Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells

Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O2)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.     

Inflammasome-independent NALP3 contributes to high-salt induced endothelial dysfunction

OBJECTIVE Na+is an important nutrient and its intake, mainly from salt(Na Cl), is essential for normal physiological function. However, high salt intake may lead to vascular injury, independent of a rise in blood pressure(BP). Canonical NALP3 inflammasome activation is a caspase-1 medicated process, resulting in the secretion of IL-18 and IL-1β which lead to endothelial dysfunction. However, some researches uncovered a direct and inflammasome-independent role of NALP3 in renal injury. Thus, this study was designed to investigate the possible mechanisms of NALP3 in high salt induced endothelial dysfunction. METHODS and RESULTS Changes in endothelial function were measured by investigating mice(C57 BL/6 J, NALP3~(-/-)and wild-type, WT)fed with normal salt diet(NSD) or high-salt diet(HSD) for12 weeks, and thoracic aortic rings from C57 BL/6 J mice cultured in high-salt medium. Changes of tube formation ability, intracellular reactive oxygen species(ROS), and NALP3 inflammasome expression were detected using mouse aortic endothelial cells(MAECs) cultured in highsalt medium. Consumption of HSD for 12 weeks did not affect BP or body weight in C57 BL/6 J mice. Endotheliumdependent relaxation(EDR) decreased significantly in C57 BL/6 J mice fed with HSD for 12 weeks, and in isolated thoracic aortic rings cultured in high-salt medium for 24 h.RESULTS from the aortic ring assay also revealed that the angiogenic function of thoracic aortas was impaired by either consumption of HSD or exposure to high-salt medium. NALP3~(-/-)mice fed with HSD showed a relatively mild decrease in EDR function when compared with WT mice. Tube length of thoracic aortic rings from NALP3~(-/-) mice was longer than those from WT mice after receiving high-salt treatment. Inhibiting NALP3 with a NALP3 antagonist, smal interfering(si) RNA experiments using si-RNALP3,and decomposing ROS significantly improved tube formation ability in MAECs under high salt medium. NALP3 expression was increased in MAECs cultured with high salt treatment and inhibiting NALP3 reversed the downregulation of p-e NOS induced by high salt in MAECs.CONCLUSION High salt intake impairs endothelial function, which is at least in part mediated by increasing NALP3 expression.     

Long term Rho-kinase inhibition ameliorates endothelial dysfunction in LDL-Receptor deficient mice

Chronic inhibition of Rho-kinase has been recently implicated in retardation of atherogenesis induced by high-fat diet in low-density lipoprotein receptor deficient (LDLR-/-) mice. However, it remains to be examined whether long-term Rho-kinase inhibition will reduce vascular dysfunction in this model. LDLR-/- mice on a high-fat diet were treated either with saline (LDLR-/-) or with the Rho-kinase inhibitor Fasudil (HA1077, 5-Isoquinolinesulfonyl homopiperazine, 100 mg/kg/day by gavage, LDLR-/- +Fasudil) for 10 weeks. Fasudil-treatment normalized endothelial function (measured by means of endothelium-dependent vasorelaxation) in LDLR-/- +Fasudil, to the level of controls (C57BL/6J). No tolerance toward Rho-kinase inhibition has been detected in Fasudil-treated animals. We conclude that long-term Rho-kinase inhibition normalizes endothelial function without development of tolerance.     

Rutaecarpine inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons

Our previous studies showed that rutaecarpine (Rut) protected against myocardial ischemia/reperfusion (I/R) injury, which was associated with activation of transient receptor potential vanilloid subtype 1 (TRPV1). Recently, TRPV1 activation was also reported to exert neuroprotective effects. The present study was to investigate the effect of Rut on hypoxia/reoxygenation (H/R)-induced apoptosis in primary rat hippocampal neurons. Three-hour hypoxia (1% O2) and consequent 24-h reoxygenation significantly increased the apoptotic death of hippocampal neurons as evidenced by increases in both TUNEL-positive cell number and caspase-3 activity. However, pretreatment with Rut (1-10microM) or caspase-3 specific inhibitor DEVD-CHO could markedly attenuate H/R-induced apoptosis in neurons. Rut markedly induced the phosphorylation of Akt and PI3K inhibitor LY294002 prevented the survival effect of Rut on neurons. Intracellular oxidative stress was significantly induced after H/R, which was inhibited by Rut and LY294002 as well as antioxidant PDTC. TRPV1 antagonist capsazepine or intracellular Ca2+ chelator BAPTA/AM could abolish these effects of Rut mentioned above. In summary, the present data suggest that Rut inhibits H/R-induced apoptosis of hippocampal neurons via TRPV1-[Ca2+]i-dependent and PI3K/Akt signaling pathway, which is related to inhibiting oxidative stress and caspase-3 activation.     

