Protective Effect And Its Potential Mechanism Of Methane-rich Saline In Mice With Renal Ischemia Reperfusion

Objective:To investigate the protective effect and its potential mechanism of methane-rich saline in mice with renal ischemia reperfusion.Methods:1.Forty-eight male C57BL/6 mice were randomly divided into six groups(n=8): Sham group,MS group,RIR group,RIR+MS 10 group,RIR+MS 20 group and RIR+MS 40 group.Renal I/R injury was produced by clamping bilateral renal pedicles for 45 min followed by reperfusion in RIR,RIR+MS 10 groups,RIR+MS 20 group and RIR+MS 40 group.While mice in the Sham group and MS group underwent laparotomy procedures without occlusion of bilateral renal pedicles.Mice in RIR+MS 10 group,RIR+MS 20 group,RIR+MS 40 group,as well as MS group received administration of 10 mL/Kg,20 mL/Kg,40 m L/Kg and 20 mL/Kg MS,respectively,by intraperitioneal injection immediately after abdominal closure.While 20 m L/Kg normal saline was given at the same time in the Sham group and RIR group.The animals were sacrificed 24 h after reperfusion.Blood samples were collected for determination of the content of serum creatinine(Cr)and BUN.The renal specimens were obtained for examination of the pathological changes(with light microscope).2.Twenty-four male C57BL/6 mice were randomly divided into four groups(n=6): Sham group,MS group,RIR group and RIR+MS group.Twenty m L/Kg NS or MS were given intraperitioneally in each group,respectively.The animals were sacrificed 24 h after reperfusion.The renal specimens were obtained for detecting of content or activities of MPO,SOD,MDA and CAT.3.Thirty-two male C57BL/6 mice were randomly divided into four groups(n=8): Sham group,MS group,RIR group and RIR+MS group.The animals were sacrificed 24 h after reperfusion.The renal specimens were obtained to prepare single cell suspensions,the macrophages were counted using Flow Cytometry.The expression of MCP-1 mRNA was detected by qPCR.Blood samples were collected for determination of the content of tumor necrosis factor-alpha(TNF-?),interleukin-6(IL-6),interleukin-10(IL-10)and interleukin-1?(IL-1?)(by ELISA).4.Twenty-four male C57BL/6 mice were randomly divided into four groups(n=6): Sham group,MS group,RIR group and RIR+MS group.The animals were sacrificed 24 h after reperfusion.The renal specimens were obtained for TUNEL examination.HK-2 cell line was used to detected the expression of caspase 3 protein level by Western blot in vitro.Results:1.Compared with the Sham group,the contents of serum Cr were significantly increased after 24 h of reperfusion in RIR group and RIR+MS 10 group,while those in MS group,RIR+MS 20 group and RIR+MS 40 group remained almost no change.Compared with RIR group,the contents of serum Cr were significantly down-regulated in RIR+MS 10 group,RIR+MS 20 group and RIR+MS 40 group.Compared with the Sham group,the contents of serum BUN were significantly increased after 24 h of reperfusion in RIR group,RIR+MS 10 group,RIR+MS 20 group and RIR+MS 40 group.While the content of BUN in MS group remained almost no change.Compared with RIR group,the contents of serum BUN were significantly down-regulated in RIR+MS 10 group,RIR+MS 20 group and RIR+MS 40 group.The pathological change of kidney showed tissue damages severely in RIR group,including tubular cell swelling,congestion,tubular dilatation,brush border loss,cytoplasmic vacuole,and nuclear loss.While the kidney damage was dose-relatived improved after treated with MS.2.Compared with the Sham or MS group,the MDA level and MPO activity in kidney tissue were significantly increased after 24 h of reperfusion in RIR group,while the activities of SOD and CAT in kidney were significantly decreased.While compared with RIR group,the MDA level and MPO activity were significantly down-regulated in RIR+MS group,while the activities of SOD and CAT were significantly up-regulated.3.Compared with the Sham or MS group,the expression of MCP-1 and macrophages count were significantly increased after 24 h of reperfusion in RIR group.While compared with RIR group,the expression of MCP-1 and macrophages count were significantly down-regulated in RIR+MS group.The contents of serum TNF-?,IL-6 and IL-10 were significantly increased after 24 h of reperfusion in RIR group compared with Sham and MS group.While compared with RIR group,the contents of serum TNF-? and IL-6 were significantly down-regulated in RIR+MS group,while the contents of cerum IL-10 were significantly up-regulated.The contents of serum IL-1? showed no significant difference among these groups.4.Compared with the Sham or MS group,the TUNEL immunostaining showed extensive TUNEL-positive staining in RIR group,predominantly in the outer medulla.While the number of TUNEL-positive cells in kidneys of RIR+MS group had reduced significantly.The expression of caspase 3 protein in HK-2 cell line were significantly increased after 24 h of reoxygenation in HR group in vitro.While compared with HR group,the expression of caspase 3 protein were significantly down-regulated in HR+MS group.Conclusion:Methane-rich saline is effective in preserving renal function against ischemia reperfusion via its anti-oxidant,anti-inflammation and anti-apoptosis activity.