Inhibitory effect of necrostatin-1 on necroptosis of retinal ganglion cells in rats with acute ocular hypertension / 中华实验眼科杂志

Objective:To investigate the inhibitory effect of specific inhibitor of necroptosis necrostatin-1 (Nec-1) on necroptosis of retinal ganglion cells (RGCs) in rats with acute ocular hypertension.Methods:Twenty-four adult male Sprague Dawley rats were randomly divided into normal control group, model control group, Nec-1 treatment group and negative control group by random number table method, with 6 rats in each group.High intraocular pressure (IOP)-induced ischemia and reperfusion model was established through anterior chamber irrigation of 0.9% sodium chloride solution in left eyes of the rats, raising the IOP to 110 mmHg (1 mmHg=0.133 kPa) for 60 minutes.Nec-1 (4 mmol/L, 2 μl) or dimethyl sulfoxide (2 μl) was intravitreally injected immediately in Nec-1 treatment group and negative control group following modeling, respectively, according to grouping.No intervention was administered to the normal control group.Paraffin sections of rat retinas of the left eyes in different groups were prepared seven days after modeling.The retinal structure was observed by hematoxylin-eosin staining, and the expression levels of thymocyte antigen-1 (Thy-1) and glial fibrillary acidic protein (GFAP) were detected via immunohistochemical staining.All animal experiments were approved by an Ethics Committee of Tianjin Union Medical Center (No.2017 Quick audit C01).Results:Seven days after modeling, compared with normal control group, the retinal nerve fiber layer was thinner in model control group and negative control group, and the RGCs were arranged loosely, and cells in the inner nuclear layer were reduced and arranged disorderly, and cells in the outer nuclear layer were normal or enlarged.Compared with model control group and negative control group, the nerve fiber layer was thickened and the number of RGCs was significantly increased in Nec-1 treatment group.The number of Thy-1-positive RGCs was decreased in model control group, negative control group and Nec-1 treatment group than normal control group, and there were more Thy-1-positive RGCs in Nec-1 treatment group than model control group and negative control group.The integrated absorbance ( A) value of GFAP protein in normal control group, model control group, negative control group and Nec-1 treatment group was 47.209±15.311, 116.220±18.194, 116.382±19.020, 92.818±10.236, respectively, showing statistically significant differences among them ( F=24.675, P<0.001). The integrated A value of GFAP protein was significantly increased in model control group, negative control group and Nec-1 treatment group than normal control group, and the integrated A value of GFAP protein in Nec-1 treatment group was lower than that in model control group and negative control group, with statistically significant differences (all at P<0.05). Conclusions:Nec-1 can promote RGCs survival by inhibiting the necroptosis of RGCs in rats with acute intraocular hypertension.

Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition / 华西口腔医学杂志

OBJECTIVES@#To investigate the role and molecular mechanism of necrostatin-1 (Nec-1), a specific programmed cell necrosis inhibitor, in promoting the oxidative stress response of macrophages under high glucose (HG) environment.@*METHODS@#Macrophages were cultured in control (5.5 mmol·L@*RESULTS@#The HG group had increased ROS level and MDA activity (@*CONCLUSIONS@#HG promotes oxidative stress on macrophages by upregulating RIP1 expression.

Protective effect of necrostatin1 on the damage of pancreas islet cells induced by TNFα / 中南大学学报(医学版)

