Taurine protects R28 cells from hypoxia/re-oxygenation-induced damage via regulation of mitochondrial energy metabolism.

Oxidative-induced damage and hypoxia/re-oxygenation (H/R) injury are common causes of irreversible visual impairment. The goals of this study were to explore the effects of taurine on R28 cells under the two damage models and the underlying mechanisms. Low doses of taurine supplementation promoted cell viability, mitochondrial membrane potential (MMP), SOD levels, ATP contents and attenuated cytotoxicity and intracellular ROS generation of the R28 cells under the two kinds of damage. The expression level of GTPBP3, a mitochondrial-tRNA (mt-tRNA) modification enzyme that catalyzes the taurine involved modification, was decreased under the two damage and taurine could reverse the reduction. After knocking down GTPBP3, the R28 cells become vulnerable to damage. The viability, cytotoxicity, MMP and intracellular ROS level of knockdown cells changed more obviously under the H/R injury than those of control cell. We also found that knockdown of GTPBP3 significantly decreased mitochondrial energy metabolism by measuring the oxidative respiration rate by the Seahorse XFe24 extracellular flux analyzer. The protection of low doses of taurine disappeared on knockdown R28 cells, indicating that GTPBP3 is crucial in the protection mechanisms of taurine. However, the impacts of the reduction of GTPBP3 level can be reversed by relatively high doses of taurine, implying the protection effects of taurine were dose-dependent, and there were more complicated mechanisms remain to be explored. This study explored a new mechanism of the neuroprotective effects of taurine, which depend on the GTPBP3-mediated taurine modification of mt-tRNAs and the promotion of mitochondrial energy metabolism.

TDCPP protects cardiomyocytes from hypoxia-reoxygenation injury induced apoptosis through mitigating calcium overload and promotion GSK-3ß phosphorylation.

TDCPP, Tris (1, 3-dichloro-2-propyl) phosphate belongs to a group of chemicals known as triester organophosphate flame retardants, It can alter calcium homeostasis at much lower concentrations in normal conditions, but the mechanism is unclear till now. Calcium overload is a leading cause of apoptosis in myocardial ischemia/reperfusion (I/R) injury, thus how to mitigate Ca2+-overload is deserved to be investigated. We therefore hypothesized that TDCPP could attenuate cardiomyocytes apoptosis in I/R injury. H/R (hypoxia/reoxygenation) experiments in vitro were used to simulate in vivo I/R injury. The present study aimed to explore the potential effect of TDCPP in cardiomyocytes after H/R injury, Ca2+ imaging technique was used to explore SOCE(store-operated calcium entry) and Ca2+-overload levels, western blot technique was used to explore the potential target, the cell morphology, cell viability and mitochondrial membrane potential were also detected. The results have shown that: TDCPP could decrease SOCE, restore H9c2 cell viability, mitigate Ca2+-overload in H/R injury and reduce the mitochondrial membrane potential. Furthermore, TDCPP decreased STIM1 expression and promoted GSK3ß phosphorylation. Collectively, for the first time, this study suggest the antiapoptosis roles of TDCPP in H/R injury are via mitigation Ca2+-overload and promoting GSK-3ß phosphorylation.

Effect of erythropoietin on the migration of bone marrow-derived mesenchymal stem cells to the acute kidney injury microenvironment.

Bone marrow-derived mesenchymal stem cells (BMSCs) preferentially migrate to the injured tissue but with limited efficiency. Here we investigated the effect of erythropoietin (EPO) treatment on the BMSC migration to the acute kidney injury (AKI) microenvironment. The possible mechanisms were also discussed. A hypoxia/re-oxygenation (HR) model of renal tubular epithelial cells (RTECs) was established to generate AKI in vitro, and a chemotaxis experiment was conducted using the transwell chamber. EPO treatment enhanced the BMSC migration to the HR-RTEC culturing chamber in a SDF-1 level-dependent manner, which was fully inhibited by the treatment of anti-SDF-1 antibody. The BMSC migration could also be partly blocked by LY294002 (phosphoinositide 3-kinase (PI3K) inhibitor) and PD98059 (MAPK inhibitor). Western blot analysis showed that phosphorylated Akt and phosphorylated MAPK in BMSCs were enhanced by EPO treatment. In the in vivo experiment, BMSCs were transplanted into the AKI mice and EPO was subcutaneously injected. The results showed that EPO injection increased the SDF-1 protein expression and BMSC accumulation in the renal tissue, which was consistent with a decent improvement of renal function. In addition, the BMSC accumulation in the renal tissue was blocked by anti-SDF-1 antibody, LY294002 or PD98059. Our data suggest that AKI microenvironment had a directional chemotactic effect on BMSCs, which could be further enhanced by the EPO treatment. The increased SDF-1 level in the AKI microenvironment and the activations of PI3K/AKT and MAPK in BMSCs were the possible mechanisms for the effect of EPO. Therefore, BMSC transplantation combined with EPO injection can be a novel and effective approach for AKI repair.

Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney.

Bone marrow-derived mesenchymal stem cells (BMSCs) can migrate to the injured kidney after acute kidney injury (AKI) with limited efficiency. This study investigated the effect of CXCR4 overexpression on BMSC migration to the AKI kidney and the possible mechanisms. CXCR4 gene-modified BMSCs (CXCR4-BMSCs) and null-BMSCs were prepared and transplanted into the AKI mice. Blood indicators, histology, expression of stromal cell-derived factor 1 (SDF-1), and BMSC migration were investigated. Hypoxia/re-oxygenation-pretreated renal tubular epithelial cells (HR-RTECs) were prepared to generate AKI in vitro. The chemotaxis experiment was performed using the transwell chamber. The phosphorylation of AKT and MAPK in the BMSCs was also investigated. The CXCR4-BMSCs showed a remarkable expression of CXCR4. The SDF-1 expression in the AKI renal tissue was increased. CXCR4-BMSCs transplantation sharply increased the accumulation of BMSCs in the renal tissue, which was consistent with a greater improvement of renal function. The in vitro experiments showed that the migration of BMSCs to the HR-RTEC culturing chamber was CXCR4-dependent, and could be fully inhibited by AMD3100, a CXCR4-specific antagonist. The migration could also be partly blocked by either LY294002 (PI3K inhibitor) or PD98059 (MAPK inhibitor). Phosphorylated Akt and MAPK were increased in the BMSCs co-cultured with HR-RTECs and their expression was the highest in the CXCR4-BMSCs, which could be recovered by AMD3100. Overexpression of CXCR4 gene could enhance BMSC migration to the kidney area after AKI. The SDF-1/CXCR4 axis via its activation of PI3K/AKT and MAPK in BMSCs could be the possible mechanisms underlying this function.

Up-regulation of CTRP12 ameliorates hypoxia/re-oxygenation-induced cardiomyocyte injury by inhibiting apoptosis, oxidative stress, and inflammation via the enhancement of Nrf2 signaling.

C1q/TNF-related protein 12 (CTRP12) has been reported to play a key role in coronary artery disease. However, whether CTRP12 plays a role in the regulation of myocardial ischemia-reperfusion injury is not fully understood. The goals of this work were to assess the possible relationship between CTRP12 and myocardial ischemia-reperfusion injury. Here, we exposed cardiomyocytes to hypoxia/re-oxygenation (H/R) to establish an in vitro cardiomyocyte injury model of myocardial ischemia-reperfusion injury. Our results showed that H/R treatment resulted in a decrease in CTRP12 expression in cardiomyocytes. The up-regulation of CTRP12 ameliorated H/R-induced cardiomyocyte injury via the down-regulation of apoptosis, oxidative stress, and inflammation. In contrast, the knockdown of CTRP12 enhanced cardiomyocyte sensitivity to H/R-induced cardiomyocyte injury. Further investigation showed that CTRP12 enhanced the levels of nuclear Nrf2 and increased the expression of Nrf2 target genes in cardiomyocytes exposed to H/R. However, the inhibition of Nrf2 markedly diminished CTRP12-overexpression-mediated cardioprotective effects against H/R injury. Overall, these data indicate that CTRP12 protects against H/R-induced cardiomyocyte injury by inhibiting apoptosis, oxidative stress, and inflammation via the enhancement of Nrf2 signaling. This work suggests a potential role of CTRP12 in myocardial ischemia-reperfusion injury and proposes it as an attractive target for cardioprotection.

Comparative transcriptomic analysis of vascular endothelial cells after hypoxia/re-oxygenation induction based on microarray technology.

