To Investigate The Protective Mechanism Of Akkermansia Muciniphila Subtype On The Mouse Model Of Non-Alcoholic Steatohepatitis-Related Hepatocellular Carcinoma Based On Proteomics Techniques

Background and ObjectivesIn contrast to long-term clinical and laboratory studies to elucidate the molecular mechanisms of chronic hepatitis virus infection(especially hepatitis B and C viruses)and alcoholic liver disease,the pathologic mechanisms of hepatocellular carcinoma(NAS-HCC)associated with non-alcoholic liver disease remain unclear.With the increasing number of patients with metabolic syndrome such as obesity,hyperglycemia and hyperlipidemia,Non-alcoholic steatohepatitis-related hepatocellular carcinoma(NASH-HCC)has become an important part of liver cancer.Akkermansia muciniphila(AKK)is an elliptical Gram-negative bacterium isolated from human feces.Animal experiments show that AKK can improve metabolic diseases such as obesity,hyperlipidemia,hyperglycemia and insulin resistance.In the present study,our group found that intragastric administration of Akkermansia Muciniphila subtype(AKKsub)reduced weight gain and inflammatory cytokines levels in mice on high fat diet(HFD).And improved olanzapine-induced hyperglycemia and insulin resistance.In addition,decreased AKK abundance was observed in both NASH-HCC and alcoholic steatohepatitis-associated liver cancer mice.However,it has not been reported whether AKKsub can also improve the incidence of hyperglycemia,hyperlipemia and liver cancer in strepzotocin high-fat diet(STZ-HFD)induced NASH-HCC model mice.Therefore,the purpose of this study was to explore the effects of AKKsub on metabolic diseases such as obesity,hyperglycemia and hyperlipemia in STZ-HFD-induced NASH-HCC model mice,as well as the occurrence and development of AKKsub in hepatocellular carcinoma.The NASH-HCC mouse model was established,and the AKK subtype(AKKsub,GP01 strain)isolated from human intestine by our research group were used for gavage treatment of the mice.MethodsEstablishment of NASH-HCC model mice:Male C57BL/6 newborn mice were injected subcutaneously with streptozocin(STZ)200μg once on day 2 of birth,and induced by high-fat diet from 4 weeks of age.The blood glucose of mice at 6 weeks of age was detected to evaluate whether the hyperglycemic model was successfully established.After modeling,the mice were divided into experimental group and control group,and were given 5×109 CFU/200μL AKKsub bacterial solution or 200μL sterile PBS every day for 16 weeks.The weight and feed intake of the mice were measured weekly.Mice were sacrificed at the age of 22 weeks,and their blood,liver,colon tissues and feces were collected.PCR,ELISA,proteomics and other experimental methods were used to analyze the changes of blood glucose and lipids,body weight intake,liver pathology and liver proteome expression.ResultsAfter AKKsub intragastric administration and death of the mice,according to the liver pathological results,the mice were divided into:experimental normal group(TB1),experimental fatty liver group(TB2),experimental adenoma group(TA1),experimental hepatocellular carcinoma group(TA2),control fatty liver group(CB2),control adenoma group(CA1),and control hepatocellular carcinoma group(CA2).The findings of this study:(1)AKKsub can significantly improve glucose metabolism in NASH-HCC mice,but cannot improve obesity and hyperlipidemia.(2)AKKsub can reduce the level of serum endotoxemia in STZ-HFD-induced NASH-HCC model mice,increase the number of mucus cells to improve intestinal permeability,and thus stabilize intestinal barrier disorders.(3)AKKsub could increase serum IL-6 level in NASH-HCC model mice.(4)AKKsub can reverse fatty lesions in the liver of NASH-HCC mice.(5)AKKsub can slow down the occurrence and development of hepatocellular carcinoma in NASH-HCC mice.(6)Proteomics analysis showed that 13 proteins were up-regulated and 24 proteins were down-regulated in the experimental group compared with the control group(T vs C).There were 110 up-regulated proteins and 90 down-regulated proteins in TB1/TA2 comparison.In TB1/CA2 comparison,there were 176 up-regulated proteins and 261 down-regulated proteins.There were 17 up-regulated proteins and 40 down-regulated proteins in the TA2/CA2 comparison.AKKsub gavage could not completely improve lipid metabolism in NASH-HCC model mice.(7)AKKsub can reduce the transcription and expression level of malic enzyme 2(ME2)of tricarboxylate cycle in liver of NASH-HCC model mice compared with those without AKKsub,and compared with those without AKKsub,AKKsub can also reduce ME2 expression level in hepatocellular carcinoma.(8)The expression of mitochondrial transport protein frataxin(FXN)was reduced in mice with HCC.(9)The expression of the tumor suppressor protein IMPORtin-11,PTEN transporter protein,was increased in mice with HCC.ConclusionIn this study,AKKsub can improve the intestinal barrier function of STZ-HFD-induced NASH-HCC model mice,reduce hyperglycemia,partially reverse fatty lesions in the liver of NASH-HCC mice,and significantly reduce the transcription and expression levels of malate 2(ME2)in the tricarboxylate cycle.Thus,the occurrence and development of liver cancer in NASH-HCC mice were slowed.In addition,the expression of mitochondrial transporter Frataxin protein was decreased in mouse liver cancer,suggesting that the expression of Frataxin might play a protective role in liver cancer.The expression of importin-11,a tumor suppressor protein PTEN transporter,was increased in mouse liver cancer,suggesting that importin-11 overexpression may promote cancer in liver cancer.