OBJECTIVES@#To investigate whether necrostatin-1 (Nec-1) can protect islet cells from the damage induced by TNF-α.@*METHODS@#After isolation and purification, the neonatal porcine islet cell clusters (NICCs) were divided into 3 groups (islets 10 000 IEQ/group): a Nec-1 group (Nec-1+TNF-α was added to the culture medium), a TNF-α group (TNF-α was added to the culture medium), and a control group (pure medium). The number of cells was observed after 48 h of co-culture. The cell death was evaluated by AO/EB staining. Insulin secretion and DNA of islets were detected by chemiluminescence and nucleic acid quantitative analysis. RT-PCR assay was used to examine the mRNA expressions of insulin gene, glueogan gene and somatostatin gene. Flow cytometry analysis was used to detect the viability of B cells.@*RESULTS@#The number of islets in Nec-1 group, TNF-α group and the control group were (8 425±2 187), (4 325±778), and (7 122±1 558) IEQ, respectively. Compared to the other two groups, the number of dead cells in TNF-α group was greatly increased. The insulin/DNA values in the Nec-1 group, TNF-α group and blank control group were (13.21±3.15), (2.47±0.45), and (7.44±0.97) mIU/mg, respectively. Compared to the TNF-α group and the control group, the mRNA relative expression levels of insulin gene (6.73±1.07), glucagon gene (10.13±1.98), somatostatin gene (8.57±1.11) were significantly increased in the Nec-1 group (all <0.05), the rate of live cells (97.32±1.87)% and live B cells (90.86±3.68)% were increased significantly in the Nec-1 group (all <0.05).@*CONCLUSIONS@#TNF-α can induce neonatal porcine islet cells damage, which is attenuated in the presence of Nec-1. Nec-1 can increase the content of endocrine cells in NICCs.

Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.

Interaction between necroptosis and apoptosis in MC3T3-E1 cell death induced by dexamethasone / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between necroptosis and apoptosis in MCET3-E1 cell death induced by glucocorticoids.@*METHODS@#MC3T3-E1 cells were incubated with 10-6 mol/L dexamethasone followed by treatment with the apoptosis inhibitor z-VAD-fmk (40 μmol/L) or the necroptosis inhibitor necrostatin-1 (40 μmol/L) for 2 h. At 72 h after incubation with dexamethasone, the cells were harvested to determine the cell viability using WST-1 assay and the rate of necrotic cells using annexin V/PI double staining; the percentage of apoptotic cells was determined using Hoechst staining. The mitochondrial membrane potential and the level of ATP in the cells were also evaluated. Transmission electron microscopy was used to observe the microstructural changes of the cells. The expressions of RIP-1 and RIP-3 in the cells were detected by Western blotting.@*RESULTS@#At a concentration of 10-6 mol/L, dexamethasone induced both apoptosis and necroptosis in MC3T3- E1 cells. Annexin V/PI double staining showed that inhibition of cell apoptosis caused an increase in cell necrosis manifested by such changes as mitochondrial swelling and plasma membrane disruption, as shown by electron microscopy; Hoechst staining showed that the percentage of apoptotic cells was significantly reduced. When necroptosis was inhibited by necrostatin-1, MC3T3-E1 cells showed significantly increased apoptosis as shown by both AV/PI and Hoechst staining, and such changes were accompanied by changes in mitochondrial membrane potential and ATP level in the cells.@*CONCLUSIONS@#In the process of dexamethasone-induced cell death, necroptosis and apoptosis can transform reciprocally accompanied by functional changes of the mitochondria.

Effect of Necrostatin-1 on the inflammation in unilateral ureter obstruction mice / 临床与实验病理学杂志

Purpose To observe the effect of Necrostatin-1 (Nec-1),a necroptosis inhibitor,on the inflammation in unilateral ureter obstruction mice.Methods Male C57BL/6J mice were randomly divided into sham operation group,unilateral ureter obstruction operation group and UUO + Nec-1 treatment group,and the mice were sacrificed at 7th day after operated.Scr and BUN were measured.The pathological changes in the kidney were observed by HE staining.Immunohistochemistry and Western blot were used to detect the protein expression of necroptosis-related indicators RIP1,RIP3,MLKL and inflammatory cytokines TNF-α,IL-1β and MCP-1.Results Compared with sham operation group,the expression of RIP1,RIP3 and MLKL protein increased in the renal tissue of UUO mice,accompanied with increased expression of TNF-α,IL-1β and MCP-1.Nec-1 treatment significantly decreased above-mentioned protein expression in UUO mice,and also reduced renal interstitial inflammation and renal tubal injury according to HE staining.Scr and BUN levels suggested improved renal function.Conclusion Nec-1 could relieve the inflammatory reaction in renal tissue of the UUO mice by inhibiting necroptosis,which may be a new target for the treatment of secondary inflammation.

