Roles of Akt pathway and cell apoptosis in leptin-mediated chronic mor-phine antinociceptive tolerance / 中国病理生理杂志

AIM:To explore the roles of Akt ( also called protein kinase B ) and active caspase-3 in the leptin-mediated chronic morphine antinociceptive tolerance in rats .METHODS: A model of chronic morphine antinociceptive tolerance was established in the SD rats .The protein levels of spinal Akt and cleaved caspase-3 were tested by Western blotting.The technique of immunohistochemical staining was used to detect the immunoreactivity positive cells of phospho -rylated ( p)-Akt and cleaved caspase-3 in the spinal cord .Double staining of immunohistochemistry was used to examine the cellular location of the p-Akt and cleaved caspase-3 positive cells.RESULTS: The chronic intrathecal injection of morphine (15 μg) for 7 d markedly upregulated the spinal protein levels of p-Akt and cleaved caspase-3 in the rats.Thirty min before injection of morphine , intrathecal injection of leptin antagonist (3μg) for 7 d significantly attenuated the upreg-ulation of the protein levels of p-Akt and cleaved caspase-3 induced by chronic morphine treatment .The p-Akt was exclu-sively observed in the spinal neurons .The cleaved caspase-3 was only localized with the spinal astrocytes .Intrathecal injec-ting the inhibitors of leptin , Akt and caspase-3 ameliorated the chronic antinociceptive tolerance .CONCLUSION: The spinal Akt pathway and active caspase-3 are involved in the leptin-mediated chronic morphine antinociceptive tolerance in rats.

Interaction between necroptosis and p38MAPK pathway mediates high glucose-induced injury in H9 c2 cardiac cells / 中国药理学通报

Aim To investigate the role of the interac-tion between necroptosis ( Nec ) and p38 mitogen-acti-vated protein kinase ( MAPK) pathway in the high glu-cose (HG)-induced H9c2 cardiac cells injury.Meth-ods The cell viability was measured by cell counter kit-8 assay .The intracellular level of reactive oxygen species ( ROS ) was tested by DCFH-DA stating fol-lowed by photofluorography .Mitochondrial membrane potential ( MMP) was detected by Rhodamine 123 stai-ning followed by photofluorography . The expression levels of receptor interaction protein 3 ( RIP3, an indi-cator of Nec ) and p38 MAPK protein were tested by Western blot assay .Results The treatment of H9c2 cardiac cells with 35 mmol? L-1 glucose ( high glu-cose, HG) for 24 h induced considerable injuries , in-cluding a decrease in cell viability , increases in ROS generation as well as MMP loss .The co-treatment of the cells with 100 μmol? L-1 necrostatin-1(Nec-1,a specific inhibitor of Nec ) and HG for 24 h or the pre-treatment of the cells with 3 μmol? L-1 SB 2 0 3 5 8 0 ( an inhibitor of p38MAPK) for 60 min before HG exposure attenuated the above injuries induced by HG .Moreo-ver, the treatment of the cells with HG for 1,3,6,9, 12 ,24 ,36 and 48 h significantly increased the expres-sion levels of RIP3, peaking at 24 h.The co-treatment of the cells with 100 μmol? L-1 Nec-1 or the pre-treatment of the cells with 3 μmol? L-1 SB203580 considerably blocked the up-regulation of RIP3 expres-sion induced by HG .On the other hand , the co-treat-ment of the cells with 100 μmol? L-1 Nec-1 alleviated the HG-induced up-regulation of the expression of p-p38MAPK.Conclusion The interaction between Nec and p38 MAPK pathway mediates the HG-induced inju-ry in H9c2 cardiac cells.