Chronic decrease in flow contributes to heart failure-induced endothelial dysfunction in rats

Chronic heart failure (CHF) impairs endothelium-dependent, nitric oxide (NO)-mediated dilation. This decreased dilation may be partly secondary to the chronic decrease in blood flow, but this hypothesis has not yet been tested. Thus, we assessed whether a localized, chronic increase in blood flow in vivo reverses endothelial dysfunction of small arteries in rats with CHF. Two months after coronary artery ligation or sham surgery, second-order side branches of the superior mesenteric artery were ligated in order to obtain persistently elevated blood flow (HF) in the adjacent first-order side branch compared with normal vessels (NF). One month later, responses to acetylcholine and flow-mediated vasodilatation (FMD) were assessed in vitro in an arteriograph. Chronic heart failure induced a decrease in mesenteric blood flow (374 +/- 25 and 305 +/- 27 micro L/min for sham and CHF, respectively; P < 0.05). Neither CHF nor the chronic increase in flow affected the responses to acetylcholine. Chronic heart failure decreased FMD (maximal response in sham and control 34 +/- 6 and 13 +/- 4%, respectively; P < 0.05). Chronic increases in blood flow did not modify FMD in sham, but restored FMD in CHF rats (28 +/- 4%; P < 0.05 vs CHF NF). The restored response was abolished by an inhibitor of NO synthesis (N(G)-nitro-l-arginine). Chronic heart failure did not affect the abundance of mesenteric endothelial NO synthase (eNOS) mRNA. A chronic increase in flow significantly increased the abundance of eNOS mRNA in sham rats, but only moderately and non-significantly in CHF rats. Thus, endothelial dysfunction of small arteries in CHF appears to be largely the consequence of the chronic decrease in flow.     

Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells

AIM: Mesenchymal stem cells (MSC) are a promising candidate for cardiac replacement therapies. However, the majority of transplanted MSC are readily lost after transplantation because of poor blood supply, ischemia-reperfusion, and inflammatory factors. We aimed to study the effects of hypoxia preconditioning (HPC) on hypoxia/reoxygenation-induced apoptosis of MSC. METHODS: Three generations of MSC were divided into 6 groups, including the normal group, hypoxia-reoxygenation (H/R) group, cyclosporine A (CsA), and the HPC 10 min, 20 min, and 30 min groups. The apoptotic index, cell viability, mitochondrial membrane potential, translocation of Bcl-2 and bax, extracellular regulated kinase (ERK), Akt, hypoxia-inducible factor 1-alpha, and the vascular endothelial growth factor (VEGF) were tested after H/R treatment. RESULTS: HPC decreased the apoptotic index and increased the viability induced by H/R. Moreover, HPC markedly stabilized mitochondrial membrane potential, upregulated Bcl-2 and VEGF expressions, and increased the phosphorylation of ERK and Akt. As a positive control, CsA has the same function as HPC, except for promoting ERK and Akt phosphorylation and upregulating VEGF. CONCLUSION: HPC had a protective effect against MSC apoptosis induced by H/R via stabilizing mitochondrial membrane potential, upregulating Bcl-2 and VEGF, and promoting ERK and Akt phosphorylation. HPC has implications for the development of novel stem cell protective strategies.     

Decreased production of neuronal NOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

BACKGROUND AND PURPOSE

Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H₂O₂ is an important endothelium-derived relaxant factor in the mouse aorta. The role of H₂O₂ and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H₂O₂ contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE^−/−).