Effects of Nec-1 on necroptosis and retinal ganglion cells in mouse model of retina ischemia reperfusion injury / 眼科新进展

Objective To observe the influence of necrostatin-1 (Nec-1) on necroptosis and retinal ganglion cells (RGC) in mice with retinal ischemia reperfusion injury (RIRI).Methods Together 60 wild-type C57 mice were randomly divided into three groups (n =20),and they were control group,experimental group and blank group.Firstly,as for investigation the effect of Nec-I on necroptosis,15 mice in the blank group left untreated,15 mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,and 15 in the control group without pretreatment.After 4 h,the RIRI model was established by anterior chamber perfusion in the latter two groups.Then retinas in 5 mice in each group were harvested 3 days after ischemia reperfusion injury for Western blot,immunofluorescence quantitative PCR and immunofluorescence staining to detect the expression of mRNA and protein of IL-1β,IL-6,TNF-α,RIP3,RIP1 and Caspase-8.Secondly,the rest of 5 mice in 20 of each group were collected and retrogradely labeled with fluoro-gold (FG) to explored the influence of Nec-1 on RGC.Mice in the blank group left untreated.Mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,while the control group was not treated anything 7 days after fluorescence labeling;after 4 h,the RIRI model was established by anterior chamber perfusion in the two groups.The retinal tissue was harvested and RGC counting was performed 3 days after ischemia reperfusion injury.Results When compared with control group,mRNA expression levels of IL-1β,IL-6,TNF-α,RIP3 and RIP1 in the experimental group were decreased significantly (all P ＜ 0.001),but Caspase-8 mRNA did not change obviously (P =0.654 8).Western blot and immunofluorescence staining showed that RIP3 expression decreased dramatically in the experimental group when compared with the control group.FG labeled RGC counting presented that RGC number of each field in experimental group was significantly larger than that in the control group (P ＜ 0.001).Couclusion Nec-1 can block necroptosis and significantly increase RGC number in mice model of experimental RIRI.

Effects of Nec-1 on necroptosis and retinal ganglion cells in mouse model of retina ischemia reperfusion injury / 眼科新进展

Objective To observe the influence of necrostatin-1 (Nec-1) on necroptosis and retinal ganglion cells (RGC) in mice with retinal ischemia reperfusion injury (RIRI).Methods Together 60 wild-type C57 mice were randomly divided into three groups (n =20),and they were control group,experimental group and blank group.Firstly,as for investigation the effect of Nec-I on necroptosis,15 mice in the blank group left untreated,15 mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,and 15 in the control group without pretreatment.After 4 h,the RIRI model was established by anterior chamber perfusion in the latter two groups.Then retinas in 5 mice in each group were harvested 3 days after ischemia reperfusion injury for Western blot,immunofluorescence quantitative PCR and immunofluorescence staining to detect the expression of mRNA and protein of IL-1β,IL-6,TNF-α,RIP3,RIP1 and Caspase-8.Secondly,the rest of 5 mice in 20 of each group were collected and retrogradely labeled with fluoro-gold (FG) to explored the influence of Nec-1 on RGC.Mice in the blank group left untreated.Mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,while the control group was not treated anything 7 days after fluorescence labeling;after 4 h,the RIRI model was established by anterior chamber perfusion in the two groups.The retinal tissue was harvested and RGC counting was performed 3 days after ischemia reperfusion injury.Results When compared with control group,mRNA expression levels of IL-1β,IL-6,TNF-α,RIP3 and RIP1 in the experimental group were decreased significantly (all P ＜ 0.001),but Caspase-8 mRNA did not change obviously (P =0.654 8).Western blot and immunofluorescence staining showed that RIP3 expression decreased dramatically in the experimental group when compared with the control group.FG labeled RGC counting presented that RGC number of each field in experimental group was significantly larger than that in the control group (P ＜ 0.001).Couclusion Nec-1 can block necroptosis and significantly increase RGC number in mice model of experimental RIRI.

