| Backgroud:Acute hepatitis,an extremely hazardous disease,which is a common clinical condition.Increasing evidences indicate that inflammatory response and oxidative stress are important mechanisms of acute hepatitis.Lipopolysaccharide(LPS)and D-galactosamine(D-Galn)induced acute liver injury is one of the most commonly used murine model resembling hepatitis virus invasion,and NF-? B participate in the LPS signal channels/TLR4 downstream cascade,which is the key way to control inflammation factor expression.Furthermore,the nuclear transcription factor Nrf2 is one of the important transcription factors regulate antioxidant stress reaction.TLR4/NF-? B and Nrf2 signaling pathway were involved in the regulation of acute hepatitis in mice induced by LPS/D-Galn.Chinese medicine Livistona chinensis,belonging to the genus of Livistona,has been widely used to clinically treat hepatitis and cancer.Our preliminary study found that the ethyl acetate fraction separated from the fruit of Livistona chinensis(EFLC)effectively ameliorated the liver injury induced by ConA.The major chemical composition of EFLC are flavonoids,but the pharmacological effects of FLCF have not previously been reported.So in this study,we evaluated the effects and mechanism of FLCF,which play an important role in the further development and utilization of Livistona chinensis.Objective:A in vitro model of LPS induced inflammation in RAW246.7 cell and a in vivo model of LPS/D-Galn induced liver injury in mice were used to investigate the pharmacological effects of FLCF and explore which underlying mechanism.Methods:1.FLCF was purified by macroporous resin and Polyamide resin column chromatographies,which content of flavonoids were detected by UV spectrophotometere and major compositions were identificated by HPLC.2.In vitro,the survival rate of RAW264.7 cells was evaluated by MTT assay to determine the safety of FLCF.The level of TNF-a,IL-6,NO and ROS was detected by analysis kits.The expression of Nrf2 was detected by immunofluorescence.The protein expression of iNOS,TLR4/NF-? B and the Nrf2/HO-1 signaling were detected by Western blotting3.The in vivo experiment was divided into two parts.In the first part,70 Babl/c mice were randomly divided into 7 groups(10 mice in each group): normal control group(NC),LPS/D-Galn group(L/D),FLCF(100 mg/kg)+L/D group(L/D+L-FLCF),FLCF(200mg/kg)+L/D(L/D+M-FLCF)group,FLCF(400mg/kg)+L/D group(L/D+H-FLCF),silymarin(100mg/kg)+L/D group(L/D+SYM),FLCF(400 mg/kg)group(H-FLCF),group.The FLCF group were orally administered with FLCF(400mg/kg)and FLCF+L/D group were pretreatment with different concentration of FLCF for 7 days,and L/D group and control group only received 0.3%(CMC-Na)pretreatment.On the day 7,the normal control group and H-FLCF group received intraperitoneal injection of saline,while other 5 groups were given intraperitoneal injection of D-Galn(700 mg/kg)and LPS(1? g/kg)to establish mice model of acute liver injury.The survival rate of mice in each group was observed in 24 h.In the second part,70 Babl/c mice were randomly divided into 7 groups(ditto,10 mice in each group),.Modeling and FLCF pretreatment were identical to previous describtion,and mice were sacrificed after modeling for 6 h.Serum samples were collected to detect the levels of ALT,AST and NO.The weight of liver and spleen were recorded,and the general morphology of liver was observed.And the pathological change of liver were evaluated by staining with H&E.The hepatic levels of GSH,SOD,MDA,TNF-? and IL-6 were measured.The expression of TLR4 and CD68 were detected by immunohistochemical method.The expression of iNOS,Bcl-2/Bax,TLR4/ NF-? B and the Nrf2/HO-1 signaling proteins in liver were detected by Western blotting.Results:1.The content of flavonoids in FLCF was 71.04%,which was mainly composed of the protocatechuic acid,protocatechuic acid,catechin,epicatechin,isoorientin,orientin,vitexin and isovitexin.2.The results of in vitro study showed that FLCF significantly down-regulated the secretion of TNF-?,IL-6 and NO in RAW264.7 cells induced by LPS.In addition,FLCF down-regulated the TLR4/NF-B signalings induced by LPS.Furthermore,FLCF upregulated the expression of Nrf2 protein and promoted its nuclear translocation to enhanced the expression of antioxidant enzyme HO-1 and significantly lowered the release of ROS,which played the role of anti-inflammatory and anti-oxidation.3.The results of study in vivo showed that FLCF significantly improved the survival rate of mice changellged with acute liver injury.FLCF obviously improved LPS/D-Galn induced liver pathological changes of mice,significantly reduced the level of serum ALT and AST,and remarkablely decreased the levels of inflammatory factors in liver involving TNF-?,IL-6 and NO.In addition,FLCF significantly increased the antioxidantive enzymes SOD and GSH,while decreased the level of MDA in liver.Furthermore,the FLCF down-regulated TLR4/NF-? B signaling pathway,but up-regulated of Nrf2/HO-1 signaling pathway and Bcl-2/Bax expression.conclusion:In summary,FLCF has anti-inflammatory and antioxidant pharmacological activities,for its protective action against acute liver injury induced by LPS/D-Galn through regulating TLR4/NF-? B,Nrf2/HO-1 signaling pathway and Bcl-2/Bax protein.Therefore FLCF may be used as a new drug to treat acute hepatitis. |
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