The Role Of NALP3 Inflammasome In The Pathogenesis Of Liver Injury With D-GalN/LPS In Mice

Background:Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection.Sepsis is characterized by high morbidity and high mortality.The liver is one of the most easily damaged organs.The severity of septic liver injury affects the prognosis of sepsis.At present,it is believed that sepsis is associated with uncontrolled inflammatory reaction,but the effect of anti-inflammatory treatment is not ideal.Therefore,it is very important to explore the molecular mechanism of sepsis in the treatment of liver injury caused by sepsis.A large number of studies have found that NALP3 inflammasome play a key role in the inflammatory response,but there are few studies on the pathogenesis of septic liver injury.Objective:To investigate the dynamic expression of NALP3 inflammasome and downstream related molecules in D-GalN/LPS induced acute liver injury model in mice,and to explore the mechanism of action of NALP3 in inflammatory liver injury.The aim of this study was to provide a new theoretical basis for the pathogenesis and treatment of septic liver injury.Methods:50 C57BL/6 mice were randomly divided into five groups:normal group,model group of 6 hours group,12 hours group,24 hours group and 48 hour group,each group had 10 mice,intraperitonealled D-GalN and LPS to made the model.The normol group was injected with equal amount of normal saline.After that,the mice were killed at the corresponding time points,and then blood samples were taken to detect the levels of ALT and AST,and the gross morphology of the liver was observed.HE staining was performed to analyze the pathological changes of the liver.Real-TimePCR was used to detect the expression of NALP3,Bcl-2 and Caspase-1 in liver tissue of mRNA.Western-Blot was used to detect the expression of NALP3,Bcl-2 and Caspase-1 in liver tissue.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of IL-18 and IL-1β in liver tissue.Results:(1)Compared with the normal group of the serum ALT and AST levels(39.33±8.31 vs 141.33±27.33),the group of 6h(468.41±155.41 vs 521.33±180.00,p<0.05)and the group of 12h(4173.00±491.67 vs 4323.67±478.14,p<0.01)were significantly increased,the group of 12 h reached peak.Compared with the group of 12 h,the group of 24h(276.83±112.12 vs 319.50±89.51,p<0.01)and the group of 48h(43.33±19.35 vs 183.17±69.47,p<0.01)were significantly decreased.(2)Compared with the normal group of the mRNA level of NALP3 and Caspase-1 in liver tissue(7.57±0.84 vs 6.85±0.71),the group of 6h(7.99±0.72vs8.49±0.28,p>0.05)and the group of 12h(11.08±0.35 和 9.78±0.96,p<0.01)were increased,the group of 12 h reached peak.Compared with the group of 12 h,the mRNA levels of NALP3 and Caspase-1 in liver tissue of mice in the group of 24h(8.36±1.01 vs 7.95±1.18,p<0.01)and the group of 48h(7.69±0.67 vs 6.96±0.57,p<0.01)were significantly reduced.(3)Compared with the normal group of the protein expression of the NALP3 and Caspase-1 in liver tissue(0.17±0.06 vs 0.25±0.06),the group of 6h(0.23±0.09 vs 0.50±0.17,p>0.05)and the expression of NALP3 and Caspase-1 in the group of 12h(0.48±0.06 vs 1.17±0.26,p<0.01)increased significantly,the group of 12 h reached peak.Compared with the group of 12 h,the expression of NALP3 and Caspase-1 in the group of 24h(0.39±0.07 vs 0.76±0.07,p<0.01)and the expression of NALP3 and Caspase-1 in the group of 48h(0.18±0.08 vs 0.37±0.08,p<0.01)decreased.(4)Compared with the normal group of its expression level of mRNA and protein of Bcl-2(5.68±0.63 vs 1.09±0.10),the group of 6h(4.84±1.09 vs 0.87±0.09,p<0.05)and the group of 12h(4.59±0.67 vs 0.59±0.12,p<0.01)decreased significantly,the group of 12 h reached minimum.Compared with the group of 12 h,The expression level of mRNA protein and in liver tissue of mice after modeling Bcl-2 in the group of 24h(5.15±0.94 vs 0.80±0.11,p<0.05)and the expression level of Bcl-2 in the group of 48h(6.04±0.32 vs 1.03±0.14,p < 0.01)increased significantly.(5)Compared with the normal group of the IL-18 andIL-1β in liver tissue of mice(63.04±13.91vs1216.66±103.90),the group of 6h(73.03±7.74 vs 1635.78±145.46,p>0.05)and the group of 12h(92.15±5.52 vs 1874.25±123.27,p<0.01)were significantly increased,the group of 12 h reached peak.Compared with the group of 12 h,the group of 24h(76.64±9.17 vs 1482.27±54.55,p<0.01)and the levels of IL-18 and IL-1β in the group of 48 hours(63.33±10.45 vs 1213.86±115.26,p<0.01)were significantly decreased.Conclusion:NALP3 inflammasome and its downstream molecules Caspase-1,IL-18 and IL-1β pathway play an important role in sepsis induced liver injury with D-GalN/LPS.