Necroptosis mediates high glucose-induced injury in human umbilical vein endothelial cells / 中国病理生理杂志

AIM: To explore whether necroptosis contributes to the high glucose (HG)-induced damage in hu-man umbilical vein endothelial cells (HUVECs).METHODS: The protein levels of receptor-interacting protein 3 (RIP3) and cleaved caspase-3 were detected by Western blot.The intracellular levels of reactive oxygen species (ROS) were deter-mined by DCFH-DA staining followed by photofluorography.Mitochondrial membrane potential (MMP) was measured by rhodamine 123 staining followed by photofluorography.RESULTS: Treatment of HUVECs with HG at different concentra-tions (10, 20 and 40 mmol/L glucose) for 24 h gradually enhanced the expression levels of RIP3.Treatment of HUVECs with HG (40 mmol/L glucose) for different time (3 h, 6 h, 9 h, 12 h and 24 h) also up-regulated the expression levels of RIP3, peaking at 9 h.Pretreatment of HUVECs with 20 μmol/L Z-VAD-FMK (an inhibitor of caspase) for 30 min before exposure to HG enhanced the expression level of RIP3.Pretreatment of HUVECs with 100 μmol/L necrostatin-1 (an inhi-bitor of necroptosis) for 1 h before exposure to HG alleviated the HG-induced injuries, such as a decrease in cell viability, an increase in ROS generation and dissipation of MMP, but up-regulated the protein level of cleaved caspase-3.CON- CLUSION: Necroptosis mediates HG-induced injury in HUVECs.There is a negative interacting between necroptosis and apoptosis.

The effect of necrostatin-1 on high mobility group protein-B1 in liver of rats with hemorrhagic shock / 中华急诊医学杂志

Objective To investigate the effect and mechanism of necrostatin-1 (Hec-1) on the level of HMGB-1 protein in liver of rats with hemorrhagic-traumatic shock.Methods A number of 96 male SD rats were divided into sham-operated group,dimethyl sulfoxide (DMSO) group and Nec-1 group (n=32in each) by randomized number method.Rat model of hemorrhagic-traumatic shock was made by fracture of femoral bone and tibia bone and exsanguination from femoral vein until 30 mmHg and maintained at 30-40 mmHg for 90 min,then the shed blood was transfused back with Ringer's solution.The rats in shamoperated group were only under anesthesia for separating and ligating blood vessels,without exsanguination to induce hemorrhagic shock and without replenishment with blood.Rats in Nec-1 group were given 1 mg/kg Nec-1 through femoral vein 5 min before replenishment with blood and Ringer' s solution,while the rats in DMSO group were given equal volume of DMSO solution instead.Eight rats in each group were sacrificed separately at 2 h,8 h,16 h and 24 h after replenishment.The serum and liver tissues of rats in each group were collected to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST),and to observe the pathological changes in liver with hematoxylin-eosin (HE) staining.The level of HMGB-1 in serum was detected by using ELISA.The cytoplasm protein and total protein expressions of HMGB-1 were assessed by using western blot analysis.Results Compared with DMSO group,levels of serum ALT at 8 h (P ＜0.05),16 h (P ＜ 0.01) and 24 h (P ＜ 0.01) in Nec-1 group were significantly lower.Level of serum AST in Nec-1 group were lower compared with DMSO group at 8 h (P ＜ 0.01),16 h (P ＜ 0.01) and 24 h (P ＜0.01).Compared with DMSO group,levels of serum HMGB-1 at 8 h (P ＜ 0.05),16 h (P ＜0.01) and 24 h (P ＜ 0.01) in Nec-1 group were significantly lower.Under light microscopy and transmission electron microscope,hepatic lobule destroyed,the blood extravasated,the immunocyte infiltrated and cellular organelle destroyed were found.Compared with DMSO group,the level of HMGB-1 protein in cytoplasm protein in Nec-1 group were significantly decreased at 8 h (P ＜ 0.01),16 h (P ＜0.01) and 24 h (P ＜0.01).The level of HMGB-1 protein in total protein in Nec-1 group were significantly decreased 8 h (P ＜ 0.05) and 24 h (P ＜ 0.05).Conclusions Nec-1 can remarkably protect the liver of rats with hemorrhagic-traumatic shock,decrease the level of HMGB-1,and protect the hepatocyte effectively.

