Necroptosis mediates chemical hypoxia-induced injury andinflammation in HT22 hippocampal cells / 中国药理学通报

Aim To investigate whether necroptosis mediates chemical hypoxia-induced HT22 mouse hippocampal cell injury and inflammation.Methods HT22 hippocampal cells were exposed to cobalt chloride (CoCl2) to establish a model of the chemical hypoxia-induced injury and inflammation.The expression level of RIP3 (an index of necroptosis) was determined by Western blot.Cell counter kit-8 (CCK-8) assay was used to test the cell viability.Lactate dehydrogenase (LDH) activity in the culture medium was measured with commercial kits.Mitochondrial membrane potential (MMP) was examined by rhodamine123 staining followed by photofluorography.The intracellular level of reactive oxygen species (ROS) was detected by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) staining followed by photofluorography.The secretion levels of interleukin-1β (IL-1β) and tumor necrosis factor-a (TNF-α) were measured by ELISA.Results Treatment of HT22 hippocampal cells with 600 μmol·L-1 CoCl2 for 36 h markedly induced cytotoxicity, leading to a decrease in cell viability to (52.0±2.65) % , indicating that chemical hypoxia-induced cellular injury model was successfully set up.Besides, CoCl2 induced considerable injuries and inflammation, evidenced by increases in LDH activity, ROS production, MMP loss, as well as the secretion levels of IL-1β and TNF-α.Co-treatment of the cells with 40~100 μmol·L-1 Nec-1 (a specific inhibitor of necroptosis) and CoCl2 markedly attenuated the decrease in viability induced by CoCl2, reaching the best anti-cytotoxicity inhibitory effect at 80 μmol·L-1.Meanwhile, the co-treatment with 80 μmol·L-1 Nec-1 blocked the above injuries and inflammatory response induced by CoCl2.In addition, treatment of HT22 hippocampal cells for 6~48 h up-regulated the expression of RIP3, and Nec-1 alleviated the up-regulation of RIP3 expression level induced by CoCl2.Conclusion Necroptosis mediates chemical hypoxia-induced HT22 hippocampal cell injury and inflammation.

Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model

BACKGROUND: Aged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells. RESULTS: We found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC. CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions

Role of heat shock protein 90 in protective effect of hydrogen sulfide against PC12 cells injuries induced by chemical hypoxia / 中国药理学通报

Aim To explore the role of heat shock protein 90(Hsp90)in hydrogen sulfide-induced protection against PC12 cells injuries elicited by cobalt chloride(CoCl_2).Methods Hydrogen sulfide preconditioning against CoCl_2-induced injury model was set up in PC12 cells.Cell viability was tested by using cell counter kit-8.Morphological changes in apoptotic PC12 cells were detected by Hoechst 33258 staining and photofluorography.Apoptotic rate was evaluated by propidium iodide(PI)staining and flow cytometry(FCM).The expression of Hsp90 was evaluated by Western blot.Results Hsp90 expression was upregulated after treatment with 400 μmol·L~(-1) sodium hydrosulfide(a H_2S donor),peaking at 3 h,returning to the basal level at 24 h.Furthermore,H_2S preconditioning obviously enhanced the upregulation of Hsp90 expression induced by CoCl_2.H_2S preconditioning markedly protected PC12 cells against injuries induced by CoCl_2,increasing the viability of cells and decreasing the percentage of apoptotic cells.17-allylamino-17-demethoxygeldanamycin(17-AAG),an inhibitor of Hsp90,antagonized H_2S preconditioning-induced Hsp90 activation and the adaptive cytoprotection.Conclusion H_2S can protect PC12 cells against CoCl_2-induced injuries,and upregulating the expression of Hsp90 may be one of the mechanisms underlying cytoprotection induced by H_2S preconditioning.

Role of survivin in PC12 cells against injuries induced by chemical hypoxia / 中国药理学通报

Aim To explore the effect of survivin in PC12 cells against injuries induced by cobalt chloride(CoCl_2).Methods PC12 cells were exposed to CoCl_2 at different doses in different time to set up the chemical hypoxia induced PC12 cells injuries model.Cell viability was tested by using cell counter kit-8.Dose-effect(200～1 000 μmol·L~(-1))and time-effect(0～48 h)relationship between hypoxia induced by CoCl_2 and the expression of survivin was evaluated by western blot.Results PC12 cells viability was inhibited significantly by CoCl_2 in a dose and time dependent manners;At the concentrations from 200 to 600 μmol·L~(-1) CoCl_2 for 24 h,survivin expression was upregulated in a dose dependent manner,peaking at 600 μmol·L~(-1) CoCl_2 treatment,exceeding this concentration of CoCl_2,with dose increasing,survivin expression decreased.At the dose of CoCl_2 up to 1 000 μmol·L~(-1),survivin did not express basically;Treatment with 600 μmol·L~(-1) CoCl_2 in different time,within the range of 0～36 h,the expression of survivin enhanced in time dependent manner.But with the extension of time,survivin expression was declining; 17-allylamino-17-demethoxygeldanamycin (2 μmol·L~(-1)),an inhibitor of Hsp90,not only decreased survivin overexpression but also obviously enhanced the injuries of PC12 cells induced by CoCl_2,which didn't damage PC12 cells alone.Conclusion Upregulation of survivin expression may be one of the endogenous defense mechanisms for PC12 cells against chemical hypoxia.