OBJECTIVE: To provide comprehensive data to understand mechanisms of vascular endothelial cell (VEC) response to hypoxia/re-oxygenation. METHODS: Human umbilical vein endothelial cells (HUVECs) were employed to construct hypoxia/re-oxygenation-induced VEC transcriptome profiling. Cells incubated under 5% O2, 5% CO2, and 90% N2 for 3 h followed by 95% air and 5% CO2 for 1 h were used in the hypoxia/re-oxygenation group. Those incubated only under 95% air and 5% CO2 were used in the normoxia control group. RESULTS: By using a well-established microarray chip consisting of 58 339 probes, the study identified 372 differentially expressed genes. While part of the genes are known to be VEC hypoxia/re-oxygenation-related, serving as a good control, a large number of genes related to VEC hypoxia/re-oxygenation were identified for the first time. Through bioinformatic analysis of these genes, we identified that multiple pathways were involved in the reaction. Subsequently, we applied real-time polymerase chain reaction (PCR) and western blot techniques to validate the microarray data. It was found that the expression of apoptosis-related proteins, like pleckstrin homology-like domain family A member 1 (PHLDA1), was also consistently up-regulated in the hypoxia/re-oxygenation group. STRING analysis found that significantly differentially expressed genes SLC38A3, SLC5A5, Lnc-SLC36A4-1, and Lnc-PLEKHJ1-1 may have physical or/and functional protein-protein interactions with PHLDA1. CONCLUSIONS: The data from this study have built a foundation to develop many hypotheses to further explore the hypoxia/re-oxygenation mechanisms, an area with great clinical significance for multiple diseases.

Reactive oxygen species and cancer: A complex interaction.

Elevated levels of Reactive Oxygen Species (ROS), increased antioxidant ability and the maintenance of redox homeostasis can cumulatively contribute to tumor progression and metastasis. The sources and the role of ROS in a heterogeneous tumor microenvironment can vary at different stages of tumor: initiation, development, and progression, thus making it a complex subject. In this review, we have summarized the sources of ROS generation in cancer cells, its role in the tumor microenvironment, the possible functions of ROS and its important scavenger systems in tumor progression with special emphasis on solid tumors.

Development of an In Vitro Cardiac Ischemic Model Using Primary Human Cardiomyocytes.

Developing experimental models to study ischemic heart disease is necessary for understanding of biological mechanisms to improve the therapeutic approaches for restoring cardiomyocytes function following injury. The aim of this study was to develop an in vitro hypoxic/re-oxygenation model of ischemia using primary human cardiomyocytes (HCM) and define subsequent cytotoxic effects. HCM were cultured in serum and glucose free medium in hypoxic condition with 1% O2 ranging from 30 min to 12 h. The optimal hypoxic exposure time was determined using Hypoxia Inducible Factor 1α (HIF-1α) as the hypoxic marker. Subsequently, the cells were moved to normoxic condition for 3, 6 and 9 h to replicate the re-oxygenation phase. Optimal period of hypoxic/re-oxygenation was determined based on 50% mitochondrial injury via 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay and cytotoxicity via lactate dehydrogenase (LDH) assay. It was found that the number of cells expressing HIF-1α increased with hypoxic time and 3 h was sufficient to stimulate the expression of this marker in all the cells. Upon re-oxygenation, mitochondrial activity reduced significantly whereas the cytotoxicity increased significantly with time. Six hours of re-oxygenation was optimal to induce reversible cell injury. The injury became irreversible after 9 h as indicated by > 60% LDH leakage compared to the control group cultured in normal condition. Under optimized hypoxic reoxygenation experimental conditions, mesenchymal stem cells formed nanotube with ischemic HCM and facilitated transfer of mitochondria suggesting the feasibility of using this as a model system to study molecular mechanisms of myocardial injury and rescue.

Antioxidant activities of aqueous extracts from 12 Chinese edible flowers in vitro and in vivo.

