HMGB1Releasc Inhibited By Compounds And Its Mechanism In Macrophages Exposed To LPS In Vitro And In Vivo

Hepatitis B virus (HBV) can cause viral hepatitis B in human beings. In order to decrease the morbidity and mortality of hepatitis B, it is important for us to take early intervening measures to reduce the inflammation of the liver. Macrophages play an important role in the process of inflammation through releasing pro-inflammatory cytokines. High mobility group box1(HMGB1) is actively secreted from the activated macrophages and is an important late inflammatory mediators.The aims of present study are to screen compounds exhibiting promising anti-inflammatory activity when lipopolysaccharide (LPS) activated macrophages was used as in vitro model, and to study the relationship between anti-inflammatory effect and inhibiting effect of HMGB1release of the selected compounds and its underlying mechanisms.This study contains the following four parts:Part Ⅰ:The compounds with promising anti-inflammatory activity were screened. Aim:To screen the compounds with promising anti-inflammatory from40compounds.Methods:RAW264.7cells were exposed to different concentration of LPS and/or compounds for24h, the levels of NO, TNF-α and/or IL-6were detected by ELIS A or Griess. The dosage range of LPS was determined and the compounds with promising anti-inflammatory were screened. Results:Ⅰ. The levels of NO, TNF-a and IL-6elevated significantly in a dose dependent manner in RAW264.7cells exposed to LPS. It was shown that macrophage model for inflammatory response to LPS was established.2. The7kinds of selected compounds, such as resveratrol (Res) and shikonin (Shik), significantly reduced NO and TNF-a.3. Res and Shik decreased NO and TNF-a in a dose dependent manner.Conclusion:1. Seven kinds of promising compounds were selected.2. Res and Shik had promising anti-inflammatory effects.Part Ⅱ:Resveratrol (Res) reduces the pro-inflammatory effects and expression of HMGB1and TLR4induced by lipopolysaccharide in RAW264.7cells. Aim:To study the anti-inflammatory effects of Res and its mechanismMethods:Murine macrophage-like RAW264.7cells (RAW264.7cells) were treated with Res and/or LPS. Levels of TNF-a, IL-6and NO were determined by ELISA or Griess, and levels of HMGB1and TLR4were examined by qRT-PCR and Western Blot assays.Results:Ⅰ. Res significantly reduced the levels of IL-6, NO and TNF-α;2. Expression levels of HMGB1and TLR4were significantly declined. Conclusion:Res could block the inflammatory effects induced by LPS in RAW264.7cells, down-regulation of HMGB1expression may be one of the mechanisms of anti-inflammatory effects caused by Res, and Res may also influence TLR4expression in the HMGB1-TLR4signaling pathway.Part Ⅲ:The inhibiting effects of Shikonin (Shik) on the lipopolysaccharide-induced release of HMGB1in RAW264.7cells via IFN and NF-κB signaling pathways. Aim:To study the anti-inflammation effect of Shik and its mechanism.Methods:RAW264.7cells were treated with Shik and/or LPS for24h, the levels of TNF-a, IL-6and NO were determined by ELISA or Griess; HMGB1, IFN-β and the ratio of nuclear to cytoplasm for NF-κB protein expression were examined by qRT-PCR, ELASA or Western Blot assays.Results:1. Shik could reduce significantly the levels of IL-6, NO and TNF-α;2. Shik significantly decreased the expression of HMGB1, IFN-β and the ratio of nuclear to cytoplasm for NF-κB protein expression;2. The inhibitors of IFN-β signaling molecule JAK and NF-κB could attenuate significantly the expression of HMGB1.Conclusion:Shik could have the anti-inflammatory effects in RAW264.7cells exposed to LPS and one of the mechanisms may be down-regulation of HMGB1expression, which was associated with the IFN-β and NF-κB signaling pathways.Part Ⅳ:The anti-inflammatory effects of resveratrol (Res) and shikonin (Shik) by HMGB1mediation in liver failure mice.Aim:To study the anti-inflammatory effects of the compounds Res and Shik in liver failure miceMethods:The liver injury in mice was observed with H&E staining in mice with acute liver failure induced by galactosamine (GalN)/LPS. The levels of ALT/AST were detected, and the levels of serum HMGBl were determined by ELISA.Results:1. The levels of ALT/AST in GalN/LPS group elevated significantly, and the liver injury was obvious;2. Compared with model group, the levels of ALT/AST in Res group remained high, and the liver injury was obvious;3. The levels of ALT/AST and liver injury in Shik group significantly decreased. However, there was no significant change in the levels of serum HMGB1.Conclusion:The mouse model of liver failure was established, Res did not have any significant anti-inflammatory effects and Shik could have anti-inflammatory effects, but could not decreased the level of HMGB1in serum.