Circadian Rhythm Disorder Affects The Prognosis Of Liver Injury Via TGF-β1 Mediated Cell Senescence

Objective: Acute liver injury(ALI)is characterized by the rapid onset of hepatocyte damage and sometimes leads to rapid deterioration of liver function and eventually results in adverse prognosis.The prognosis of liver injury depends on the regression of inflammation and the regeneration of liver cells.This process is affected by a variety of internal and external factors including biological rhythm.The aim of this study was to explore the effect of circadian disorder(CD)on the severity of acute liver injury by establishing a mouse model of circadian disturbance concomitant with liver injury.The TGF-β/Smad mediated autonomous senescence pathway was investigated and the potential therapeutic effect of TGF-β receptor Ⅰ inhibitor was studied.The results will provide a new perspective for the clinical management of patients with acute liver injury.Methods: 1.Establishment of animal models.The CD mouse model was established by continuously exposure to artificial light.The effectiveness of the model was evaluated from three aspects: the general status of animal,changes in behavior,and the changes in clock gene in m RNA level.ALI mouse models were established by three D-Gal N/LPS dose gradients(200/2.5,400/5,800/10)to determine the optimal dose for building ALI model.2.The role of TGF-β/Smad pathway in the CD+ALI mice.The CD+ALI mice model was established by continuous light exposure combined with D-Gal N/LPS intraperitoneal injection.The survival conditions of control group(control),circadian dysrhythmia group(CD),acute liver injury group(ALI)and dysrhythmia combined with liver injury group(CD + ALI)were observed and compared.Serum transaminase,IL-6 and TNF-α were detected.The severity of liver injury in each group was compared by histological examination,TUNEL fluorescence staining.The expression of P53,P21,γ-H2 AX,SASP and other senescence related components in liver tissue were detected by q RT-PCR and Western blot.The senescence level of liver cells was evaluated by cell senescence associated β-galactosidase(SA-β-gal)staining.q RT-PCR and Western blot were used to detect the expression of TGF-β/Smad signal components in liver tissue of CD+ALI mice.P53/P21 homologous double labeled immunofluorescence staining was used to verify the existence of senescence.The expression of TGF-β1 m RNA in CD mice was detected by q RT-PCR at multiple time points.3.The effect of TGF-β receptor Ⅰ inhibitor on CD+ALI.After the establishment of CD mice model,TGF-β receptor Ⅰ inhibitor was given before the establishment of liver injury model,and the severity of liver injury and the senescent level of liver cells were detected by the above methods to further evaluate the potential therapeutic value of TGF-β signal inhibition in CD+ALI mice.Results: 1.Establishment of animal models.Continuous illumination for one month can lead to significant behavioral changes in mice,with the changes of the rhythms of clock,bmal1,nr1d1,per1,per2,per3 and cry1 in liver tissue,indicating successfully establishment of dysrhythmia mice model.The combined intraperitoneal injection of 400 mg / kg D-Gal N and 5 μg/kg LPS was the optimal dose for ALI model,under which the mice died 5.5 hours after administration and the 72 hour-survival rate reached 50%.Serum ALT and AST were significantly higher than those in the control group at the peak of liver injury.HE staining showed typical histological presentation of ALI,including irregular hepatic lobules and liver plate,hepatocyte swelling,hepatocellular hemorrhagic necrosis,and infiltration of inflammatory cells.2.TGF-β/Smad pathway and hepatocyte senescence were upregulated in CD+ALI.Compared with ALI mice,CD + ALI mice showed more severe liver injury.Death occurred 4.5 hours after administration in CD+ALI group with the 72-hour survival rate lowered to 20%.The serum levels of ALT,AST,IL-6 and TNF-α were significantly higher than those in ALI group(P < 0.01).The results of HE staining in liver tissue showed that there were more hemorrhagic necrosis and inflammatory cell infiltration in CD+ALI mice,and more TUNEL positive cells.In CD+ALI mice,the transcription levels of p21,TGF-β1,IL-1α and cyclin E were significantly up-regulated,while the m RNA level of cyclin D was down-regulated.The protein levels of P21,Cyclin D and Cyclin E were consistent with the changes of m RNA level.The expression levels of γ-H2 AX in CD+ALI were significantly higher than those of ALI group.The p53 and its phosphorylated and acetylated proteins were higher in CD+ALI than control group,but there was no significant difference between the ALI and CD+ALI group.In CD+ALI group,the expression of β-galactosidase was higher than the other two groups.3.TGF-β receptor Ⅰ inhibitor LY2157299 alleviated the liver injury and hepatocyte senescence in CD+ALI mice.Compared with CD+ALI group,the levels of serum ALT,AST,IL-6 and TNF-α at the peak of inflammation were significantly decreased in treatment group(P < 0.01).P21,TGF-β1,IL-1α and cyclin E were significantly lower than those in ALI group,while Cyclin D was higher,suggesting the improvement of cell senescence.Conclusion: Circadian rhythm disorder induced by continuous light exposure can lead to poor prognosis of ALI via TGF-β/Smad mediated senescence pathway.TGF-β receptor Ⅰ inhibitor LY2157299 can alleviate hepatocyte senescence and liver injury in CD mice with ALI.