| Objective To investigate the effect and mechanism of Necrostatin-1 suppress inflammatory cytokines in liver in hemorrhagic-traumatic shock rats.Methods This study was divided into two parts.PartⅠThe model was SD rats suffered hemorrhagic-traumatic shock, followed by fluid resuscitation.A number of 30 male SD rats were divided into sham-operated group, DMSO group and Nec-1 group with 10 rats in each group by digital random number generators. Rats in sham-operated group were only reveived anesthesia by Chloral hydrate, separated and ligateing left common carotid artery, without trauma induced hemorrhagic and reperfusion. Rats in Nec-1 group were suffered hemorrhagic-traumatic shock, 5 minutes before reperfusion they were injected Nec-1(1 mg/kg) by femoral vein, and the rats in DMSO group were injected the same doses DMSO 5 minutes before reperfusion through the femoral vein. Ten rats were excuted at 24 hour after reperfusion in each group. The serum and liver tissues of each group were collected. We detected serum alanine ALT and AST. The pathology changes in liver were observed by hematoxylin-eosin(HE) staining. The cell organelle damage was observed by transmission electron microscope. The degree of hepatocellular apoptosis was detected by Tunel kit. The MIP-1α m RNA and MCP-1 m RNA in the liver after 24 h resperfusion was determined by RT-PCR. The expression of MIP-1α and MCP-1 in serum was detected by ELISA. Part Ⅱ The model was also SD rats suffered hemorrhagic-traumatic shock, followed by fluid resuscitation.A number of 96 male SD rats were divided into sham-operated group, DMSO group and Nec-1 group with 32 rats in each group by digital random number generators. Rats in sham-operated group were only reveived anesthesia by Chloral hydrate, separated and ligateing left common carotid artery, without trauma induced hemorrhagic and reperfusion. Rats in Nec-1 group were suffered hemorrhagic-traumatic shock, followed by fluid resuscitation,5 minutes before reperfusion they were injected Nec-1(1 mg/kg) by femoral vein, and the rats in DMSO group were injected the same doses DMSO 5 minutes before reperfusion through the femoral vein. Eight rats were executed at 2 h,8 h,16 h and 24 h hours after reperfusion in each group. The serum and liver tissues of each group were collected. We detected serum alanine ALT and aspartate AST. The pathology changes in liver were observed by hematoxylin-eosin(HE) staining. The expression of HMGB-1 in serum was detected by ELISA. The cytoplasm protein and total protein expressions of HMGB-1 were assessed by western blot analysis. The expression of RIP3 was detected by western boltting.Results PartⅠUnder light microscopy, it was noted that hepatic lobule destroyed, the blood extravasated, hepatocyte ballooning degeneration and the immunocyte infiltrated in DMSO group. The damage was obviously ameliorated in Nec-1 group. Under the ransmission electron microscope, cell membrane structures was almost complete in DMSO group. Structural failure of mitochondria and endoplasmic reticulum was observed in DMSO group. And the damage was obviously ameliorated in Nec-1 group. There is no different between DMSO group and Nec-1 in the degree of hepatocellular apoptosis by Tunel detection(P > 0.05). Levels of serum ALT(248.05±67.55 、 127.2±20.04 vs.73.46±23.75),AST(823.74±89.04 、416.67±100.46 vs.236.36±67.79),MIP-1α(0.249±0.149、0.182±0.099 vs.0.063±0.026) and MCP-1(981.519±60.483、714.424±55.092 vs.310.517±15.877) at 24 hours in Nec-1 group were significantly decreased compared with model group and DMSO group( P < 0.05). The expressions of MIP-1α m RNA(7.134±2.811 、2.924±1.153 vs.1.000±0.000) protein and MCP-1 m RNA(7.319±0.517 、5.554±0.435 vs.1.000±0.000) in Nec-1 group were significantly decreased(P<0.05). PartⅡUnder light microscopy, it was noted that hepatic lobule destroyed, the blood extravasated, hepatocyte ballooning degeneration and the immunocyte infiltrated in DMSO group. The damage was obviously ameliorated in Nec-1 group. Compared with DMSO group, levels of serum ALT,serum AST and serum HMGB-1 at 8 h,16 h and 24 h in Nec-1 group were significantly decreased. Compared with DMSO group,the expressions of HMGB-1 protein in cytoplasm protein in Nec-1 group were significantly decreased at 8 h(0.025±0.003 、 0.184±0.007 vs.0.124±0.013),16 h(0.022±0.004 、 0.400±0.019 vs.0.318±0.030) and 24 h(0.022±0.003 、0.341±0.051 vs.0.245±0.014). The expressions of HMGB-1 protein in total protein in Nec-1 group were significantly decreased 8 h(1.010±0.042 、1.348±0.046 vs.1.298±0.073) and 24 h(1.053±0.048、1.438±0.634 vs.1.374±0.060). Compared with DMSO group,the expression of RIP3 at 8 h(0.169±0.023 、0.673±0.042 vs.0.543±0.126),16 h(0.178±0.022、1.044±0.115 vs.0.836±0.090) and 24 h(0.186±0.023 、 1.446±0.141 vs.1.266±0.128) in Nec-1 group were significantly decreased.Conclusion 1. Nec-1 can protect the liver of rats with hemorrhagic-traumatic shock, decrease the expression of MIP-1α m RNA and MCP-1 m RNA, reduce the infiltration of immunocyte in hepatocyte. 2. Nec-1 can alleviate the damage of liver in rats with hemorrhagic-traumatic shock, decrease the expression of HMGB-1 and RIP3, protect the hepatocyte effectively. |
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