Effect Of Arkadia On The Degradation Of Snon Protein In Liver Fibrosis In Vivo And Vitro

Background/aims: Liver fibrosis is the common phase of the development of various chronic liver diseases and liver cirrhosis or liver function, but its pathogenesis has not been fully clarified, and the overall effect is still not ideal. Therefore, we need to further explore the molecular mechanism of the occurrence and development of hepatic fibrosis, to provide effective experimental evidence for the target to intervene the development of liver fibrosis and to reverse the liver fibrosis.Transforming growth factor-β1, TGF-β1, is the most effective and powerful cytokine in promoting liver fibrosis at present. Smad signal is the main channel to play its role. And the SnoN protein can block the TGF-β1/Smad signal and inhibit the hepatic fibrosis. Our previous study found that SnoN protein is reduced progressively in the fibrotic liver from different liver fibrosis animal model, and in liver tissues from patients with hepatitis B virus. Our study also found SnoN is downregulated in fibrotic liver by a mechanism independent of gene transcription stability.Arkadia, an E3 ubiquitin ligase that is implicated in the degradation of SnoN, was expressed in the liver tissus. However, the role and mechanism underlying Arkadia degradation SnoN in the fibrotic liver remains unknown. So we started this study from two aspects: in vivo and in vitro, to research the molecular mechanism of Arkadia degradation by SnoN, Our study is of great significance on illustration the mechanism of liver fibrosis. The study findings will help to understand the role and mechnism of Arkadia in regulating SnoN protein expression in liver fibrosis.Methods: ① Experanmental study in vivo: Fifty healthy male Wistar rats(six to eight weeks of age, 180 to 200 g) were housed in a temperature-and humidity-controlled facility under a 12 h light-dark cycle and given free access to a standard laboratory diet and tap water. Rats were randomly divided into normal control(n =7) and liver fibrosis model groups(n = 43). The liver fibrosis model group received intraperitoneal injections of 0.15 m L/100 g 50% CCl4 in olive oil(5:5, v/v) twice weekly for eight weeks, and rats in the normal control group received intraperitoneal injections of the same volume of physiological saline over the same period. The liver fibrosis reversion phase was then investigated over the four weeks subsequent to completing the full course of intraperitoneal injections. Rats were sacrificed at 2, 4, 6, 8, 10 and 12 weeks. Seven rats were sacrificed per time point, All rats from the normal group were sacrificed during the eighth week. And the success of the rat liver fibrosis model was determined by observing the gross specimens, HE staining, Masson staining and serum biochemical determination. And the expression of Arkadia, SnoN, FN, TGF-β1, Collagen-I and α-SMA was detected by Blotting Western technology. Real time PCR to detect the expression of Arkadia and FN and the expression of Arkadia, SnoN and FN was detected by immunohistochemistry.②Experanmental study in vitro: HSC-T6 cells were pretreated with TGF-β1 with the concentration 10ng/ml and collected at 2, 6, 12, 24 or 48 hours(h) or treated with different concentrations of TGF-β1 for 12 h and then collected the cells. And we use cell immunofluorescence method to detecte the expression of Arkadia, SnoN and FN. We use Real time PCR and real time PCR to detect the expression of Arkadia and FN.Results: The results including gross specimens, HE staining, Masson staining, and serum biochemical indicated that the model of chemical liver fibrosis induced by carbon tetrachloride and its reversal model were successful; the expression of mRNA and FN was up-regulated in the fibrosis liver tissue. And the expression of FN, TGF-β1, Collagen- I and α-SMA increased with the progression of fibrosis. The expression of SnoN protein in hepatic fibrosis tissue was decreased, while the expression of Arkadia protein increased. And in the HSC-T6 cells stimulated by TGF-β1, the expression of Arkadia and FN increased with the increase of the concentration and time of TGF-β1, and the expression of 10ng/ml and 12 h were the most.Conclusions: The expression of Arkadia protein in liver tissue increased, and the expression of SnoN decreased. Liver fibrosis can be reversed after the cessation of injury, and the expression of Arkadia and SnoN protein will also change accordingly.