AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning(RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transpl...AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning(RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation(OLT), ischemic postconditioning(IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine(Cr) and creatinine kinase-myocardial band(CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species(ROS), H2O2, mitochondrial membrane potential(ΔΨm) and total nitric oxide(NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase(e NOS) was analyzed by reverse transcription-polymerase chain reaction(RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions(P < 0.05). ROS(P < 0.001) including H2O2(P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC(P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion(I/R) injury was significantly attenuated in the RIPerC group(P < 0.001). A marked increase of NO content and phosphoserine eN OS, both in protein and mR NA levels, was observed in liver graft of the RIPer C group as compared with the OLT group(P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group(P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group(P < 0.01).CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/e NOS/NO pathway.展开更多
AIM: To optimize the perfusates used for hypothermicmachine perfusion(HMP).METHODS: Sprague-Dawley rats were assigned randomly to three groups(n = 12 per group) that received either saline, University of Wisconsin col...AIM: To optimize the perfusates used for hypothermicmachine perfusion(HMP).METHODS: Sprague-Dawley rats were assigned randomly to three groups(n = 12 per group) that received either saline, University of Wisconsin coldstorage solution(UW) or histidine-tryptophan-ketoglutarate solution(HTK) as the perfusate. Each group was divided into two subgroups: static cold storage(SCS) and HMP(n = 6 per subgroup). The liver graft was retrieved according to the method described by Kamada. For the SCS group, the graft was directly placed into cold perfusate(0-4?℃) for 6 h after liver isolation while the portal vein of the graft was connected to the perfusion machine for the HMP group. Then the perfusates were collected at different time points for analysis of aspartate aminotransferase(AST), alanine transaminase(ALT) and lactate dehydrogenase(LDH) levels. Liver tissues were obtained for evaluation of histology, dry/wet weight(D/W) ratio, and malondialdehyde(MDA) and adenosine-triphosphate(ATP) levels. The portal vein pressure and velocity were monitored in real time in all HMP subgroups.RESULTS: Comparison of HMP and SCS: Regardless of the perfusate, HMP improved the architecture of donor graft in reducing the congestion around sinusoids and central vein and maintaining sinusoid lining in morphology; HMP improved liver function in terms of ALT, AST and LDH, especially during the 3-6 h period(SCS vs HMP using saline: ALT3, 225.00 ± 105.62 vs 49.50 ± 18.50, P = 0.047; LDH3, 1362.17 ± 563.30 vs 325.75 ± 147.43, P = 0.041; UW: LDH6, 2880.14 ± 948.46 vs 2135.00 ± 174.27, P = 0.049; HTK, AST6, 307.50 ± 52.95 vs 185.20 ± 20.46, P = 0.041); HMP decreased MDA level(saline, 2.79 ± 0.30 vs 1.09 ± 0.09, P = 0.008; UW, 3.01 ± 0.77 vs 1.23 ± 0.68, P = 0.005; HTK, 3.30 ± 0.52 vs 1.56 ± 0.22, P = 0.006). Comparison among HMP subgroups: HTK showed less portal vein resistance than UW and saline(vs saline, 3.41 ± 0.49 vs 5.00 ± 0.38, P < 0.001; vs UW, 3.41 ± 0.49 vs 4.52 ± 0.63, P = 0.007); UW reduced edema most efficiently(vs saline, 0.68 ± 0.02 vs 0.79 ± 0.05, P = 0.013), while HTK maintained ATP levels best(vs saline, 622.60 ± 29.11 vs 327.43 ± 44.66, P < 0.001; vs UW, 622.60 ± 29.11 vs 301.80 ± 37.68, P < 0.001).CONCLUSION: HMP is superior to SCS in maintaining both architecture and function of liver grafts. Further, HTK was found to be the optimal perfusate for HMP.展开更多
AIM To investigate the protective mechanism of mitofusin-2(Mfn2) in rat remote ischemic perconditioning(RIC) models and revalidate it in alpha mouse liver-12(AML-12) hypoxia cell lines.METHODS Sprague-Dawley rats were...AIM To investigate the protective mechanism of mitofusin-2(Mfn2) in rat remote ischemic perconditioning(RIC) models and revalidate it in alpha mouse liver-12(AML-12) hypoxia cell lines.METHODS Sprague-Dawley rats were divided into three groups(n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting(WB) and quantitative real-time(q RT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture(hypoxia) and anoxic incubator tank culture with Mfn2 knockdown(hypoxia + Si), and data of q RT-PCR, WB, mitochondrial membrane potential(ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected.RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group(P < 0.05). q RTPCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2(MICUs) axis was changed(P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis(P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group(P < 0.005). Finally, q RT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups(P < 0.005).CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.展开更多
基金Supported by National Natural Science Foundation of China,No.81421062the Science and Technology Bureau of Zhejiang Province,China,No.2016C33145+1 种基金the National Natural Science Foundation of China,No.81470891the 863 National High Technology Research and Development Program of China for young scientist No.2015AA020923
文摘AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning(RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation(OLT), ischemic postconditioning(IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine(Cr) and creatinine kinase-myocardial band(CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species(ROS), H2O2, mitochondrial membrane potential(ΔΨm) and total nitric oxide(NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase(e NOS) was analyzed by reverse transcription-polymerase chain reaction(RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions(P < 0.05). ROS(P < 0.001) including H2O2(P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC(P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion(I/R) injury was significantly attenuated in the RIPerC group(P < 0.001). A marked increase of NO content and phosphoserine eN OS, both in protein and mR NA levels, was observed in liver graft of the RIPer C group as compared with the OLT group(P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group(P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group(P < 0.01).CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/e NOS/NO pathway.