Effect of autophagy on necroptosis of renal tubular epithelial cells in sub-total nephrectomy rats / 中国病理生理杂志

[ ABSTRACT] AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy ( SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS:Male Sprague-Dawley rats were randomly assigned to control group ( n=6 ) and SNx group ( n=42 ) .The rats in SNx group were subjected to SNx.Sham surgery was performed in the rats in control group.The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks ( n=6) and the other rats in SNx group were divided into SNx+vehi-cle group, SNx+necrostatin-1 (Nec-1) group and SNx+3-methyladenine (3-MA) group.The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species ( ROS) in the rat kidney were determined by Western blot and DCFH-DA staining.The death of renal tubular epi-thelial cells in the SNx rats was observed by TUNEL staining and electron microscopy.Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen ( BUN) , serum creatinine ( SCr) and the pathological changes of the renal tissues were ana-lyzed.RESULTS:The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx (P cells were decreased in the SNx rats treated with Nec-1 and 3-MA (P<0.01), but 3-MA did not reduce the increased con-centration of ROS.In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr (P<0.05), glomeruloscle-rosis index and tubulointerstitial injury score (P<0.01).CONCLUSION:Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats.Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.

The effect of necrostatin-1 on expression of liver monocyte chemotactic protein-1 in septic rats / 中华危重病急救医学

Objective To investigate the effect of necrostatin-1 (Nec-1) on the expression of liver monocyte chemotactic protein-1 (MCP-1) in septic rats and its mechanism. Methods Forty-eight male Sprague-Dawley (SD) rats were randomly divided into sham group, model group, and Nec-1 group by randomized digital number method, with 16 rats in each group. The model of sepsis was reproduced by cecal ligation and puncture (CLP). Rats in sham group received anesthesia, and flipping the cecum followed by closure of the abdomen without ligation of the cecum. Rats in Nec-1 group were given 1 mg/kg Nec-1 [25 mg Nec-1 solution dissolved in 2.5 mL of dimethyl sulfoxide (DMSO)] through caudal vein 30 minutes before operation, while the rats in model group were given 0.1 mL/kg of DMSO only. Blood from abdominal aorta and liver tissue in each group were collected at 0 hour and 8 hours after operation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined with automatic biochemistry analyzer. The pathological changes in liver were observed under light microscope using hematoxylin-eosin (HE) staining. The serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme linked immunosorbent assay (ELISA). The MCP-1 mRNA expression in the liver was determined by reverse transcription-polymerase chain reaction (RT-PCR). Results There was no significant differences in the levels of serum ALT, AST, TNF-α, IL-6 and expressions of liver MCP-1 mRNA at 0 hour among three groups, and the liver cellular structure was normal. At 8 hours, compared with sham group, the expressions of serum ALT, AST, TNF-α, IL-6 and liver MCP-1 mRNA were significantly increased in model group and Nec-1 group [ALT (U/L): 172.35±21.88, 129.67±18.20 vs. 60.04±11.74, AST (U/L): 511.03±34.92, 363.51±25.25 vs. 254.83±31.04, TNF-α(ng/L): 603.96±24.18, 483.87±26.60 vs. 265.74±15.14, IL-6 (ng/L): 975.62±65.37, 712.09±45.47 vs. 310.42±13.88, MCP-1 mRNA (2-ΔΔCt): 7.09±0.18, 5.51±0.45 vs. 0.99±0.06, all P < 0.05]. Levels of the above parameters in Nec-1 group at 8 hours were significantly decreased compared with those of model group (all P < 0.05). Under light microscopy, it was noted that the structure of hepatic lobules was destroyed, with exacerbation of immunocyte infiltration at 8 hours in model group. At 8 hours, it was found that Nec-1 alleviated the pathological damage in Nec-1 group. Conclusion Nec-1 can protect the liver of rats with sepsis, lower the expression of serum TNF-α and serum IL-6 and liver MCP-1 mRNA, and obviously reduce the damage of inflammation.

