| Background Acute liver failure(ALF)is a group of clinical symptoms in which a large number of hepatocytes are necrotic in a short time,leading to severe functional damage of the liver,hepatic encephalopathy,jaundice,coagulopathy,multiorgan failure and other complications.ALF has a rapid course of disease and high clinical mortality,and there is still a lack of specific drugs or special treatment.The etiology of ALF is complex and its pathogenesis has not been fully elucidated.In the early stage of ALF,a large amount of neutrophils rapidly infiltrate into the liver tissue,releasing a variety of pro-inflammatory cytokines and chemokines,inducing liver inflammation and aggravating hepatocyte injury.Neutrophil elastase(NE)is an immune-regulating serine protease released by neutrophils and participates in the formation of neutrophil extracellular traps(NETs).Recent studies have shown that NETs have a variety of pathophysiological functions such as antibacterial and pro-inflammatory.At present,the role and mechanism of NE and NETs mediating ALF have not been reported.Tian-Huang Formula(THF)is a kind of traditional Chinese medicine formula created by Professor Guo Jiao of Guangdong Pharmaceutical University,composed of panax notoginseng and Coptis chinensis.Previous studies have found that THF can reduce blood glucose,blood lipid and cholesterol.According to the theory of traditional Chinese medicine,heat toxicity and blood stasis are the core pathogenesis of ALF.The role and mechanism of intervention of THF in ALF are still unclear.The purpose of this study was to investigate the effect and mechanism of THF on NEmediated NETs formation and intervention of ALF.Objectives 1.To study the intervened effect of pharmaceutical inhibitor of NE on mouse ALF and reveal the pathophysiological function of NE regulating ALF;2.To study the effect of intervention of NETs inhibitor on mouse ALF;3.Based on NE-mediated NETs formation,the role and mechanism of the intervention of THF in ALF were studied to provide basic research data support for the prevention and treatment of ALF by traditional Chinese medicine.Methods 1.Study on the effect and mechanism of NE inhibitor on experimental ALF in mice Mice were intraperitoneally injected with D-Gal N /LPS(0.75 mg/g;2.5 μg/g)to induce ALF.The confirmed ALF mice were intraperitoneally injected with NE inhibitor Sivelestat(150 mg/kg),while the control group were given blank solvent.The effect and mechanism of NE inhibitor on ALF in mice were studied by evaluating the degree of liver necrosis and liver function,detecting the degree of neutrophil infiltration in liver,the formation of NETs,the expression of m RNA of pro-inflammatory factors and hepatocyte apoptosis genes.2.Study on the effect and mechanism of using inhibitors to block the formation of NETs and intervene ALF GSK484 is a kind of inhibitor of PAD4,which plays a key role in the formation of NETs.In this study,NETs inhibitor GSK484(20 mg/kg)and equivalent blank solvent control were injected intraperitoneally into ALF mouse models.By evaluating the degree of liver necrosis and liver function,detecting the degree of neutrophil infiltration in the liver and the expression of m RNA of inflammatory factors,the effect and mechanism of NETs inhibitor on experimental ALF in mice were studied.3.Study on the effect and mechanism of Tian-Huang Formula on mouse ALF Experimental ALF mice were established by intragastric administration of 300 mg/kg of THF,and the control group were given equal saline.THF regulating the function of NETs was evaluated by detecting the formation of NETs in liver.To study the effect and mechanism of THF on experimental ALF in mice,the degree of liver necrosis and liver function,neutrophil infiltration,the m RNA expression of inflammatory gene and antioxidant gene were evaluated.Results 1.Pharmaceutical NE inhibitor significantly improved experimental ALF in mice Compared with ALF blank control group,the necrosis of liver tissue was significantly reduced in the treatment group with NE inhibitor Sivelestat,and the activities of ALT and AST,indicators of damage to hepatocytes,were decreased by 60% and 30%,respectively.Results of IHC and WB showed that Sivelestat significantly reduced the abundance of neutrophil marker,MPO in liver tissues of ALF mice.The results of immunofluorescence showed that Sivelestat significantly reduced the formation of NETs in liver tissues of ALF mice.The results of RT-PCR showed that m RNA of pro-inflammatory cytokines of TNF-α,RIP1,RIP3 and HMGB1 and others were significantly down-regulated in the treatment group with NE inhibitor Sivelestat.2.GSK484 inhibited the formation of NETs in liver tissue induced by ALF and improved ALF in mice The results of immunofluorescence of liver tissues showed that GSK484 markedly inhibited the formation of NETs induced by ALF in wild-type mice.Compared with the ALF blank control group,the necrotic area of liver tissue was significantly reduced in the NETs inhibitor GSK484 treatment group,and the activities of ALT and AST,indicators of injury of hepatocytes,were decreased by 25% and 30%,respectively.The results of IHC showed that GSK484 evidently reduced the abundance of neutrophil marker,MPO in liver tissues of ALF mice.The results of Q-PCR showed that NETs inhibitor GSK484 down-regulated the expression of m RNA of TNF-α in the liver of ALF mice.3.Tian-Huang Formula attenuated experimental ALF in mice by regulating NETs The results of immunofluorescence showed that THF significantly reduced the formation of NETs in liver of ALF mice.Compared with ALF blank control group,the necrotic area of liver tissue was reduced by 75% in the treatment group with THF.Serum ALT activity decreased by 35% and AST decreased by 15%.The abundance of MPO protein was significantly reduced in liver tissue of the treatment group with THF.The results of RTPCR showed that THF significantly down-regulated the m RNA expression of pro-inflammatory genes of IL-1β,IL-6 and up-regulated the m RNA expression of antioxidant gene of G6 pdh.Conclusions1.NE inhibitor Sivelestat can improve mouse ALF by regulating the formation of NETs,reducing liver inflammation and apoptosis of hepatocytes;2.NETs inhibitor GSK484 has significant therapeutic effects on ALF mice;3.Tian-Huang Formula improve mouse ALF via significantly inhibiting the formation of NETs in mouse liver with ALF,decreasing liver inflammation and improving the antioxidant capacity of hepatocytes.In conclusion,the results in the present study reveal that NE and NETs play an important pathophysiological function in mediating mouse ALF,supporting NE and NETs as potential targets for the development of new drugs(including TCM)of ALF.Meanwhile,this study revealed that Tian-Huang Formula has the effect of regulating NETs and improving mouse ALF,supporting further research on the effect and mechanism of this formula in treating ALF. |
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