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced inflammation mediated by necroptosis in H9c2 cardiac cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the role of ATP-sensitive potassium ( KATP ) channels in the inhibitory effect of hydrogen sulfide ( H2 S) on high glucose ( HG)-induced inflammation mediated by necroptosis in H 9c2 cardiac cells. METHODS:The expression levels of receptor-interacting protein 3 ( RIP3; an indicator of necroptosis ) and cyclooxyge-nase-2 (COX-2) were determined by Western blot.The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α( TNF-α) were detected by ELISA .RESULTS:After H9c2 cardiac cells were treated with 35 mmol/L glucose ( HG) for 24 h, the expression of RIP3 was significantly increased .Pre-treatment of the cells with 100 μmol/L diazoxide ( DZ; a KATP channel opener) or 400 μmol/L NaHS (a donor of H2S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG.Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a KATP channel blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3.On the other hand, co-treatment of the cells with 100μmol/L necrostatin-1 ( a specific inhibitor of necroptosis ) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses , evidenced by decreases in the expression of COX-2 and secretion levels of IL-1βand TNF-α.However , pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION:KATP channels play an important role in the inhib-itory effect of H2 S on HG-induced inflammation mediated by necroptosis in H 9c2 cardiac cells.

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells / 中国病理生理杂志

AIM:To investigate the roles of ATP-sensitive potassium ( KATP ) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide ( H2 S) on the cardiomyocyte injury.METHODS:The expres-sion level of KATP channel protein was tested by Western blot.The cell viability was measured by CCK-8 assay.The number of apoptotic cells was observed by Hoechst 33258 nuclear staining.Mitochondrial membrane potential ( MMP) was exam-ined by JC-1 staining.RESULTS:After the H9c2 cells were treated with 35 mmol/L glucose ( high glucose, HG) for 1~24 h, the protein level of KATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h.Pretreatment of the cells with 400μmol/L NaHS ( a donor of H2 S) prior to exposure to HG for 12 h con-siderably blocked the down-regulation of KATP channels induced by HG.Pretreatment of the cells with 100 μmol/L mito-chondrial KATP channel opener diazoxide, 50μmol/L non-selective KATP channel opener pinacidil or NaHS obviously inhibi-ted HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss.Pretreatment with 100μmol/L mitochondrial KATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective KATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS.CONCLUSION:KATP channels mediate the inhibitory effect of H2 S on HG-induced cardiac injury.

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway / 中国病理生理杂志

AIM:Tostudywhe ther theangiotens in-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardio-myocytes against high glucose (HG)-induced injury by inhibiting nuclear factor-κB (NF-κB) pathway.METHODS:The cell viability was measured by CCK-8 assay.The intracellular levels of reactive oxygen species ( ROS) were detected by DCFH-DA staining .The number of apoptotic cells was tested by Hoechst 33258 nuclear staining .Mitochondrial membrane potential ( MMP) was examined by JC-1 staining.The levels of NF-κB p65 subunit and cleaved caspase-3 protein were de-termined by Western blotting.RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose (HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated ( p) NF-κB p65, peaking at 60 min.Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30μmol/L and HG for 24 h inhibited HG-induced cy-totoxicity, evidenced by an increase in cell viability .On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage , indicated by decreases in the number of apoptotic cells , cleaved caspase-3 level, ROS generation and MMP loss .However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor (Mas receptor).Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG.CONCLUSION:Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway .

Edaravone protects H9 c2 cells against doxorubicin-induced cardiotoxicity / 中国药理学通报

Aim To explore whether edaravone (EDA), a novel free radical scavenger, protects H9c2 cardiac cells against doxorubicin ( DOX )-induced car-diotoxicity. Methods H9c2 cells were treated with 5μmol·L-1 DOX to establish a model of DOX cardio-toxicity. Cell viability was examined by cell counter kit ( CCK-8 ) . Changes in morphology and amount of ap-optotic cells were detected by Hoechst 33258 staining;intracellular level of reactive oxygen species ( ROS ) was measured by DCFH-DA staining and photofluorog-raphy;mitochondrial membrane potential ( MMP) was observed by rhodamine 123 ( RH123 ) staining and photoflurograph; the expression level of caspase-3 was determined by Western blot assay. Results Pretreat-ment of H9 c2 cells with 20 , 40 and 80 μmol · L-1 EDA for 60 min markedly inhibited cytotoxicity in-duced by 5 μmol · L-1 DOX, respectively, as evi-denced by an increase in cell viability. The protective effect induced by 40 μmol · L-1 EDA was maximal. Pretreatment of H9 c2 cells with 40 μmol · L-1 EDA for 30 , 60 , 90 and 120 min significantly attenuated DOX-induced cytotoxicity, respectively, having a max-imal protection at 60 min. Furthermore, pretreatment of H9 c2 cells with 40 μmol · L-1 EDA for 60 min be-fore exposure to 5 μmol · L-1 DOX for 24 h obviously reduced cardiac injuries, as evidenced by decreases in the DOX-induced intracellular ROS generation, num-ber of apoptotic cells, and expression of cleaved caspase-3, as well as loss of MMP. Conclusions EDA can protect H9 c2 cardiac cells against DOX-in-duced cardiotoxicity, this protection may be associated with inhibition of ROS production and preservation of MMP.

