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P37 Role of endoplasmic reticulum stress in the regulation endogenous hydrogen sulfide for hepatocyte apoptosis induced by LPS
Abstracts / Nitric Oxide 39 (2014) S16–S49
of organ function were measured. At 30 h, there was 20% mortality in CSE / mice, while no mortality in the wild-type controls. By 36 h, the mortality increased to 50% in CSE / mice, while it remained low (10%) in the wild-type controls. However, by 46 h, the two survival curves converged, and showed a similar, 60% mortality in both groups. Analysis of the plasma markers of organ injury revealed slight trends for a CLP-induced increase in the liver injury markers ALT and AST and the pancreatic injury marker amylase in wild-type mice. More pronounced increases were noted in the same parameters in the CSE / mice. Blood urea nitrogen levels were elevated 3-fold over baseline in wild-type mice subjected to CLP and 4-fold over baseline in CSE / mice. There was an impairment of endothelium-dependent relaxation responses in the thoracic aorta after CLP, which was more pronounced in the CSE / mice. These data indicate that CSE, and its product, H2S, exerts protective effects against the development of sepsis, especially in the early stages of the disease. http://dx.doi.org/10.1016/j.niox.2014.03.085
P36 Exogenous hydrogen sulfide promotes upregulation of HO-1 expression in rat hepatocytes induced by LPS Yan-sha Wang, Ying-lei Ji, Yi-Chang Liu, Zhen-yong Gu Department of Forensic Medicine, Medical School of Nantong University, Nantong 226001, China The study was designed to investigate the effects of exogenous hydrogen sulfide (H2S) on expression of heme oxygenase-1 (HO-1) in rat hepatocytes induced by LPS. The cultured rat hepatocyte line BRL cells were treated with different dose of LPS and H2S donor NaHS respectively. In separate expriment, hepatocytes were pre-treated with NaHS for 24 h or post-treated with different dose of NaHS for 1 h then incubated with LPS for another 12 h. The expression of HO-1 protein was detected with Western blotting.Compared with vehicle, LPS induced profound upregulation of HO-1 protein expression in a dose- and time-dependent manner. Both of pre-conditioning and post-conditioning with NaHS led to upregulation of HO-1 protein expression in a dose-dependent manner,further HO-1 protein expression were more in pre-conditioning than in post-conditioning with NaHS. However,compared with LPS, pre-conditioning or post-conditioning with NaHS at 50, 100 and 200 micromol/L together with LPS induced the marked upregulation of HO-1 protein expression,up-regulating to 31.54%, 44.05%, 66.07% and 17.19%, 22.52%, 60.53%, respectively. It is well known that HO-1 is an antioxidant protein, and its product carbon monooxide also has an antioxidant capacity. Thses results showed pre-conditioning and post-conditioning with exogenous H2S promoted LPS-induced upregulation of HO-1 protein in hepatocytes synergily, indicating that HO-1 or carbon monooxide mediated the biological effects in part of exogenous H2S. http://dx.doi.org/10.1016/j.niox.2014.03.086
P37 Role of endoplasmic reticulum stress in the regulation endogenous hydrogen sulfide for hepatocyte apoptosis induced by LPS Ying-lei Ji, Yan-sha Wang, Yi-Chang Liu, Zhen-yong Gu Department of Forensic Medicine, Medical School of Nantong University, Nantong 226001, China The study was carried out to investigate the role of endoplasmic reticulum stress (ERS) in regulation of endogenous hydrogen sulfide (H2S) for hepatocyte apoptosis induced by lipopolysaccharide (LPS).