Vascular Response of Isolated Rat Thoracic Aorta after Chemical Hypoxia/Reoxygenation / 대한마취과학회지

BACKGROUND: Hypoxia/reoxygenation (H/R) results in formation of toxic reactive oxygen species (ROS), which can impair the vascular pathophysiology. Nitric oxide (NO) is an important free radical in many physiological or pathological processes including H/R injury. The loss of NO after H/R might be one of the major causes of an impaired vascular response. METHODS: Isolated rat aortic rings were prepared and NaCN was used to induce chemical hypoxia. The NaCN concentration and the hypoxia/reoxygenation time were determined by the responsiveness of phenylephrine (Phe), sodium nitroprusside (SNP) and acetylcholine (Ach). A cumulative doses of Phe and SNP (10(-9)-10(-5.5) M) were added to construct the vascular contraction and relaxation curves. The cumulative doses of Ach (10(-9)-10(-5) M) were added to construct the relaxation after precontraction with Phe (10(-6) M). The effects of the N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and the superoxide dismutase (SOD, 50 unit) pretreatment during chemical H/R were evaluated. RESULTS: The NaCN concentration and H/R time were 1 mM, 30 minutes/5 minutes, respectively. Chemical hypoxia reduced the Phe-induced vascular contraction significantly. However chemical H/R increased the Phe-induced contraction significantly, and impaired the relaxation by SNP and Ach. A pretreatment with L-NAME increased the Phe-induced contraction and impaired the relaxation by SNP as well as Ach. The SOD pretreatment reduced the Phe-induced increased vascular contraction after NaCN-induced chemical H/R. CONCLUSIONS: NO plays a key role in endothelial-dependent relaxation and the recovery of the augmented contractility by vasoconstrictors after chemically-induced H/R.

Roles of hypoxia regulated genes/protein in neuroprotection against hypoxia injury in differentiated SH-SY5Y cells preconditioned with CoCl_2 / 第三军医大学学报

Objective To investigate the roles of hypoxia regulated genes/protein in chemical hypoxia preconditioning in differentiated SH-SY5Y cells.Methods Differentiated SH-SY5Y cells were randomly divided into control group,chemical hypoxic preconditioning group(50 ?mol/L CoCl2 preconditioning for 3 h,normal culture for 1 h,then in 2%O2 for 28 h) and hypoxia group(in 2%O2 for 28 h).RT-PCR was used to examine the mRNA expressions of VEGF,GLUT-1,EPO,LDH-A.Further evaluation of the potential neuroprotective effect of VEGF was done by investigating the effect of recombinant human VEGF on subsequent hypoxia injury.Results The mRNA levels of GLUT-1,EPO increased in the preconditioning group as compared with those in the hypoxia group(P

Regulation of magnesium release by cAMP during chemical hypoxia in the rat heart and isolated ventricular myocytes

Chemically induced hypoxia has been shown to induce a depletion of ATP. Since intracellular free Mg2+ ((Mg2+)i) appears to be tightly regulated following cellular energy depletion, we hypothesized that the increase in (Mg2+)i would result in Mg2+ extrusion following hormonal stimulation. To determine the relation between Mg2+ efflux and cellular energy state in a hypoxic rat heart and isolated myocytes, (Mg2+)i, ATP and Mg2+ content were measured by using mag-fura-2, luciferin-luciferase and atomic absorbance spectrophotometry. Mg2+ effluxes were stimulated by norepinephrine (NE) or cAMP analogues, respectively. Mg2+ effluxes induced by NE or cAMP were more stimulated in the presence of metabolic inhibitors (MI). Chemical hypoxia with NaCN (2 mM) caused a rapid decrease of cellular ATP within 1 min. Measurement of (Mg2+)i confirmed that ATP depletion was accompanied by an increase in (Mg2+)i. No change in Mg2+ efflux was observed when cells were incubated with MI. In the presence of MI, the cAMP-induced Mg2+ effluxes were inhibited by quinidine, imipramine, and removal of extracellular Na+. In addition, after several min of perfusion with Na+-free buffer, a large increase in Mg2+ efflux occurred when Na+-free buffer was switched to 120 mM Na+ containing buffer. A similar Mg2+ efflux was observed in myocytes. These effluxes were inhibited by quinidine and imipramine. These results indicate that the activation of Mg2+ effluxes by hormonal stimulation is directly dependent on intracellular Mg2+ contents and that these Mg2+ effluxes appear to occur through the Na+-dependent Na+/Mg2+ exchange system during chemical hypoxia.

Study on chemical hypoxia-mimetic (CoCl_2) agent-induced inflammatory reaction in human keratinocytes / 中国药理学通报

Aim To explore the effect of chemical hypoxia-mimetic agent,cobalt chloride(CoCl2)on inflammatory reaction in human keratinocytes(HaCat cells).Methods After HaCat cells were treated with CoCl2 at different concentrations to set up a chemical hypoxia-induced cell model of skin injury,cell viability,intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP),the levels of both interleukin 6(IL-6)and interleukin 8(IL-8)as well as the expression of heme oxygenase-1(HO-1)were detected.Results The viability of HaCat cells was reduced by CoCl2 at the concentrations from 500 to 3 000 ?mol?L-1,and the higher CoCl2 doses,the lower cell viability was.CoCl2 induced oxidative stress reaction(increasing ROS production and decreasing MMP).CoCl2 induced inflammatory reaction,enhancing the release of IL-6 and IL-8.CoCl2 at concentrations from 1 000 to 3 000 ?mol?L-1 upregulated HO-1 expression in HaCat cells.Conclusion CoCl2 induces not only oxidative stress,but also inflammatory reaction,increasing the release of both IL-6 and IL-8,as well as HO-1 expression.