL-1β and IL-6 expression in Group B than Group C （P 〈 0.01, the expression of IL-10 was higher in Group C than that in Group B. Conclusion Highpressured mixed gas （PO2：3200 hPa ＋ PCO： 800 hPa = 4000 hPa） preservation can reduce cold ischemia and reperfusion injury of donor heart, therefore to maintain myocardial viability and prolong preservation time of donor heart.