S27
The cultured rat hepatocyte line BRL cells were treated with LPS and ERS inducer thapsigargin (TG), ERS inhibitor 4-phenylbutyric acid (4PBA) respectively or in a different combination manner. RNA interference technique was used to silence the expression of CBS and CSE. H2S level, cell viability and apoptosis rate were measured. The expressions of CBS and CSE mRNA and protein, GRP78,CHOP, caspase-12 and caspase-3 were detected with RT-PCR and Western blotting. Compared with vehicle, LPS caused a decrease in cell viability, an increase in apoptosis rate and significantly increased expression of GRP78 protein in a dose- and time-dependent manner. Almost similar increases were observed in expression of CHOP,caspase-12 and caspase-3 proteins. TG led to a marked decrease in cell viability and an increase in apoptosis rate. Furthermore, TG exacerbated while 4-PBA alleviated LPS-induced hepatocyte injuries. LPS induced a up-regulation of CBS-H2S and CSEH2S systems. Both of CBS siRNA and CSE siRNA reversed the increased endogenous H2S formation and up-regulation of CBS and CSE expressions, inhibited the decreased cell viability and increased apoptosis rate and obviously decreased expressions of GRP78, CHOP, caspase-12, caspase-3 induced by LPS. Results showed that endogenous H2S can aggravate LPS-induced hepatocyte apoptosis through enhancement of ERS. http://dx.doi.org/10.1016/j.niox.2014.03.087
P38 PKCepsilon-ERK1/2-STAT3 signal pathway contributes to regulation of endogenous hydrogen sulfide for hepatocyte apoptosis induced by lipopolysaccharide Jun Yan, Ying-lei Ji, Yan-sha Wang, Yi-Chang Liu, Zhen-yong GU Department of Forensic Medicine, Nantong University, Nantong 226001, China The study was designed to elucidate roles of PKCepsilon, ERK1/2, STAT3 in regulations of endogenous H2S in rat hepatocyte apoptosis induced by LPS. The cultured rat hepatocytes were treated with vehicle or different dose of LPS respectively. In the separate experiments, hepatocytes were treated with CBS inhibitor AOAA,CSE inhibitor PAG or PKC epsilon, ERK1/2, STAT3 specific inhibitors SCP0213, A6355, S31201,respectively or in a different combination manner. PKC epsilon, ERK1/2 and STAT3 phospharylations, PKCepsilon membranous translocation, STAT3 nuclear translocation were detected with Western blotting. Apoptosis rates were detected with flow cytometry. PKC epsilon, ERK1/2 and STAT3 phosphorylation expression in LPS-treated hepatocytes were significantly up-regulated, obviously in 30 min compared with vehicle,furthermore membranous tranlocation of PKCepsilon and nuclear tranlocation of STAT3 were found. Compared with LPS, AOAA or PAG combined with LPS respectively or jointly could cause further more obvious up-regulation in phosphorylative levels of PKCepsilon, ERK1/2 or STAT3 protein expression. SCP0213, A6355 or S31-201 combined with LPS respectively could cause increased apoptosis rates in 4 h and 12 h compared with LPS alone. When hepatocytes were treated with SCP0213, A6355 or S31-201 combined with AOAA or PAG respectively, apoptosis rates increased in 12 h but no significant changes in 4 h compared with AOAA or PAG + LPS treatments.Compared with LPS,SCP0213 + LPS could cause down-regulation in phosphorylative levels of ERK1/2 or STAT3 protein expressions, A6355 + LPS could cause down-regulation in the phosphorylative level of STAT3 protein expression without change in PKCepsilon, S31– 201 + LPS could not cause any changes of phosphorylative level of protein expression in PKCepsilon or ERK1/2. In conclusion,PKCepsilonERK1/2-STAT3 pathway was involved in regulations of endogenous H2S for LPS-induced hepatocyte apoptosis. http://dx.doi.org/10.1016/j.niox.2014.03.088
of organ function were measured. At 30 h, there was 20% mortality in CSE / mice, while no mortality in the wild-type controls. By 36 h, the mortality increased to 50% in CSE / mice, while it remained low (10%) in the wild-type controls. However, by 46 h, the two survival curves converged, and showed a similar, 60% mortality in both groups. Analysis of the plasma markers of organ injury revealed slight trends for a CLP-induced increase in the liver injury markers ALT and AST and the pancreatic injury marker amylase in wild-type mice. More pronounced increases were noted in the same parameters in the CSE / mice. Blood urea nitrogen levels were elevated 3-fold over baseline in wild-type mice subjected to CLP and 4-fold over baseline in CSE / mice. There was an impairment of endothelium-dependent relaxation responses in the thoracic aorta after CLP, which was more pronounced in the CSE / mice. These data indicate that CSE, and its product, H2S, exerts protective effects against the development of sepsis, especially in the early stages of the disease. http://dx.doi.org/10.1016/j.niox.2014.03.085
P36 Exogenous hydrogen sulfide promotes upregulation of HO-1 expression in rat hepatocytes induced by LPS Yan-sha Wang, Ying-lei Ji, Yi-Chang Liu, Zhen-yong Gu Department of Forensic Medicine, Medical School of Nantong University, Nantong 226001, China The study was designed to investigate the effects of exogenous hydrogen sulfide (H2S) on expression of heme oxygenase-1 (HO-1) in rat hepatocytes induced by LPS. The cultured rat hepatocyte line BRL cells were treated with different dose of LPS and H2S donor NaHS respectively. In separate expriment, hepatocytes were pre-treated with NaHS for 24 h or post-treated with different dose of NaHS for 1 h then incubated with LPS for another 12 h. The expression of HO-1 protein was detected with Western blotting.Compared with vehicle, LPS induced profound upregulation of HO-1 protein expression in a dose- and time-dependent manner. Both of pre-conditioning and post-conditioning with NaHS led to upregulation of HO-1 protein expression in a dose-dependent manner,further HO-1 protein expression were more in pre-conditioning than in post-conditioning with NaHS. However,compared with LPS, pre-conditioning or post-conditioning with NaHS at 50, 100 and 200 micromol/L together with LPS induced the marked upregulation of HO-1 protein expression,up-regulating to 31.54%, 44.05%, 66.07% and 17.19%, 22.52%, 60.53%, respectively. It is well known that HO-1 is an antioxidant protein, and its product carbon monooxide also has an antioxidant capacity. Thses results showed pre-conditioning and post-conditioning with exogenous H2S promoted LPS-induced upregulation of HO-1 protein in hepatocytes synergily, indicating that HO-1 or carbon monooxide mediated the biological effects in part of exogenous H2S. http://dx.doi.org/10.1016/j.niox.2014.03.086
P37 Role of endoplasmic reticulum stress in the regulation endogenous hydrogen sulfide for hepatocyte apoptosis induced by LPS Ying-lei Ji, Yan-sha Wang, Yi-Chang Liu, Zhen-yong Gu Department of Forensic Medicine, Medical School of Nantong University, Nantong 226001, China The study was carried out to investigate the role of endoplasmic reticulum stress (ERS) in regulation of endogenous hydrogen sulfide (H2S) for hepatocyte apoptosis induced by lipopolysaccharide (LPS).
