Study On The Protective Effects Of Circadian Rhythm And A Novel Functional Glucan Against Mammalian Hepatic Injury

Hepatic injury due to its universality and diversity of incentives is widespread in the population. Clinical data had disclosed a number of pathological features of liver injury, during which the association with the circadian rhythm is increasingly being paid attention by researchers. Circadian rhythm is the phenomenon that various physiological activities exhibit rhythmic oscillation in vivo. Large number of reports had demonstrated that circadian rhythm disorder links with many diseases occurrence, including liver disease, however, until now, the association between circadian clock and liver injury is limited to the clinical phenotype data. The underlying molecular mechanism that how does liver injury occcurrence affect the molecular clock system and how does clock system affect the incidence of liver injury have not been reported yet. Based on this, the present study systematically investigates the impact of circadian clock on liver damage occurrence and its mechanism using gene knockout mice. On the other hand, although in the past decades, advances in the understanding of the pathogenesis of liver injury have been impressive, there are still few safety and cheap drugs used to treat liver disease. Thus, the other aim of the current study is to assess the possible protective effect of a novel beta-glucan-salecan on liver injury and the related mechanisms systematically, which provide the theoretical basis for salecan to become a potential therapeutic drug that treat liver damage.In the present study, we first detected the impact of a typical chronic liver damage-hepatic fibrosis on the rhythmic expression of hepatic clock genes to determine the molecular linkage between circadian system and hepatic injury. Results from real-time PCR revealed that compared with normal mice, the mesors of Clock and Perl were significantly higher in fibrosis mice. The amplitudes of the variations in Bmall and Perl were significantly lower in fibrosis group. Additionally, the acrophases of Clock, Perl and Cryl were delayed in fibrosis mice compared with control mice. Especially, the rhythmic expression of Cry2 was totally disrupted in fibrotic liver. In addition, in fibrosis animals, the mesors of both PPARa and POR were significantly lower and the amplitudes of both genes were markedly attenuated. There were no significant differences in rhythmic parameters of Per2 in both groups, which may imply that the rhythmic expression of Per2 gene could be an important contribution mechanism to liver fibrosis development. To verify this hypothesis, we further set up various liver injury models in WT and Per2-/- mice to investigate the impact of Per2 on the development of liver injury and the related mechanism. CCl4 induced acute liver injury experiment showed that serum ALT and AST activities were elevated in Per2-null mice compared with wide-type mice at 24 hours after CCl4 treatment, which was in agreement with the observation of significantly larger areas of centrilobular necrosis in the livers of Per2-null mice. Furthermore, HPLC analysis demonstrated the intercellular ATP content was lower in Per2-null mice than in wild-type mice, Per2-/- liver exhibited enhanced ATP biosynthesis inhibitor-Ucp2 gene expression level. These data indicated that Per2 could attenuate CCl4 induced acute hepatotoxicity through decreasing Ucp-2 gene expression to maintain intercellular ATP content. Hepatic fibrosis was induced in WT and Per2-/- mice by repetitive intraperitoneal CCl4 injection for 4 weeks. Masson's trichrome staining and liver immunohistochemistry for the activated HSC marker a-SMA were performed, the results showed that Per2-/- mice displayed much more collagen deposition and a-SMA protein expression. Furthermore, serum ALT and AST activity were significantly elevated in fibrotic Per2-/- mice than fibrotic WT mice. On the other hand, compared to WT mice, Per2-/- mice exhibited less efficiency in fibrosis resolution. TUNEL and a-SMA double staining result revealed that the number of apoptotic HSC was significantly decreased in Per2-/- mice than WT mice. Transfected Per2 cDNA into cultured HSC accelerated cell apoptosis. Our data showed that the clock gene Per2 attenuates liver fibrosis by decreasing liver injury development and more importantly, it could accelerate collagen depression by promoting HSC apoptosis. Bile duct ligation induced obstructive cholestasis experiment also demonstrated the hepato-protective effect of Per2. Following 10-day BDL, livers from Per2-/- mice exhibited markedly increased extent of bile infarct and extracellular matrix (ECM) deposition compared with WT mice. Cholestatic liver injury-promoting gene-PAI was markedly upregulated in Per2-/- mice. Collectively, our data had disclosed that Per2 plays a protective role against liver injury, preliminary offer the molecular association between circadian system and hepatic injury.Salecan, produced by a new strain Agrobacterium sp. ZX09 is a novel water-soluble extracellularβ-glucan that isolated by our group recently. Mice were given salecan or PBS for 4 days, and ethanol was than administered orally 1 h after the last injection to induce acute alcoholic liver injury. Our results indicated that pretreatment with salecan significantly ameliorated hepatic damage induced by ethanol, as the evidence of markedly reduced serum aminotransferase activities, TC, TG content and hepatic TC, TG content. Histological results indicated that obvious swelling and hydropic degeneration of hepatocytes was occurred in PBS group, while salecan pretreatment markedly improved the histopathological changes. Furthermore, salecan administration remarkably alleviated the formation of oxidative stress induced by ethanol ingestion. The mRNA level of PPARa which is a major gene responsible for fatty acid oxidation was significantly increased after salecan pretreatment. The expression of DGAT1 that was an important gene responsible for triacylglycerol synthesis was markedly decreased after salecan administrated. These findings suggested that salecan could attenuate acute liver injury induced by ethanol treatment. Moreover, CCl4 induced acute liver injury experiment showed that mice pretreated with salecan for 4 days showed a marked and significant reduction of serum ALT, AST and LDH activities compared to the PBS group at 24 and 48h post CCl4 injection. Histopathological examination of the livers revealed that hepatocellular degeneration and necrosis were significantly attenuated at early stage during CCl4 intoxication and liver recovery was markedly accelerated at later stage in salecan pre-administrated mice. Furthermore, the expression of anti-oxidant genes was significantly elevated in salecan treated group. Interestingly, the administration of salecan remarkably enhanced hepatocyte proliferation monitored by PCNA immunohistochemistry in the recovery phase after CCl4 injection. Taken together, these results demonstrated that salecan exhibits protective action on acute hepatic injury induced by CCl4 through attenuating oxidative stress at early stage and accelerating hepatocyte regeneration during later stage. Thus, salecan may potentially represent a novel, protective strategy against liver injury.