The antioxidant function of edible flowers have attracted increasing interest. However, information is lacking on the impact of edible flowers on oxidative injury including hypoxia-re-oxygenation and hyperlipidemia. The antioxidant activities of aqueous extracts from 12 Chinese edible flowers were assessed in four different antioxidant models, including total antioxidant capacity (TAC), oxygen radical absorbance capacity (ORAC), scavenging hydroxyl radical capacity (SHRC) and scavenging superoxide anion radical capacity (SSARC). Subsequently, the potential antioxidant effects on rat cardiac microvascular endothelial cells (rCMEC) treated with hypoxia-re-oxygenation and hyperlipidemia rats induced by high-fat diet were also evaluated. The highest TAC, ORAC, SHRC and SSARC were Lonicera japonica Thunb., Rosa rugosa Thunb., Chrysanthemum indicum L. and Rosa rugosa Thunb., respectively. Most aqueous extracts of edible flowers exhibited good antioxidant effects on injury of rCMEC induced by hypoxia-re-oxygenation. In addition, the aqueous extracts of Lonicera japonica Thunb., Carthamus tinctorius L., Magnolia officinalis Rehd. et Wils., Rosmarinus officinalis L. and Chrysanthemum morifolium Ramat. could suppress the build-up of oxidative stress by increasing serum superoxide dismutase, glutathion peroxidase, and reducing malonaldehyde concentration in hyperlipidemia rats. These findings provided scientific support for screening edible flowers as natural antioxidants and preventative treatments for oxidative stress-related diseases.

Effects of calcium-sensing receptors on apoptosis in rat hippocampus during hypoxia/re-oxygenation through the ERK1/2 pathway.

OBJECTIVES: To explore the effects of calcium-sensing receptors (CaSR) on apoptosis in rat hippocampus during hypoxia/re-oxygenation (H/R). METHODS: After post-culturing of isolated rat hippocampus, the cultures were subjected to H/R, meanwhile gadolinium chloride (GdCl3, agonist of CaSR) and NPS 2390 (antagonists of CaSR) was added to reperfusion solution. The number of hippocampal neuron, cell proliferation assay and apoptosis rate was determined by inverted microscope, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometer (FCM). Besides, caspase-3, Bax, cytochrome C (Cyt-c), extracellular signal-regulated protein kinase (ERK) 1/2, pERK1/2, P38 and pP38 were analyzed by western blotting. RESULTS: The hippocampal neuron number and cell viability were significantly decreased after H/R treatment, and were further significantly reduced when co-treatment with CaSR agonist GdCl3. But the effects of GdCl3 were attenuated by NPS-2390. Whereas, apoptosis rate, the expression level of caspase-3, Bax and Cyt-c were all significantly increased under H/R condition, and was further significantly increased by GdCl3, but were reversed by NPS-2390 (P < 0.05). Moreover, there were no significant differences in expression of ERK1/2, P38 and pP38 among different groups. However, the expression of pERK1/2 was significantly increased after H/R treatment, but was significantly reduced by NPS 2390 (P < 0.05). CONCLUSION: The results suggest that CaSR might play significant roles in the induction of hippocampus apoptosis in rat during H/R through phosphorylation of ERK1/2.

Noradrenaline enhances angiotensin II responses via p38 MAPK activation after hypoxia/re-oxygenation in renal interlobar arteries.

AIM: Hypoxia and sympathetic activation are main factors in the pathogenesis of acute kidney injury (AKI). We tested the hypothesis that noradrenaline (NE) in combination with hypoxia aggravates the vasoreactivity of renal arteries after hypoxia/re-oxygenation (H/R). We tested the role of adrenergic receptors and p38 MAPK using an in vitro H/R protocol. METHODS: Mouse interlobar arteries (ILA) and afferent arterioles (AA) were investigated under isometric and isotonic conditions respectively. The in vitro protocol consisted of 60-min hypoxia and control condition, respectively, 10-min re-oxygenation followed by concentration-response curves for Ang II or endothelin. RESULTS: Hypoxia reduced the response to Ang II. Hypoxia and NE (10(-9)  mol L(-1) ) together increased it in ILA and AA. In ILA, NE alone influenced neither Ang II responses under control conditions nor endothelin responses after hypoxia. Prazosin or yohimbine treatment did not significantly influence the NE+hypoxia effect. The combination of prazosin and yohimbine or propranolol alone inhibited the effect of NE+hypoxia. BRL37344 (ß3 receptor agonist) mimicked the NE effect. In contrast, the incubation with ß3 receptor blocker did not influence the mentioned effect. Phosphorylation of p38 MAPK and MLC(20) was increased after H/R with NE and Ang II treatment. The selective p38 MAPK inhibitor SB202190 blocked the NE+hypoxia effect on the Ang II response. CONCLUSION: The results suggest an interaction of NE and hypoxia in enhancing vasoreactivity, which may be important for the pathogenesis of AKI. The effect of NE+hypoxia in ILA is mediated by several adrenergic receptors and requires the p38 MAPK activation.