EXPERIMENTAL APPROACH

Changes in isometric tension were recorded on a myograph; simultaneously, NO and H₂O₂ were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms.

KEY RESULTS

Aortas from ApoE^−/− mice showed impaired vasodilatation paralleled by decreased NO and H₂O₂ production. Inhibition of nNOS with L-Arg^NO2-L-Dbu, knockdown of nNOS and catalase, which decomposes H₂O₂ into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H₂O₂ in wild-type animals, but had no effect in ApoE^−/− mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE^−/− mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE^−/− mice.

CONCLUSIONS AND IMPLICATIONS

Our data show that endothelial nNOS-derived H₂O₂ production is impaired and contributes to endothelial dysfunction in ApoE^−/− aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.     

Excessive stimulation of poly(ADP-ribosyl)ation contributes to endothelial dysfunction in pre-eclampsia

1. Pre-eclampsia is a serious pregnancy disorder associated with widespread activation of the maternal vascular endothelium. Recent evidence implicates a role for oxidative stress in the aetiology of this condition. 2. Reactive oxygen species, particularly superoxide anions, invokes endothelial cell activation through many pathways. Oxidant-induced cell injury triggers the activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). 3. We have studied whether the loss of endothelial function in pre-eclampsia is dependent on PARP activity. Endothelium-dependent responses of myometrial arteries were tested following exposure to either plasma from women with pre-eclampsia or normal pregnant women in the presence and absence of a novel potent inhibitor of PARP, PJ34. Additional effects of plasma and PJ34 inhibition were identified in microvascular endothelial cell cultures. 4. In myometrial arteries, PARP inhibition blocked the attenuation of endothelium-dependent responses following exposure to plasma from women with pre-eclampsia. In endothelial cell cultures, plasma from pre-eclamptics induced measurable oxidative stress and a concomitant increase in PARP activity and reduction in cellular ATP. Again, these biochemical changes were reversed by PJ34. 5. These results suggest that PARP activity plays a pathogenic role in the development of endothelial dysfunction in pre-eclampsia and promotes PARP inhibition as a potential therapy in this condition.     

Effects of hypoxia/reoxygenation and cytokines on adhesion of leukocytes to cerebral microvascular endothelial cells 1

目的：研究缺氧／再给氧（Ｈ／Ｒ）对中性粒细胞（Ｎｅｕ）和单核细胞（Ｍｏｎ）与诱导的培养牛脑微血管细胞粘附作用的影响．方法：牛脑微血管内皮细胞（ＣＭＥＣ）和平滑肌细胞（ＣＭＳＭＣ）经Ｈ／Ｒ处理或经ＴＮＦα和ＩＬ１α刺激,Ｍｏｎ和Ｎｅｕ与ＣＭＥＣ和ＣＭＳＭＣ粘附的细胞数目用流式细胞仪测定．结果：ＣＭＥＣ先缺氧２ｈ再经复氧与ＴＮＦα（２μｇ·Ｌ－１）作用２ｈ,Ｍｏｎ和Ｎｅｕ与ＣＭＥＣ的粘附率分别提高到３５０％±０９％和３６０％±０６％（ＴＮＦα处理组分别是２８９％±１１％和２８８％±１３％）．ＣＭＳＭＣ也按上述处理,Ｍｏｎ和Ｎｅｕ的粘附率分别显著提高为４９９％±０４％和４３９％±１４％（ＴＮＦα处理组分别为３４０％±１９％和３４０％±１３％）．ＣＭＥＣ和ＣＭＳＭＣ经ＩＬ１α刺激,可得到类似结果．结论：Ｈ／Ｒ提高Ｍｏｎ和Ｎｅｕ与ＴＮＦα和ＩＬ１α诱导的ＣＭＥＣ和ＣＭＳＭＣ的粘附作用．     

