Researching The Protective Effect Of Lycium Barbarum Polysaccharides On Carbon Tetrachloride-induced Acute Liver Injury In Mice By KEAP1/IKKβ/NF-κB Signaling Pathway

Objective This study was to investigate the protective effect of Lycium Barbarum Polysaccharides（LBP）against carbon tetrachloride（CCl₄）induced acute liver injury（ALI）in mice by KEAP1/IKKβ/NF-κB Signaling Pathway,so as to provide the experimental basis for the treatment of ALI and the development of related medicines.Methods Forty ICR male mice were randomly divided into five groups:normal control group,model group,LBP groups with different doses(100 mg·kg^-1、200 mg·kg^-1、400mg·kg^-1).LBP powder was dissolved in normal saline at the corresponding concentration and then gavage,the normal control group and the model group were respectively given the same dose of normal saline,the process lasted 14 days with once a day.At one hour after the last administration,mice received an intraperitoneal injection of CCl₄solution dissolved peanut oil,while control group injected the same dose of peanut oil via intraperitoneal way.At 24hours after the administration of CCl₄,the body weight of mice were detected,collected orbital blood,and the collected liver tissue was weighed and then to calculate the varation of liver indices（liver mass/body weight）of every mouse.The changes of alanine amminotransferase（ALT）and aspartate aminotransferase（AST）and expressions of tumor necrosis factor-α（TNF-α）,interleukin-1（IL-1β）and interleukin-6（IL-6）were measured by ELISA to observe the levels of inflammation.The histopathological changes of liver were boserved by HE staining.The expression condition of keich-like ECH-associated protein 1（KEAP1）,inhibitor of nuclear factors-κB kinase（IKKβ）,nuclear factor-κB（NF-κB）,TNF-α,IL-1βand IL-6 in hepatocytes,immunohistochemistry（IHC）analysis was performed.Immunofluorescence（IF）used to evaluate the dynamic relationship between KEAP1,IKKβand NF-κB in liver tissue.The m RNA transcriptions’expression levels of KEAP1,IKKβ,NF-κB,TNF-α,IL-1βand IL-6 in the mouse liver homogenates were monitored by real-time fluorescence quantiative PCR（RT-q PCR）.The protein activities of KEAP1,IKKβ,NF-κB,apoptosis associated speck-like protein domain（ASC）,nucleotid-binding oligomerization domain receptor family pyrin domain containing protein 3（NLRP3）,cysteine aspartate specific protease-1（caspase-1）,TNF-α,IL-1βand IL-6 in liver tissues were detceted through Western blot.Results（1）The results of liver indexes showed that the body weight in model group lowered,liver weight and liver indexes increased than the normal control group（p<0.05）;while LBP group at different doses were the opposite of the indicators than the model group（p<0.05）.（2）The ELISA test’s results showed that the expressions of ALT,AST,TNF-α,IL-1βand IL-6 in model group were higher than that of the normal control group（p<0.05）;while those indexes in the LBP group at different doses were lower than the model group（p<0.05）.（3）Histopathological evaluation of HE staining indicated that morphological structure of hepatocytes in normal control group was no significant abnormal;the arrangement of hepatic lobule disordered and cells were varying degrees of infiltration of inflammatory cells in model group;above the pathological morphology indicators of different concentration of LBP group were improved to varying degrees.（4）IHC results show that the expression of KEAP1 in the model group was lower than normal control group,（p<0.05）,while the expressions of IKKβ,NF-κB,TNF-α,IL-1βand IL-6 were higher（p<0.05）;compared with the model group,the KEAP expression of in the LBP groups with different doses enhanced（p<0.05）,IKKβ,NF-κB,TNF-α,IL-1βand IL-6 expressions decreased（p<0.05）.（5）The IF revealed that:compared with normal control group,the positive expression of KEAP1 in cytoplasm of liver cell in model group decreased,and the positive expression of IKKβin cytoplasm and the positive expression of NF-κB in nucleus increased;compared with model group,the positive expression of KEAP1 in cytoplasm accelearated,the positive expressions of IKKβin cytoplasm and NF-κB in nucleus reduced in LBP groups with different doses.（6）RT-q PCR’s results showed that the KEAP1 m RNA expression in the model group decreased than normal control group（p<0.05）,while m RNA expressions of IKKβ,NF-κB,TNF-α,IL-1βand IL-6 increased（p<0.05）;compared with model group,the KEAP1 m RNA expression increased and m RNA expressions of IKKβ,NF-κB,TNF-α,IL-1βand IL-6 reduced in different doses of LBP groups（p<0.05）.（7）The results of WB indicated that the expression of KEAP1 protein in the model group decreased than normal group（p<0.05）,while the protein levels of IKKβ,NF-κB,ASC,NLRP3,caspase-1,TNF-α,IL-1βand IL-6 increased（p<0.05）;compared with model group,the expression of KEAP1protein in different doses of LBP groups raised（p<0.05）,while the protein levels of IKKβ,NF-κB,ASC,NLRP3,caspase-1,TNF-α,IL-1βand IL-6 alleviated（p<0.05）.Conclusion1.LBP may effectively improve the level of damage and reduce the expression of NF-κB and other inflammatory factors to CCl₄-induced ALI in mice.2.The mechanism of protective effect of LBP on CCl₄-induced ALI in mice may be related to enhancing the expression of KEAP1 and attenuating the activity of IKKβ/NF-κB signaling pathway.