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Aim To research dynamically the changes of endogenous cystathionine- β-synthase/hydrogen sul-fide system in PC12 cells injury induced by rotenone. Methods Rotenone-induced injury in PC12 cells ( characteristic of dopaminergic neurons) was used as a PD cell model. The expression of CBS was evaluated by Western blot. Intracellular CBS activity and H2 S production were detected by Methylene blue spectro-phot-ometric method. The viability of PC12 cells was measured by CCK-8 assay. GSH detection kit was used to detect the intracellular GSH content. Results In the groups of 6 and 12 hours, the expression and activ-ity of CBS were elevated, and the production of H2 S was increased. In the groups of 24 and 48 hours, CBS expression and activity were significantly decreased, and the amount of H2 S was significantly reduced. Ap-plication of 1. 5 μmol·L-1 rotenone for different time (6-48h) could decrease the cell viability and intra-cellular GSH contents in a time-dependent manner. Conclusions The expression and activity of endoge-nous CBS, stimulated by rotenone, are elevated firstly and then decreased. The generation of H2 S, stimulated by rotenone, is increased and then reduced significant-ly, which may be related to PC12 cells against oxida-tive stress damage induced by rotenone.
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Objective To estimate the influences of N-acetyl cysteine (NAC) on a chemical hypoxiamimetic agent CoCl2 induced-injury to, and expressions of inflammatory factors by, an immortal human skin keratinocyte line HaCaT. Methods HaCaT cells were treated with CoCl2 of 2000 μmol/L for 4 hours to set up a chemical hypoxia-induced cell model of skin injury. NAC of various concentrations ( 1000, 2000, 3000 μmol/L)was used to pretreat HaCaT cells for 2 hours prior to the establishment of cell model. After these treatments,cell viability was detected by cell counting kit 8 (CCK-8), the levels of interleukin 6 and 8 (IL-6 and -8) and tumor necrosis factor α (TNF-α) in culture supernatant by ELISA kits, mitochondrial membrane potential (MMP) by rhodamine 123 (Rh123) staining and photofluorography, intracellular reduced glutathione (GSH)content by glutathione detection kit. Results An obvious decline was observed in HaCaT cell viability after pretreatment with various concentrations of NAC for 2 hours. The treatment with CoCl2 of 2000 μmol/L for 4 hours induced an elevation in the supernatant levels of IL-6, IL-8 and TNF-α and a decrease in GSH content and MMP, while the pretreatment with NAC for 2 hours retarded the CoCl2-induced increase in IL-6 and IL-8 levels as well as decrease in GSH content and MMP. Conclusion The reactive oxygen species (ROS) scavenger NAC can protect against CoCl2-induced injury to and inflammatory reaction in HaCaT cells, which may be associated with a decrement in oxidative stress.
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[Objective] To explore the cytoprotecfion of hydrogen sulfide (H_2S) against cobalt chloride (CoCl_2)-induced apeptosis in PC12 cells and the underlying mechanisms. [Methods] CoCl_2 (a chemical hypoxia-mimetic agent) was used to establish the chemical hypoxia-induced PC12 cell injuries model. Sodium hydrosulfide (NaHS) was used as a H_2S donor. The viability of PC12 cells was measured by CCK-8 assay. The percentage of apeptotic cells was assessed by propidium iodide stain flow cytometry (FCM). The morphological change of apeptotic cells was tested by using the chromatin dye Hoechst 33258. The mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 staining and photofluorography. The level of reactive oxygen species (ROS) in PC12 cells was measured by DCFH-DA staining and photofluorography. [Results] CoCl_2 induced a decrease in cell viability and an increase in percentage of apeptosis in PC12 cells along with dissipation of MMP as well as overproduction of ROS. When PC12 cells were treated with Naris 30 min before CoCl_2 treatment a decrease in viability of PC12 cells induced by 600 μmol/L CoCl_2 was concentration-dependently blocked by NaHs (100, 200, and 400 μmol/L). Pretreatment with NaHS at 200 and 400 μmol/L obviously reduced the apepetotic percentage of PC12 cells induced by 600 μmol/L CoCl_2 and inhibited the dissipation of MMP and overproduction of ROS. [Conclusion] H_2S protected PC12 cells against CoCl_2-induced apeptosis, which may be associated with the inhibition of H_2S on the dissipation of MMP and overproduction of ROS induced by CoCl_2.
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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.
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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.
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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.