Nec-1 Group Research Articles

The effect of necrostatin-1 on expression of liver monocyte chemotactic protein-1 in septic rats

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. 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). 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. 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.

Necrostatin-1 inhibits Hmgb1-IL-23/IL-17 pathway and attenuates cardiac ischemia reperfusion injury.

Ischemia reperfusion (IR) injury is a major issue in cardiac transplantation and inflammatory processes play a major role in myocardial IR injury. Necrostatin-1 (Nec-1) is a small molecule capable of inhibiting RIP1 kinase activity and attenuates inflammation-mediated tissue injury. In our study, hearts of C57Bl/6 mice were flushed and stored in cold Bretschneider solution for 8h and then transplanted into syngeneic recipients. We found that Nec-1 decreased cardiomyocyte necrosis and recruitment of neutrophils and macrophages. Troponin T (TnT) production on 24h after myocardial IR injury was reduced by Nec-1 administration. Cardiac output at 60mmHg of afterload pressure was significantly increased in hearts with Nec-1 administration and the cardiac allograft survival in Nec-1-treated animals was significantly prolonged (MST=90days in IR+Nec-1 group, P<0.05 as compared with IR group, MST=83.5days). Nec-1 treatment attenuated ROS generation and increased expression of NOS2 and COX-2. The expression of Hmgb1, IL-23, and IL-17A were also decreased with Nec-1 administration. Furthermore, the decreased TnT expression induced by Nec-1 was abrogated with exogenous Hmgb1 administration. In conclusion, Nec-1 played a protective role in cardiomyocyte IR injury, and this was associated with inhibited Hmgb1-IL-23/IL-17 pathway.

Protective effect of necrostatin-1 on the liver of rats with trauma induced hemorrhagic shock

To investigate the effects of necrostatin-1( Nec-1) on the liver of rats with trauma induced hemorrhagic shock. 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 a nd 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, and the rats in Nec-1 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-α (TNF-α) and interleukin -1β (IL-1β) in the liver were determined 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. Compared to the 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>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-β 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-β 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-β 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]. 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.