缺氧／再给氧和细胞因子对白细胞与脑微血管内皮细胞粘附作用？…

目的：研究缺氧／再给氧（Ｈ／Ｒ）对中性粒细胞（Ｎｅｕ）和单核细胞（Ｍｏｎ）与诱导的培养牛脑微血管细胞粘附作用的影响。方法：牛脑微血管内皮细胞（ＣＭＦＣ）和平滑肌细胞（ＣＭＳＭＣ）经Ｈ／Ｒ处理或经ＴＮＦ－α和ＩＬ－１α刺激，Ｍｏｎ和Ｎｅｕ与ＣＭＥＣ和ＣＭＳＭＣ粘附的细胞数目用流式细胞仪测定，结果：ＣＭＥＣ先缺氧２ｈ再经复氧与ＴＮＦ－α（２μｇ．Ｌ＾－１）作用２ｈ，Ｍｏｎ和Ｎｅｕ与ＣＭＥＣ的粘附率分别     

Effect of Trolox C on hypoxia/reoxygenation-induced injury in isolated perfused rat liver

Livers isolated from 18 hours fasted rats were subjected to N(2) hypoxia (for 45 min) followed by reoxygenation (for 45 min). The perfusion medium used was Krebs-Henseleit bicarbonate buffer (KHBB, pH 7.4). Lactate and alanine were added as gluconeogenic and ureagenic substrates and Trolox C was also added to perfusate. Oxygen consumption, lactate dehydrogenase (LDH), alanine transaminase (ALT), total glutathione, oxidized glutathione, bile flow, glucose and urea were measured. After hypoxia oxygen consumption significantly dropped but Trolox C had no influence on this decrease. ALT and LDH were significantly increased by hypoxia/reoxygenation. This increase was markedly attenuated in the presence of Trolox C. The total glutathione and oxidized glutathione efflux increased following hypoxia, which were prevented by the treatment of Trolox C. Bile flow rate decreased following hypoxia/reoxygenation but did not continue to decrease in the reoxygenation phase by Trolox C. Following hypoxia/reoxygenation glucose and urea releases decreased. Trolox C had no influence on inhibition of glucose and urea production. These results suggest that Trolox C protected the liver cells against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model.     

Effect of antioxidants on hypoxia/reoxygenation-induced injury in isolated perfused rat liver.

Isolated perfused livers from rats fasted overnight were subjected to 30 min. of hypoxia followed by reoxygenation for 60 min., resulting in marked cytotoxicity as evidenced by an enhanced release of cytosolic enzymes (lactate dehydrogenase: 14-fold over controls, glutamate-pyruvate-transaminase: 12-fold over controls) and glutathione (twofold over controls) into the perfusate, by calcium accumulation (by a factor of 1.4) in the tissue and by an 80% inhibition of bile secretion. Virtually no mitochondrial injury became apparent and no evidence for lipid peroxidation could be found. In the presence of ascorbate, an augmentation of hepatic injury was observed. This might be due to the pro-oxidant activity of ascorbate in the presence of ionized iron, which is easily released from high molecular weight stores under reductive (e.g. hypoxic) conditions. The water soluble vitamin E analogue trolox C as well as propyl gallate clearly protected the liver against hypoxia/reoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model. Due to their water solubility and their high efficacy as free radical scavengers, these antioxidants might be of therapeutic value.     

Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells

17-fold) increase in DNA fragmentation. Fluorescence microscopy, using DNA binding dyes, demonstrated that cell death following hypoxia/reoxygenation was due predominantly to apoptosis and not necrosis. Furthermore, reoxygenation, but not hypoxia alone, caused a time-dependent increase in the activation of JNK as monitored by western blot analysis using a phospho-specific JNK antibody. In contrast, p38 mitogen-activated protein kinase was activated following hypoxia, but this activation was not augmented during reoxygenation. Exposure of human kidney cells to a 2'-methoxyethyl mixed backbone antisense oligonucleotide directed against human JNK1 (JNK1 AS) resulted in a potent suppression of JNK mRNA and protein expression, whereas a 6-base mismatch control oligonucleotide was without effect. Moreover, a significant diminution of reoxygenation-induced apoptosis was observed in cells exposed to JNK1 AS but not to the mismatch control oligonucleotide. Taken together, these results strongly indicate that activation of the JNK signaling cascade is a major mechanism whereby hypoxia/reoxygenation induces apoptosis.     