S27
The cultured rat hepatocyte line BRL cells were treated with LPS and ERS inducer thapsigargin (TG), ERS inhibitor 4-phenylbutyric acid (4PBA) respectively or in a different combination manner. RNA interference technique was used to silence the expression of CBS and CSE. H2S level, cell viability and apoptosis rate were measured. The expressions of CBS and CSE mRNA and protein, GRP78,CHOP, caspase-12 and caspase-3 were detected with RT-PCR and Western blotting. Compared with vehicle, LPS caused a decrease in cell viability, an increase in apoptosis rate and significantly increased expression of GRP78 protein in a dose- and time-dependent manner. Almost similar increases were observed in expression of CHOP,caspase-12 and caspase-3 proteins. TG led to a marked decrease in cell viability and an increase in apoptosis rate. Furthermore, TG exacerbated while 4-PBA alleviated LPS-induced hepatocyte injuries. LPS induced a up-regulation of CBS-H2S and CSEH2S systems. Both of CBS siRNA and CSE siRNA reversed the increased endogenous H2S formation and up-regulation of CBS and CSE expressions, inhibited the decreased cell viability and increased apoptosis rate and obviously decreased expressions of GRP78, CHOP, caspase-12, caspase-3 induced by LPS. Results showed that endogenous H2S can aggravate LPS-induced hepatocyte apoptosis through enhancement of ERS. http://dx.doi.org/10.1016/j.niox.2014.03.087
P38 PKCepsilon-ERK1/2-STAT3 signal pathway contributes to regulation of endogenous hydrogen sulfide for hepatocyte apoptosis induced by lipopolysaccharide Jun Yan, Ying-lei Ji, Yan-sha Wang, Yi-Chang Liu, Zhen-yong GU Department of Forensic Medicine, Nantong University, Nantong 226001, China The study was designed to elucidate roles of PKCepsilon, ERK1/2, STAT3 in regulations of endogenous H2S in rat hepatocyte apoptosis induced by LPS. The cultured rat hepatocytes were treated with vehicle or different dose of LPS respectively. In the separate experiments, hepatocytes were treated with CBS inhibitor AOAA,CSE inhibitor PAG or PKC epsilon, ERK1/2, STAT3 specific inhibitors SCP0213, A6355, S31201,respectively or in a different combination manner. PKC epsilon, ERK1/2 and STAT3 phospharylations, PKCepsilon membranous translocation, STAT3 nuclear translocation were detected with Western blotting. Apoptosis rates were detected with flow cytometry. PKC epsilon, ERK1/2 and STAT3 phosphorylation expression in LPS-treated hepatocytes were significantly up-regulated, obviously in 30 min compared with vehicle,furthermore membranous tranlocation of PKCepsilon and nuclear tranlocation of STAT3 were found. Compared with LPS, AOAA or PAG combined with LPS respectively or jointly could cause further more obvious up-regulation in phosphorylative levels of PKCepsilon, ERK1/2 or STAT3 protein expression. SCP0213, A6355 or S31-201 combined with LPS respectively could cause increased apoptosis rates in 4 h and 12 h compared with LPS alone. When hepatocytes were treated with SCP0213, A6355 or S31-201 combined with AOAA or PAG respectively, apoptosis rates increased in 12 h but no significant changes in 4 h compared with AOAA or PAG + LPS treatments.Compared with LPS,SCP0213 + LPS could cause down-regulation in phosphorylative levels of ERK1/2 or STAT3 protein expressions, A6355 + LPS could cause down-regulation in the phosphorylative level of STAT3 protein expression without change in PKCepsilon, S31– 201 + LPS could not cause any changes of phosphorylative level of protein expression in PKCepsilon or ERK1/2. In conclusion,PKCepsilonERK1/2-STAT3 pathway was involved in regulations of endogenous H2S for LPS-induced hepatocyte apoptosis. http://dx.doi.org/10.1016/j.niox.2014.03.088