Changes of endogenous cystathionine-β-synthase/hydrogen sulfide system in PC12 cells injury induced by rotenone / 中国药理学通报

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.

ERK1/2 pathway mediates the protective effect of hydrogen sulfide against cisplatin-induced injury in human marrow mesenchymal stem cells / 中华骨科杂志

Objective To explore the protection and mechanism of hydrogen sulfide (H2S) preconditioning against injury induced by cisplatin in human marrow mesenchymal stem cells (HMMSCs).Methods HMMSCs were treated by cisplatin at 0,5,10,20,40,60,80 mg/L concentrations for 24 h respectively and were exposed to cisplatin at 20 mg/L concentrations for 0,6,12,24,36,48 h respectively.HMMSCs were pretreated with NaHS at 0,0.4,0.6,0.8,1.0 mmol/L respectively for 30 min before exposed to cisplatin at 20 mg/L concentrations for 24 h.HMMSCs were treated by U0126 or combined with human epidermal growth factor (HEGF) together for 30 min before they were preconditioned with NaHS for 30 min.The cell survival rate,cell apoptosis rate,the expression of p-ERK1/2 and t-ERK1/2 were recorded.Results The cell survival rate decreased to 71.72％±2.72％,59.41％±5.44％,50.37％±4.55％,38.97％±2.92％,30.11％±4.64％ and 21.71％±5.35％ respectively after cells were treated with cisplatin at 5,10,20,40,60,80 mg/L concentrations respectively,and the differences were statistically significant compared with 0 mg/L group.The cell viability fell to 70.30％±6.20％,61.63％±2.70％,51.29％±3.13％,38.72％±3.66％ and 27.57％±2.32％ after HMMSCs were treated with cisplatin at 20 mg/L for 6,12,24,36,48 h respectively,and the differences were statistically significant compared with 0 h group.The cell viability increased to 65.99％±2.67％,72.93％±5.44％,75.10％±4.71％ and 76.56％± 5.25％ when HMMSCs got pretreatment of NaHS,and the differences had statistical significance compared with cisplatin group.The cell apoptotic rate decreased from 35.29％±2.77％ to 18.62％±0.97％ when HMMSCs were pretreated with NaHS at 0.6 mmo/L.Treatment of HMMSCs with cisplatin at 20 mg/L for 24 h reduced p-ERK1/2 expression.The pretreatment of NaHS could inhibit the inhibitory action to the expression of p-ERK1/2 induced by cisplatin.Pretreatment with U0126 or HEGF inhibited or promoted the protection and the upregulated expression ofp-ERK1/2 caused by NaHS pretreatment.Conclusion The preconditioning of H2S can protect cell damage caused by cisplatin via activating the ERK1/2 pathway of HMMSCs.

Phosphorylation of NF-κB P65 subunit mediates chemical hypoxia-induced inflammatory injury in HaCaT cells / 中华皮肤科杂志

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride (CoCl2-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit8 (CCK-8), the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours (P ＜ 0.05, 0.01, 0.01 ). The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells,despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.

Protective effect of N-acetyl cysteine against chemical hypoxia-induced injury to an immortal human skin keratinocyte line HaCaT / 中华皮肤科杂志

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.

Effects of Hydrogen Sulfide on Apoptosis of PC12 Cells Induced by Chemical Hypoxia / 中山大学学报(医学科学版)

[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.

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.