Pathological upregulation of galectin-3 contributes to ischemia/reperfusion-induced cardiac dysfunction

OBJECTIVE Myocardial ischemia/reperfusion(I/R) injury is caused by the restoration of the coronary blood flow following an ischemic episode. Accumulating evidence suggests that galectin-3, a β-galactosidebinding lectin, acts as a biomarker in heart disease. The aim of this study is to explore whether galectin-3 affects the susceptibility of the heart to I/R injury. METHODS Male C57 BL/6 J mice were subjected to myocardial30 min/24 h ischemia/reperfusion. Then, RNA sequencing(RNA-seq) was performed to identify the differentially expressed genes in the I/R group compared with the sham group. Echocardiography, H&E and TTC staining,TUNEL, a 12 h/1 h hypoxia/reoxygenation(H/R), mitochondrial membrane potential(MMP), Western blotting and pharmacological intervention were conducted to evaluate pathological damage or to elucidate the mechanisms of galectin-3 affects the susceptibility of the heart to I/R injury in vivo and in vitro. RESULTS RNA-seq analysis identified that Lgals3(galecin-3) plays an indispensable role in IR-induced cardiac damage. Immunostaining and immunoblot assays confirmed that the expression of galectin-3 was markedly increased in myocardial I/R injury both in vivo and in vitro. Echocardiographic analysis showed that cardiac dysfunction in experimental I/R injury was significantly attenuated by galectin-3 inhibitors including pectin(1%, ip) from citrus and binding peptide G3-C12(5.0 mg·kg-1, ip). Galectin-3 inhibitor-treated mice exhibited smaller infarct sizes and decreased tissue injury. Furthermore, TUNEL staining showed that galectin-3 inhibition suppressed I/R-mediated cardiomyocyte apoptosis.MMP and mitochondrial permeability transition pore(m PTP) levels were well-preserved and I/R-induced changes of mitochondrial cyto c, cytosol cyto c, caspase-9,caspase-3, Bcl-2 and Bax in the galectin-3 inhibitor-treated groups were observed. CONCLUSION Our findings indicate that the pathological upregulation of galectin-3 contributes to I/R-induced cardiac dysfunction and that galectin-3 inhibition ameliorates myocardial injury, highlighting its therapeutic potential.     

The protective effect of genistein postconditioning on hypoxia/reoxygenation-induced injury in human gastric epithelial cells

Aim:

The aim of this study was to investigate the protective effect of genistein postconditioning on hypoxia/reoxygenation-induced injury in human gastric epithelial cells and to begin a tentative discussion on the mechanism behind this protection.

Methods:

A model of hypoxia/reoxygenation-induced injury was established in the human gastric epithelial cell line (GES-1). All cells in our present study were randomly divided into five groups: a normal control group (N), a hypoxia/reoxygenation group (H/R), a genistein postconditioning group (GP), a capsazepine+genistein postconditioning group (C+GP) and a DMSO vehicle postconditioning group (DM). The methods used included MTT assays to test cell viability, flow cytometric analyses to quantify the percentage of cell apoptosis, Western blot analyses to measure the protein expression of calcitonin gene-related peptide (CGRP), Bcl-2, and Bax, and immunocytochemistry assays to detect the expression of CGRP in each group.

Results:

The MTT assays indicated that the cell viabilities of the groups were 100.0%±0%, 51.4%±4.1%, 66.7%±2.0%, 56.1%±2.8%, and 50.7%±2.4%, respectively. Compared with the H/R group, the viability of the GP group was significantly increased (P<0.01). Flow cytometric analysis showed that the cell apoptosis percentage of each group was 2.28%±0.44%, 12.17%±2.15%, 5.40%±1.22%, 10.43%±1.37%, and 11.02%±2.19%, respectively. Western blot analysis demonstrated that CGRP, Bcl-2, and Bax were expressed in normal human gastric epithelial cells. Compared with the H/R group, the GP group exhibited increased expression of CGRP and Bcl-2 and decreased expression of Bax. Immunocytochemistry assays indicated that the number of CGRP-positive cells in the GP group was significantly increased.

