The Effect And Mechanisms Of Ursolic Acid On Age-associated Nash In Rats

Background Non-alcoholic steatohepatitis(NASH)is a progressive form of non-alcoholic steatohepatitis(NAFLD),characterized by liver fat deposition and inflammation.Aging has a recognized role in NASH progression.Ursolic acid(UA)can improve abnormalities of lipid metabolism and inflammation in rodents.However,few studies have reported the relationship between UA and age-associated NASH.Purpose To investigate the effects of UA against non-alcoholic steatohepatitis in natural aging rats,and explore the possible mechanisms.Experimental approach Twenty-five 22-month-old male SD rats were randomly divided into 3 groups: the old control group(Aged,n=9),the low-dose UA group(UA-L,10 mg/kg,n=8)and the high-dose UA group(UA-H,50 mg/kg,n=8),eight 3-month-old male SD rats were as the control group(Young,n=8).After adaptive feeding in the animal feeding center for one week,UA concentration was given according to 5mL/kg by gavage once a day for 7 weeks.At the end of week 6,an oral glucosetolerance test(OGTT)was performed.At the end of week 7,the rats were fasted overnight(free water),weighed and sacrificed.The plasma alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),total cholesterol(TC),glucose and nonesterified fatty acid(NEFA),insulin concentrations were either measured enzymatically or evaluated by ELISA.Lipid deposition and morphological changes in liver were observed by Oil Red O and HE staining.Real-time quantitative polymerase chain reaction(qPCR),Western blot and immunohistochemistry were used to detect the expression of liver genes and proteins.Proteomics and metabolomics were used to explore the potential targets of UA on agerelated NASH;Further,HepG2 cells and RAW246.7 cells were used to design the in vitro experiment.Oleic acid(OA)was used to induce the NASH in vitro model.Normal control group(Con),model group(OA)and medication group(UA)were set up.Western blot and immunofluorescence were used to detect the expression of related proteins.Key results UA could reduce plasma TG concentration,insulin level and HOMA-IR index and liver Oil Red O staining area as well as the TG and the aggregation of inflammatory cells in liver of naturally aging rats,indicating UA-induced amelioration of NASH.Meanwhile,iTRAQ labeled LC-MS-MS proteomic analysis results showed that the differential proteins were enriched in immune system process,immune response,defense response,leukocyte migration and leukocyte chemotaxis.Among thedifferential proteins contained in these GO terms,we selected S100A8 and S100A9 proteins for further exploration.It has been demonstrated that S100A8/A9 can specifically bind with arachidonic acid(AA)to transport to the target cells,and then activate NF-κB proinflammatory signaling pathways.Metabolomics analysis showed that UA could reverse the high expression of AA in the liver of aged rats.In vitro experiments displayed that UA could reverse OA-induced lipid deposition and the increase of S100A8,S100A9,NF-κB as well as the decrease of IL-10 in HepG2 cells(liver hepatocellular cells)and RAW264.7(mouse leukemia cells of monocyte macrophage).Conclusions UA could improve lipid deposition,insulin resistance in the liver of naturally aging rats,and alleviate non-alcoholic steatohepatitis to a certain extent,which may be related to inhibiting the release of S100A8/A9 in macrophages and hepatocytes and its downstream NF-κB inflammatory response pathway.