Research of Adaptive Protection of Preconditioning with Hydrogen Peroxide in Bone Marrow Mesenchymal Cells Against Apoptosis / 中山大学学报(医学科学版)

[Objective] To investigate the effects of preconditioning with low concentration of hydrogen peroxide (H2O2) on oxidative stress-induced BMSC apoptosis.[Methods] In vitro separation,purification,culture,and amplification of bone marrow mesenchymal stem cells were performed.BMSC were insulted with 0,50,100,200,300,400,500 μmol/L H2O2 and the effect of different consentration of H2O2 on BMSC was detected by Flow cytometry (FCM).And then cells were preconditioned with different consentraion of H2O2.(FCM) was used to determine the protective role of H2O2 preconditioning on BMSC apoptosis,BMSC chromatin distribution changes were observed by Hoechst33324;BMSC Caspase-3 and Bcl-2 gene changes were detected by RT-PCR.[Results] Analysis of BMSC apoptosis by flow cytometry showed that H2O2 induced BMSC apoptosis in a dose-dependent manner,and pretreatment of the cells with low concentration of H2O2 prevented subsequent stimulation with high H2O2.RT-PCR results showed that preconditioning with low concentration of H2O2 reduced the BMSC Caspase-3 gene expression but increased Bcl-2 gene expression.[Conclusion] Preconditioning with low concentration of H2O2 has an adaptive role in BMSC,and its mechanism may be related to inhibit abnormal gene expression of Caspase-3 and increase the gene expression of Bcl-2.

Roles of heat shock protein 90 in the blockage of H2S against cardiomyocyte injuries induced by chemical hypoxia / 中国病理生理杂志

AIM: To explore the roles of heat shock protein 90 (HSP90) in the blockage of hydrogen sulfide (H2S) against chemical hypoxia-mimetic agent (cobalt chloride, CoCl_2)-induced oxidative stress injuries in H9c2 cardiac cell. METHODS: H9c2 cells were treated with CoCl_2 to set up the chemical hypoxia-induced the model of cardiomyocyte injury. Sodium hydrosulfide (NaHS, a H2S donor) was added into medium for 30 min before CoCl_2 treatment. ATP content was detected by high performance liquid chromatogram (HPLC). Mitochondrial membrane potential (MMP) was measured by rhodamine123 (Rh123) staining and photofluorography. The activity of superoxide dismutase (SOD) was observed using a SOD kit. The expression of heme oxygenase-1 (HO-1) was evaluated by Western blotting. RESULTS: CoCl_2 at concentration of 600 μmol/L significantly reduced SOD activity, ATP level and MMP, and enhanced the expression of HO-1 in H9c2 cells. Pretreatment with 400 μmol/L NaHS dramatically inhibited the cytotoxicity induced by CoCl_2, increased SOD activity, ATP level and MMP, decreased HO-1 expression. 17-allylamino-17 demethoxygeldanamycine(17AAG), an inhibitor of HSP90, obviously blocked the inhibitory effect of H2S on the CoCl_2-induced cytotoxicity, reduced the levels of ATP and MMP, increased HO-1 expression. However, no significantly influence on SOD activity was observed. CONCLUSION: HSP90 may mediate the cardioprotection of H2S via inhibiting the oxidative stress induced by chemical hypoxia.

Construction of human osteoblast-like cell model with the down-regulated estrogen receptor α subunit gene by RNA interference / 中华老年医学杂志

Objective To construct the human osteoblast-like cell model with estrogen receptor α(ERα)subunit gene knocked down. Methods According to the computer-aided design(CAD),ERα-specific small interference RNA(siRNA)gene was synthesized and cloned into the expression vector pSilencer 4.1-CMV. The recombinant ERα siRNA plasmid was transfected into human osteoblast-like cell line MG63 by lipofectin,the cloned MG63 ceils were selected by hygromycin,and the cloned MG63 cell was cultured more than 20 passages after transfection.The expression of ERα mRNA in MG63 cells was detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression and location of ERα protein were identified by immunocytochemistry.Compared with control groups,proliferation rate,growth cycle and expression did not show significant difference.Results The recombinant eukaryote plasmid vector was constructed.Furthermore,the recombinant plasmid knocked down ERα protein in human osteoblast-like cells. Conclusions The human osteoblast-like cell model with RNAi-knocked down ERα gene is constructed successfully.This model appears to be very useful for the future research on ERα biological characters and on molecular mechanism of bone metabolism.

