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Study On The Mechanism Of VX-765 In Acute Liver Failure

Posted on:2024-04-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:M J JiaoFull Text:PDF
GTID:1524307157962819Subject:Internal medicine
Abstract/Summary:PDF Full Text Request
Objective: Acute liver failure(ALF)is a clinical pathological syndrome characterized by massive or sub-massive necrosis of liver cells caused by various factors such as viruses,drugs,and alcohol,resulting in severe decompensation of liver function.Clinical symptoms often include bleeding,jaundice,liver coma,high mortality rate and poor prognosis.Significant systemic inflammatory responses and the release of a large number of pro-inflammatory cytokines occurs in the process and finding new therapeutic methods has always been a challenge in ALF research.As a new type of programmed cell death,pyroptosis is mainly mediated by cysteinyl aspartate-specific proteinase-1.In the classic signal pathway of pyroptosis,when cells are stimulated by related signals,caspase-1 is activated and consequently mediates the activation of GSDMD.After activation,GSDMD releases its N-terminal domain and punches holes in the cell membrane,causing the rapture of cell membrane and the release of cell contents and proinflammatory cytokines(IL-1β and IL-18)which lead to pyroptosis.In this process,IL-1β and IL-18 are activated by caspase-1.The secretion and release of proinflammatory cytokines IL-1β and IL-18 can further aggravate the body’s inflammatory responses,presenting an inflammatory cascade amplification effect.VX-765 is a commonly used pyroptosis inhibitor that can effectively and selectively inhibit the activity of caspase-1.Although a large number of studies have shown that VX-765 can play a protective role in various diseases by inhibiting inflammatory responses,the role and mechanism of VX-765 in the development of acute liver failure remains unclear.The purpose of this study is to investigate whether VX-765 has a protective effect on acute liver failure and its mechanism.Methods: Healthy male C57BL/6 mice(SPF grade)aged 8-12 weeks were selected for animal experiments.The animal model of acute liver failure was established by intraperitoneal injection of D-galactosamine(D-Gal N)and lipopolysaccharide(LPS)into mice,with a dose of 700mg/kg of D-Gal N and10μg/kg of LPS respectively.VX-765(100 mg/kg)was injected intraperitoneally into mice 2 hours prior to D-Gal N/LPS treatment in ALF mice.PPARα si RNA(50 μM/kg)and control si RNA(50 μM/kg)were injected into mice through the tail vein 24 hours before D-Gal N/LPS administration.Six hours after D-Gal N/LPS injection,the mice were anesthetized with chloral hydrate.Blood was taken from the inferior vena cava and liver tissue was obtained.A portion of the liver tissue was fixed with 10%neutral formalin solution and sent for pathological examination.To observe the severity of liver tissue damage,histopathological analysis was applied by hematoxylin and eosin(HE)staining and an automatic biochemical analyzer was used to detect the levels of serum transaminases ALT and AST in mice.Paraffin sections of liver tissue from patients with hepatitis B virus-related acute liver failure(HBV-ALF),chronic hepatitis B(CHB)and from normal control group were selected respectively for immunohistochemical staining.Human normal hepatocyte LO2 cells were selected for the in vitro experiments,and LPS(20 ng/ml)was used to stimulate LO2 cells to induce cell inflammation.VX-765(50 μM)and PPARα Si RNA(50 n M)were administered 2 hours and 24 hours respectively before LPS administration and cells were collected 6 hours after LPS stimulation for related experiments.The levels of inflammatory cytokines,pyroptosis-associated proteins and PPARαwere detected using quantitative reverse transcription-polymerase chain reaction(q RT-PCR)and western blotting.Results:1.As acute liver failure progressed,the degree of damage to liver tissue and the expression of proinflammatory cytokines and pyroptosis-related proteins gradually increased.2.VX-765 could improve the survival rate of mice with acute liver failure,reduce the degree of liver tissue damage and alleviate inflammatory responses during the progression of ALF,thereby providing a protective effect against ALF.Compared with the ALF model group(D-Gal N/LPS),the intervention group(VX-765+D-Gal N/LPS)significantly improved the survival rate of mice,reduced the degree of pathological damage to liver tissue,and decreased serum transaminase(ALT,AST)levels.The m RNA and protein expressions of NF-κB and proinflammatory cytokines IL-1β and IL-18 were reduced with a statistically significant difference.3.The results of cell experiments showed that VX-765 also had anti-inflammatory effects in vitro and could inhibit LPS-induced cellular inflammatory responses.Compared with the model group(LPS group),the m RNA and protein expression of NF-κB and proinflammatory cytokines IL-1β and IL-18 decreased in the VX-765 intervention group(VX-765+LPS).With increasing VX-765 concentrations(10 μM、 25 μM、 50 μM),IL-1β,IL-18 and NF-κB expression levels decreased gradually in a dose-dependent manner and the difference was statistically significant.4.In clinical practice,as liver tissue damage in patients gradually worsens and ALF pogresses,the expression levels of proinflammatory cytokines(IL-1β,IL-18)and pyroptosis-related protein caspase-1 increased while PPARα expression decreased.5.VX-765 could upregulate PPARα expression both in vivo and in vitro.PPARα m RNA and protein expression levels of liver tissue in VX-765 intervention group(VX-765+D-Gal N/LPS)significantly increased compared to the ALF model group(D-Gal N/LPS).In vitro experiments have shown that PPARα expression could be upregulated gradually with increasing concentrations of VX-765(10 μM、25 μM、50 μM)in a dose dependent manner with a statistically significant difference.6.Inhibition of PPARα expression could reduce the protective effect of VX-765 on ALF mice,decrease the survival rate of mice,exacerbate the degree of liver tissue damage and inflammatory responses.When PPARαsi RNA was applied to inhibit PPARα,the survival rate of mice in the VX-765+PPARα si RNA+D-Gal N/LPS group decreased significantly with darker liver tissues and more severe pathological damage compared with the VX-765+Control si RNA+D-Gal N/LPS group.Liver cells showed massive necrosis,liver cords dissociated and diffuse congestion occurred in liver sinuses,accompanied by amounts of inflammatory cell infiltration,increasing transaminase levels(ALT,AST)and elevated expression levels of NF-κB and proinflammatory cytokines IL-1β and IL-18 with a significant difference statistically.7.In vitro experiments showed that PPARα inhibition could reverse the anti-inflammatory effect of VX-765 and exacerbate the LPS-induced cellular inflammatory response.Compared with the VX-765+Control si RNA+LPS group,the expression levels of NF-κB and proinflammatory cytokines IL-1βand IL-18 increased apparently and the different was statistically significant when PPARα si RNA was applied to inhibit PPARα.Conclusions:1.As ALF progresses,inflammatory responses and pyroptosis deteriorate gradually.2.VX-765 can inhibit pyroptosis and reduce inflammatory responses to protect against ALF.3.The protective effect of VX-765 on acute liver failure can be achieved by upregulating PPARα expression,while the protective effect can be reversed by inhibiting PPARα.4.VX-765 might become a possible therapeutic strategy for ALF.
Keywords/Search Tags:Acute liver failure, VX-765, Pyroptosis, Peroxisome proliferator-activated receptor α
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