Protective effect of necrostatin-1 on the liver of rats with trauma induced hemorrhagic shock / 中华危重病急救医学

Objective To investigate the effects of necrostatin-1 (Nec-1) on the liver of rats with trauma induced hemorrhagic shock.Methods Trauma induced hemorrhagic shock model was produced by adopting the left femur,tibia fracture and soft tissue injury,bleeding and reperfusion in male Sprague-Dawley (SD) rats.A total of 22 rats were divided into model group and Nec-1 group with 11 rats in each group by randomized digital number method and the 72-hour mortality was observed.In addition,72 rats were randomly divided into sham group,model group,Nec-1 group with 24 rats in each group.Rats in sham group were only received anesthesia,separating and ligating blood vessels,without trauma induced hemorrhagic and reperfusion,and the rats in Nec-1 group were received 1 mg/kg Nec-1 through femoral vein 5 minutes before reperfusion,while the rats in model group were received the same amount of solvent.The serum and liver tissues of each group were collected at 2,4,8 hours after reperfusion.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by automatic biochemistry analyzer.The pathology changes in liver were observed by hematoxylin-eosin (HE) staining.The mRNA expressions of tumor necrosis factor-oα (TNF-α) and interleukin-1β (IL-1β) in the liver were detcrmined by reverse transcription-polymerase chain reaction (RT-PCR).The protein expressions of receptor interaction of protease 1/3 (RIP1/RIP3) were also assessed by Western Blot analysis.Results Compared with model group,Nec-1 significantly reduced the 72-hour mortality [18.18％ (2/11) vs.63.64％ (7/11),P=0.040].Two hours after trauma induced hemorrhagic shock and reperfusion,the expressions of ALT and AST in model group were significantly increased compared with those in sham group [ALT (U/L):110.21 ±22.32 vs.80.98 ± 19.94,AST (U/L):364.29 ±64.83 vs.279.76 ±70.64,both P＜0.05],and reached the peak at 8 hours [ALT (U/L):387.41 ± 47.11 vs.82.76 ± 22.44,AST (U/L):973.35 ± 77.51 vs.261.49 ±52.03,both P＜0.01].Levels of serum ALT and AST in Nec-1 group were significantly decreased compared with model group [ALT (U/L) 4 hours:144.64± 33.79 vs.213.96± 36.21,8 hours:159.48 ± 43.57 vs.387.41 ± 47.11; AST (U/L) 4 hours:398.78 ± 59.48 vs.630.61 ± 59.93,8 hours:427.38 ± 80.75 vs.973.35 ± 77.51,all P＜0.01].Under light microscopy,it was noted that the hepatic sinus expansion,liver cells degeneration,necrosis,as well as infiltration of abundant inflammatory cells were observed.But the pathology changes in hepatic tissues were significantly mitigated in Nec-1 group.Along with the time extension,the mRNA expressions of TNF-α and IL-1β and the protein expressions of RIP1 and RIP3 were markedly up-regulated.Compared with model group,difference in the mRNA expressions of TNF-α and IL-1β in hepatic tissues in Nec-1 group were statistically significant,and the most obvious difference was at 8 hours [TNF-α mRNA:1.457 ± 0.081 vs.2.317 ± 0.062,IL-1β mRNA:0.690 ± 0.087 vs.1.812 ± 0.112,both P＜0.01].But there was no statistically significant difference in RIP1 and RIP3 between Nec-1 group and model group [RIP1 protein 8 hours:0.561 ± 0.033 vs.0.587 ± 0.036,RIP3 protein 8 hours:0.976 ± 0.040 vs.1.044 ± 0.115,both P＞0.05].Conclusion Nec-1 may be remarkable protect effect on the liver of rats with trauma induced hemorrhage shock and reperfusion,and the intrinsic mechanisms need further investigation.