基金Supported by National Science and Technology Major Project,No.2012ZX10002-017Natural Science Foundation of China for Innovative Research Group,No.81121002+4 种基金National Natural Science Foundation of China,No.81000137 and No.81470891The Qianjiang Talent Program of Zhejiang Province,China,No.2012R10045the Scientific Research Program for the Returned Overseas Chinese Scholars,Ministry of Health,China,No.J20112008National High Technology Research and Development Program of China for Young Scientists(863 Program),No.2015AA020923Ministry of Education,Zhejiang Province,China,No.Y201328095
文摘AIM: To optimize the perfusates used for hypothermicmachine perfusion(HMP).METHODS: Sprague-Dawley rats were assigned randomly to three groups(n = 12 per group) that received either saline, University of Wisconsin coldstorage solution(UW) or histidine-tryptophan-ketoglutarate solution(HTK) as the perfusate. Each group was divided into two subgroups: static cold storage(SCS) and HMP(n = 6 per subgroup). The liver graft was retrieved according to the method described by Kamada. For the SCS group, the graft was directly placed into cold perfusate(0-4?℃) for 6 h after liver isolation while the portal vein of the graft was connected to the perfusion machine for the HMP group. Then the perfusates were collected at different time points for analysis of aspartate aminotransferase(AST), alanine transaminase(ALT) and lactate dehydrogenase(LDH) levels. Liver tissues were obtained for evaluation of histology, dry/wet weight(D/W) ratio, and malondialdehyde(MDA) and adenosine-triphosphate(ATP) levels. The portal vein pressure and velocity were monitored in real time in all HMP subgroups.RESULTS: Comparison of HMP and SCS: Regardless of the perfusate, HMP improved the architecture of donor graft in reducing the congestion around sinusoids and central vein and maintaining sinusoid lining in morphology; HMP improved liver function in terms of ALT, AST and LDH, especially during the 3-6 h period(SCS vs HMP using saline: ALT3, 225.00 ± 105.62 vs 49.50 ± 18.50, P = 0.047; LDH3, 1362.17 ± 563.30 vs 325.75 ± 147.43, P = 0.041; UW: LDH6, 2880.14 ± 948.46 vs 2135.00 ± 174.27, P = 0.049; HTK, AST6, 307.50 ± 52.95 vs 185.20 ± 20.46, P = 0.041); HMP decreased MDA level(saline, 2.79 ± 0.30 vs 1.09 ± 0.09, P = 0.008; UW, 3.01 ± 0.77 vs 1.23 ± 0.68, P = 0.005; HTK, 3.30 ± 0.52 vs 1.56 ± 0.22, P = 0.006). Comparison among HMP subgroups: HTK showed less portal vein resistance than UW and saline(vs saline, 3.41 ± 0.49 vs 5.00 ± 0.38, P < 0.001; vs UW, 3.41 ± 0.49 vs 4.52 ± 0.63, P = 0.007); UW reduced edema most efficiently(vs saline, 0.68 ± 0.02 vs 0.79 ± 0.05, P = 0.013), while HTK maintained ATP levels best(vs saline, 622.60 ± 29.11 vs 327.43 ± 44.66, P < 0.001; vs UW, 622.60 ± 29.11 vs 301.80 ± 37.68, P < 0.001).CONCLUSION: HMP is superior to SCS in maintaining both architecture and function of liver grafts. Further, HTK was found to be the optimal perfusate for HMP.
基金Supported by Science and Technology Innovation Talents Support Plan,Department of Education,Henan Province,China,No.17HASTIT044China Postdoctoral Science Foundation,No.2017M610374
文摘AIM To investigate the protective mechanism of mitofusin-2(Mfn2) in rat remote ischemic perconditioning(RIC) models and revalidate it in alpha mouse liver-12(AML-12) hypoxia cell lines.METHODS Sprague-Dawley rats were divided into three groups(n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting(WB) and quantitative real-time(q RT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture(hypoxia) and anoxic incubator tank culture with Mfn2 knockdown(hypoxia + Si), and data of q RT-PCR, WB, mitochondrial membrane potential(ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected.RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group(P < 0.05). q RTPCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2(MICUs) axis was changed(P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis(P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group(P < 0.005). Finally, q RT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups(P < 0.005).CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.
