The Study Of Recombinant Human CXCL8(3-72)/G31P Impact On Inflammation, Liver Cell Proliferation And Fibrosis Of Nonalcoholic Fatty Liver Of Mice

Background: Fatty liver is caused by one or more incentives to hepatic steatosis andlipid accumulation is characterized by clinical pathological syndrome.According to thepresence of excessive alcohol consumption that would become divided into alcoholicfatty liver (AFLD) and nonalcoholic fatty liver disease (NAFL).NAFLD includes aspectrum of disease ranging from intrahepatic fat accumulation (steatosis) to variousdegrees of necrotic inflammation and fibrosis (NASH),and even the furtherdevelopment of liver cirrhosis and hepatocellular carcinoma (HCC). Chronicinflammatory disease of NAFLD are highly relevant with type2diabetes andcardiovascular disease.Therefore, the high prevalence rate of NAFLD become a humanhealth burden in the world. However, there is no direct and effective way to treat thisdisease.Therefore, the treatment of nonalcoholic fatty liver disease should focus on themechanical research of the animal model, the clinical trials based on cellular activities,and the combination of lifestyle intervention with drug treatment for the main signalingpathways, lipid metabolism,oxidative stress, inflammatory reaction.Objective：NAFLD is a chronic inflammatory disease induced by multi factors.TheNAFLD various stages of development in the chronic inflammatory are based onsoluble cytokines and chemokines to the regulation of inflammatory cell function andsurvival.Interleukin-8(IL-8) as a kind of inflammatory factor of interleukins closelyrelate to the initiation and progression of NAFLD.G31P is a high affinity, non-activeanalogues of human IL-8screening through human IL-8gene site-directedmutagenesis,which makes it an ideal IL-8receptor antagonist.Recent studies confirmedthe G31P can bind to CXCR1/CXCR2effectively, and have a significant effect ontreatment of neutrophil-related infections.It has been confirmed that G31P has couldinhibit the expression of vessel wall inflammatory factors in hyperlipidemia mice meanwhile it play a good role of prevention and therapy of coronaryatherosclerosis.This finding possibly provide new ideas for of prevention and treatmentof non-alcoholic fatty liver with G31P. This research mainly observe that G31P affect onliver tissues histology, blood fat and the expression of TNF-α,IFN-γ,IL-8,KC inserum and the interference to the liver tissue cells proliferation and initiation of liverfibrosis in NAFLD mice induced by feeding with fat diet.Methods:1）Model Building:30Male BALB/c mice (6-week-old) were randomlydivided into the high fat diet fed group (n=12, high fat diet, treated with normal saline)and the G31P treatment group (n=12, high fat diet, treated with G31P) and the controlgroup (n=6, standard show fed, treated with normal saline). Mice were fed for eightmonths;Observe the pathological changes in liver with HE staining2) Collect bloodfrom eyes of mice to prepare serum through centrifugation.Detect the index of CHO,TG,HDL, LDL, ALT in serum3)Detect the content of KC(IL-8) in serum with ELISA,themRNA expression levels of KC(IL-8) and its receptor(CXCR2) with RT-PCR,theexpression of CXCR2in liver tissue with immunohistochemistry4)The mRNAexpression levels of the inflammatory factors(TNF-α,IFN-γ) in liver tissue withRT-PCR5)The detection of mouse liver tissue propagation-related factors, Detection theexpression of MEF2A,PCNA in liver tissue with immunohistochemistry,the mRNAexpression levels of MEF2A,PCNA with RT-PCR6)The detection of mouse liverfibrosis-related factors:detect the expression of MMP-2,MMP-9in liver tissue withimmunohistochemistry,the mRNA expression levels of MMP-2,MMP-9in livertissue with TR-PCR.Results:1) Have a success on the construction of NAFLD after made mice maintained withWestern diet for8months meanwhile used G31P interfere continuously to construct theG31P therapy group. The weights of G31P group decreased markedly compared to theNAFLD group. G31P significantly improved liver steatosis and inflammation inNAFLD model mice by histopathology.2) The G31P significantly reduce high-fat diet-induced serum CHO, LDL, TG contentand serum glutamic alanine aminotransferase (ALT) levels，and Serum HDL levelsincreased.3) The G31P reduce the NAFLD content of serum KC (IL-8) and KC (IL-8) expressionof the liver tissue, the G31P inhibition of KC (IL-8) receptor CXCR2expression in liver tissue,and finding that the expression of CXCR2decreased83%4) Detection of the expression of inflammatory factors in livers tissue:The expressioncontent of TNF-α,IFN-γdecreased markedly compared with model group.5) The G31P reduce expression of PCNA,MEF2A in mRNA levels which relate to livercell proliferation,and decrease expression of PCNA,MEF2A in liver tissue, finding thatthe expression content of MEF2A of G31P therapy group decreased41%compared withNAFLD model group, PCNA decreased64%compare with that in NAFLD modelgroup.6) Immunohistochemistry to detect the expression of MMP-2, MMP-9in liver tissuesconcluded that expression of MMP-2decreased91%, expression of MMP-9decreased86%compared with NAFLD model group by G31P administration. RT-PCR detectedexpression of MMP-2and MMP-9on mRNA level concluded that expression ofMMP-2mRNA and MMP-9mRNA decreased significantly of G31P therapy comparedwith NAFLD model group.Conclusion:1) G31P can improve the symptoms of high-fat diet-inducedhyperlipidemia and liver injury2) G31P can inhibit the inflammation of the liveroccurred in the non-alcoholic fatty liver3）The G31P inhibit the proliferation and liverfibrosis in non-alcoholic fatty liver hepatocytes.