Conclusion:

Genistein postconditioning has a protective effect on hypoxia/reoxygenation-induced injury in human gastric epithelial cells. The mechanism by which genistein exerts this protection may be via activation of cellular vanilloid receptor subtype 1, resulting in the generation of an endogenous protection substance, CGRP.     

Hydroxysafflor yellow A inhibits hypoxia/reoxygenation-induced cardiomyocyte injury via regulating the AMPK/NLRP3 inflammasome pathway

Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent that alleviates myocardial ischaemia/reperfusion injury (MIRI), but the exact mechanisms remain elusive. The aim of this study was to investigate the potential protective effect of HSYA against MIRI through mechanisms related to NLRP3 inflammasome regulation. In this study, hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes were treated with HSYA or the AMPK inhibitor, compound C (CC). Our results showed that HSYA pretreatment improved cardiomyocyte viability, maintained mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased caspase-3 activity, and inhibited NOD-like receptor 3 (NLRP3) inflammasome activation during H/R injury. Moreover, the inhibition of AMPK activation by the CC inhibitor partially abolished the effects of HSYA treatment, including suppressing the upregulation of NLRP3 inflammasome components (NLRP3, caspase-1 and interleukin-1β) and promoting autophagy (LC3-II/LC3-I and p62). In conclusion, the protective mechanism of HSYA in H/R-induced cardiomyocyte injury is associated with inhibiting NLRP3 inflammasome activation through the AMPK signalling pathway.     

内质网应激相关蛋白1过表达对缺氧/复氧诱导的H9c2心肌细胞凋亡的影响

目的 研究内质网应激相关蛋白1(SERP1)过表达对缺氧/复氧(H/R)诱导的大鼠胚胎H9c2心肌细胞凋亡的影响.方法 2018年1—12月将中国科学院上海细胞库大鼠H9c2心肌细胞系建立H/R损伤大鼠心肌细胞模型,采用pCMV6质粒为载体构建内质pCMV6-网应激相关蛋白1(pCMV6-SERP1)过表达重组质粒,分...     

The protective effect of genistein postconditioning on hypoxia/ reoxygenation-induced injury in human gastric epithelial cells

Aim： The aim of this study was to investigate the protective effect of genistein postconditioning on hypoxia/reoxygenation- induced injury in human gastric epithelial cells and to begin a tentative discussion on the mechanism behind this protection. Methods： A model of hypoxia/reoxygenation-induced injury was established in the human gastric epithelial cell line （GES-1）. All cells in our present study were randomly divided into five groups： a normal control group （N）, a hypoxia/ reoxygenation group （H/R）, a genistein postconditioning group （GP）, a capsazepine＋genistein postconditioning group （C＋GP） and a DMSO vehicle postconditioning group （DM）. The methods used included MTT assays to test cell viability, flow cytometric analyses to quantify the percentage of cell apoptosis, Western blot analyses to measure the protein expression of calcitonin gene-related peptide （CGRP）, Bcl-2, and Bax, and immunocytochemistry assays to detect the expression of CGRP in each group. Results： The MTT assays indicated that the cell viabilities of the groups were 100.0%±0%, 51.4%±4.1%, 66.7%±2.0%, 56.1%±2.8%, and 50.7%±2.4%, respectively. Compared with the H/R group, the viability of the GP group was significantly increased （P〈0.01）. Flow cytometric analysis showed that the cell apoptosis percentage of each group was 2.28%±0.44%, 12.17%±2.15%, 5.40%±1.22%, 10.43%±1.37%, and 11.02%±2.19%, respectively. Western blot analysis demonstrated that CGRP, Bcl-2, and Bax were expressed in normal human gastric epithelial cells. Compared with the H/R group, the GP group exhibited increased expression of CGRP and Bcl-2 and decreased expression of Bax. Immunocytochemistry assays indicated that the number of CGRP-positive cells in the GP group was significantly increased. Conclusion： Genistein postconditioning has a protective effect on hypoxia/reoxygenation-induced injury in human gastric epithelial ceils. The mechanism by which genistein exerts this protection may be via activation of cell