Nur77 Inhibits Proliferation Of Hepatocytes Via NF-?B/STAT3 Signaling Pathway In The Early Stage Of Liver Regeneration

Background and objective:The liver has a strong regenerative ability.After removing 2/3 of the liver of the mouse,the residual liver tissue can restore its original size in about 10 days and maintain the original function of the liver.Liver regeneration is an important protection mechanism that guarantees the liver to maintain metabolic homeostasis.However,when the liver suffers destructive damage or chronic liver damage,the liver's regenerative ability is significantly destroy,and it is no longer possible to repair itself through regeneration,resulting in acute and chronic liver failure.Our country has the largest population of hepatitis B and liver cancer in the world.The burden of liver disease is heavy.Acute and chronic liver insufficiency and liver failure caused by various liver diseases are major problems that clinicians need to solve.Studying the molecular regulation mechanism of liver regeneration will provide potential biological therapeutic targets for the treatment of chronic hepatitis,liver fibrosis,liver failure and other liver diseases,and has important scientific and clinical significance.Nur77 is a receptor protein encoded by NR4A1.Since no endogenous ligand is found,it is considered to be an orphan nuclear receptor.Together with Nurr1 and Nor1,it constitutes the NR4 A nuclear receptor family.The NR4 A nuclear receptor family plays an important role in regulating cell proliferation,apoptosis and energy metabolism in various tissues and cells.It has been reported in the literature that Nurr1 is significantly upregulated early after hepatectomy.Nor1 and its target genes CCND1 and VCAM-1 were up-regulated during the cell proliferation phase after hepatectomy.The previous study of our group found that Nur77 knockout mice had accelerated hepatocyte proliferation and early liver regeneration after Partial Hepatectomy(PHx),but inflammation damage and apoptosis significantly increased during liver regeneration.Gene expression of inflammatory mediators and chemokines is also significantly up-regulated.Liver regeneration is a complex and precise process jointly regulated by multiple steady-state mechanisms such as inflammatory damage-repair,proliferation-apoptosis,etc.The team speculates that Nur77 is regulating liver Regenerative immune plays an important role in the regulation of homeostasis.In this study,PHx was used to construct a liver regeneration model,and Nur77-specific agonist Csn-B was used to activate Nur77 activity,to detect changes in inflammatory factors and inflammation-related signaling pathway molecules in liver tissue,and to explore the molecular mechanism of Nur77 inhibition of liver regeneration.Materials and Method:72 8-to-12-week-old C57BL/6 mice were randomly divided into 3 groups: sham operation group(Sham group),DMSO group and experimental group(Csn-B group),24 mice in each group,30 minutes before surgery The Csn-B group was intraperitoneally injected with the Nur77 targeted agonist Csn-B(13mg/kg,LD50),and the Sham group and the DMSO group were injected with the same amount of DMSO solvent.Liver tissues were collected at 3 h,24 h,48 h,and 72 h after surgery,and the expression levels of Nur77,liver-to-body weight ratio,Ki-67 positive cell ratio,KC activated cell ratio,mitotic phase ratio,The m RNA levels of IL-6,IL-10,IL-12 and the protein levels of ?H2AX,Cleaved Capase3,Cleaved Capase8,cell cycle related proteins,phosphorylated p65 and phosphorylated STAT3.HL-7702 and NCTC1469 hepatocytes were cultured with Nur77 agonist Csn-B and antagonist DIM-C-p Ph OH to detect CCK-8 cell proliferation and flow cytometry to detect cell cycle.Results:1.We measure the expression level of Nur77 during the liver tissues of PHx mice by q RT-PCR and Western blot.The expression level of Nur77 was rapidly induced in the Csn-B group and DMSO group at the early stage of PHx,and the Csn-B group Nur77 m RNA expression level was 3h.It was 4 times that of the DMSO group(36.78±2.75 vs 9.56±1.90,P<0.001),and was 2.6 times that of the DMSO group at 24h(16.16±2.40 vs 6.12±1.16,P<0.001),and the expression level of Nur77 protein was higher than that of DMSO The group was increased by 2 times at 3h and 24h(P<0.001).At 48 h,Nur77 decreased to similar levels in the Sham group.2.The Csn-B group of mice after PHx showed a lower liver weight ratio than the DMSO group.HE tissue pathology and immunohistochemical detection of the mitotic phase and Ki-67 positive cells in the Csn-B group were significantly lower The protein expression levels of Cyclin D1,Cyclin B,and Cyclin E in the DMSO group were significantly lower than those in the DMSO group by Western blot detection in the Csn-B group,suggesting that Nur77 inhibited the proliferation of liver cells and liver regeneration after PHx.3.The expression of ?H2AX protein was detected by immunohistochemistry and Western blot.The ratio of immunohistochemical ?H2AX positive cells and the expression level of ?H2AX protein in the Csn-B group were significantly lower than those in the DMSO group.Western blot detected the expression of Cleaved Capase3 and Cleaved Capase8,Csn-Cleaved Capase3 protein expression level of mice in group B was significantly lower than that of DMSO group at 3h and 72 h after PHx operation,and Cleaved Capase8 protein expression level was significantly lower than that of DMSO group mice at 3h,24 h,48h and 72 h.HE histopathological detection of focal necrosis of liver lobule in Csn-B group was significantly less than that in DMSO group.3.q RT-PCR detection found that the expression of IL6 and IL12 in the Csn-B group was significantly lower than that in the DMSO group,while IL12 was significantly higher than that in the DMSO group.Immunohistochemical detection revealed that the proportion of Fs/80 positive areas in Csn-B group was significantly lower than that in DMSO group at 3h and 24 h.Phosphorylated NF-?B and phosphorylated STAT3 in Csn-B group were detected by Western blot.The protein expression level was lower than that in the DMSO group,suggesting that Nur77 inhibited the inhibition of NF-?b/STAT3 signaling pathway and KC cell activation.4.Treatment of HL-7702 and NCTC1469 hepatocytes with Nur77 agonist Csn-B and antagonist DIM-C-p Ph OH,cell proliferation was detected by CCK-8 and cell cycle was detected by flow cytometry.Nur77 agonist Csn-B was found Inhibits the proliferation of hepatocytes,while the antagonist DIM-C-p Ph OH promotes hepatocyte proliferation.Conelusions:1.After PHx in mice,Nur77 is rapidly induced in the early stage of liver regeneration.Nur77 targeting agonist Csn-B can significantly induce Nur77 expression after PHx.2.Nur77 can significantly inhibit the proliferation of liver cells and liver regeneration in mice after PHx surgery.3.Nur77 can significantly inhibit DNA damage and apoptosis in liver tissue of mice after PHx operation.4.Nur77 may inhibit the liver regeneration process by inhibiting the NF-?B/STAT3 pathway and Kupffer cell activation in the initial stage of liver regeneration after PHx mice.5.In vitro cell experiments,the Nur77 agonist Csn-B inhibits the proliferation of hepatocytes,while the antagonist DIM-C-p Ph OH promotes hepatocyte proliferation.