Estrogen reduces apoptosis induced by H_2O_2 in PC 12 cells / 基础医学与临床

Objective To observe the effect of estrogen on H2O2-induced apoptosis and to explore the mechanism of protection by estrogen.Methods Setting up the experimental model of apoptosis induced by H2O2 in PC12 cells. The viability of cells was assessed by MTT assay. The activity of lactate dehydrogenase was detected by colorimetry. Hoechst 33258 was used as indicator of apoptosis. Apoptotic cells were measured by flow cytometry (FCM) with propidium iodide stain. The activity of caspase-3 was detected by colorimetry. Results H2O2 significantly decreased the viability of cell, increased LDH release and promoted apoptosis as well as caspase-3 activity in PC12. Estrogen markedly reduced these changes. Conclusion Estrogen reduces apoptosis induced by H2O2, and its mechanisms may be explained by inhibition of activation of caspase-3.

Effect of lymphocytes modified by brain-derived neurotrophic factor gene on the proliferation and the H_2O_2-induced apoptosis of PC12 cell / 中国药理学通报

AIM To study the expression of brain-derived neurotrophic factor (BDNF) in lymphocytes modified by BDNF gene and its effect on the proliferation and the H 2O 2-induced apoptosis of PC12 cells for the transfer of ex vivo therapeutic gene into central nervous system (CNS) tissue with atraumatic means. METHODS Recombined BDNF cDNA plasmid was transfected by LipofectAMINE into the packing cell line PA317 and G418-resistant clones with highest titer was selected. Rat lymphocytes were infected repeatedly with virus supernatant. The expression of BDNF in rat lymphocytes was assayed by FCM and immunohistochemistry. The proliferation of PC12 cells was evaluated by MTT assay. H 2O 2-induced apoptosis of PC12 cells was determined by PI stain flow cytometry. RESULTS BDNF expressed in rat lymphocytes genetically modified and the concentration of BDNF in the conditioned medium of engineered lymphocytes had a linear correlation with the proliferation of the PC12 cells. The supernatant of lymphocytes modified by BDNF gene decreased the apoptosis of PC12 cells induced by H 2O 2. CONCLUSION Rat lymphocytes genetically modified can express and secret active BDNF in vitro.

The adaptive cytoprotection of H_2O_2 preconditioning against oxidative-stress damage in PC12 cell / 中国药理学通报

AIM To study the adaptive cytoprotection of H 2O 2 preconditioning against oxidative stress damage in PC12 cell and the relationships between brain derived neurotrophic factor (BDNF) and the adaptive cytoprotection of H 2O 2 preconditioning. METHODS The viability of PC12 cells was evaluated by MTT assay. Apoptosis was detected by PI stain flow cytometer. Expression of BDNF was analyzed by immuo flow cytometer. RESULTS After H 2O 2 preconditioning, the survival of PC12 cells exposure to H 2O 2 (20～60 ?mol?L -1 ) was increased and the apoptosis of PC12 cells induced by H 2O 2 (20 or 30 ?mol?L -1 ) was inhibited and the expression of BDNF in PC12 cells was enhanced. CONCLUSION H 2O 2 preconditioning is protective against the damage of PC12 cells induced by H 2O 2 and its mechanisms may involve up modulation of the expression of BDNF.

H_2S:the third endogenous gaseous transmitter / 中国药理学通报

It is hypothesized that H 2S is the third endogenous signaling gasotransmitter, after NO and CO. Endogenous H 2S may regulate some aspects of synaptic activity via cAMP mediated pathways and function as a neuromodulator or transmitter. H 2S is an endogenous vasorelaxant factor that activates K ATP channels and hyperpolarizes membrane potential of vascular SMCs. By directly acting on vascular SMCs, H 2S may reduce the extracellular calcium entry and relax vascular tissues.