| Background and PurposeNonalcoholic steatohepatitis(NASH)is an inflammatory form of nonalcoholic fatty liver disease(NAFLD).It is characterized by hepatocyte swelling,liver steatosis,inflammation and fibrosis.NAFLD is a liver manifestation of metabolic syndrome,with a global prevalence of 25%-26%,of which about 21% of NAFLD patients may have NASH,and 40% of NASH patients may develop liver fibrosis or even cirrhosis.As a serious form of NAFLD disease progression,NASH has become one of the most concerned chronic liver diseases.However,at present,the explanation on the pathogenesis of NASH is still not completely clear.Therefore,there is an urgent need to explore the pathogenesis of NASH.Trace elements are elements that account for less than 1/10000 of the total body weight in the human body.they have immunomodulatory,antimicrobial activity and insulin sensitization,and participate in many metabolic pathways such as enzymes,hormones,vitamins and nucleic acids.Studies have shown that the lack or excess of trace elements can damage energy metabolism,resulting in metabolic syndrome such as impaired glucose tolerance,dyslipidemia and oxidative stress.Therefore,the metabolic disorder of trace elements and the disorder of glucose and lipid metabolism and oxidative stress may be involved in the occurrence and development of NASH.At present,there is no study on the changes of trace elements in liver under the condition of NASH.In order to understand the status of trace elements in NASH liver,the model of NASH mice was established by MCD diet.The content of trace elements in liver was determined by ICP-MS,the changes of trace elements in liver and oxidative stress were observed,and the relationship between the content of trace elements in liver and the content of TG and TC in liver and the indexes of oxidative stress MDA,T-AOC and total SOD in liver were discussed.And to explore the role of the changes of trace elements in the evolution of the course of NASH,in order to provide important reference value for the clinical diagnosis and treatment of NASH.MethodForty male SPF C57BL/6J mice(22±2g)were randomly divided into two groups according to their body weight,that is,the control group(Control)and the model group(MCD),were given methionine sufficient MCS feed and MCD feed respectively.According to the feeding time of 3 and 9 weeks,each group was subdivided into 4 groups: 3-week control group(Control3),3-week MCD group(MCD3),9-week control group(Control9)and 9-week MCD group(MCD9).Each group was fed with isocaloric pairs and fasted for 12 hours after the expiration of 3and 9 weeks respectively according to the feeding time.The body weight of the mice was weighed before anesthesia,and the blood was taken through the celiac vein under anesthesia.The serum was separated and stored in the refrigerator at-20℃ for testing.After the blood was collected,the whole liver was perfused with 4 ℃ precooled normal saline at the heart tip and the complete liver was isolated.The shape of the liver was observed,the wet weight of the liver was weighed and the liver index was calculated.Part of the liver was fixed with 4% paraformaldehyde for hematoxylin eosin(HE)staining,oil red O staining and NAFLD activity score.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),serum iron(SI),total iron binding capacity(TIBC),serum triglyceride(TG)and serum total cholesterol(TC);were detected by automatic biochemical analyzer.total antioxidant capacity(T-AOC),malondialdehyde(MDA)and superoxide dismutase(SOD)activity of liver tissue were detected by kit.The content of TG and TC in liver tissue was detected by chloroform-methanol method combined with kit.The contents of iron(Fe),copper(Cu),zinc(Zn),manganese(Mn),selenium(Se),chromium(Cr),cadmium(Cd),molybdenum(Mo),arsenic(As),barium(Ba),cobalt(Co),nickel(Ni),strontium(Sr),vanadium(V)and tin(Sn)in liver tissues were determined by inductively coupled plasma mass spectrometry(ICP-MS).Results(1)General condition: Compared with Control group,the body weight of mice fed with MCD diet decreased significantly(P<0.001),and that of mice fed with 3weeks and 9 weeks decreased significantly(P<0.001).Compared with Control group,liver wet weight and liver index of mice fed with MCD for 3 weeks decreased significantly(P<0.001),but there was no significant difference at 9 weeks.(2)Liver histopathology and NAFLD activity score: Compared with the Control group,the liver of mice fed with MCD diet for 3 weeks showed cell swelling,round fat vacuoles in the cytoplasm,loose and lightly stained cytoplasm,a small number of hepatocytes with mild steatosis,no obvious balloon degeneration,and occasional inflammatory cell infiltration near the central vein.Extensive steatosis,focal infiltration of inflammatory cells and balloon degeneration of hepatocytes could be seen in the liver tissue of mice fed with MCD diet for 9 weeks.The result of NAFLD activity score showed that the NAS score of MCD group was higher than that of Control group.With the extension of modeling time,the NAS score increased and reached the level of NASH 9 weeks later.(3)Serum indexes: Compared with Control group,serum ALT and AST of mice fed with MCD diet for 3 and 9 weeks increased significantly,while serum TC and TG decreased significantly,while serum ALT and AST levels of mice fed with MCD diet for 9 weeks were higher than those of mice fed with MCD diet for 3 weeks,while serum TG and TC were lower than those of 3 weeks.Compared with Control group,serum SI and TS levels decreased significantly after 3 and 9 weeks of MCD feeding.(4)Liver oxidative stress index: Compared with Control group,liver MDA of mice fed with MCD diet for 3 and 9 weeks increased significantly(P<0.001),while liver T-AOC and total SOD activity decreased significantly(P<0.001).Liver MDA content of mice fed with MCD for 9 weeks was significantly higher than that of 3weeks(P<0.001),while T-AOC and total SOD activity was significantly lower than that of 3 weeks(P<0.001).(5)Liver TG and TC levels: Compared with Control group,the content of TG in liver of mice fed with MCD for 3 weeks increased significantly(P<0.001),and the contents of TG and TC in liver increased significantly after 9 weeks of feeding(P<0.001).(6)The content of trace elements in liver: Compared with Control group,the contents of Cu,Zn and Mo in liver of mice fed with MCD diet for 3 weeks were significantly decreased(P<0.001).The contents of V,Mn,Co and Se in liver of mice fed with MCD diet for 9 weeks also decreased significantly(P<0.001).The trend of Cu,Zn,Mo and Fe in liver was consistent with that of 3 weeks.Compared with 3weeks,the contents of V,Cu,Zn,Se,Mo,Cd and Ba in liver of mice fed with MCD for 9 weeks decreased significantly,while the content of Fe increased significantly.(7)Correlation of trace elements in liver with lipid content and oxidative stress index: Partial correlation analysis showed that the contents of Cu,Zn,Mn,Se,Cd,Mo,Co and V in the liver were negatively correlated with the contents of TG and TC in the liver,while the content of Fe in the liver was positively correlated with the contents of TG and TC in the liver.Cu,Zn,Mn,Se,Mo,Co and V elements in liver were negatively correlated with liver MDA content,while liver Fe content was positively correlated with liver MDA content(P<0.005),liver Cu,Zn,Se,Mo and Co contents were positively correlated with T-AOC and total SOD activity,while liver Fe content was negatively correlated with T-AOC(P<0.001).Conclusion(1)The NASH mouse model was successfully induced by MCD feed for 9 weeks,and the pathological changes of liver in this model could reflect the basic pathological changes of NASH.(2)The disturbance of many trace elements occurred in the liver of mice fed with MCD diet for 3 weeks,the contents of Cu,Zn and Mo decreased and the content of Fe increased,while the disorder of trace elements in liver aggravated and the contents of V,Mn,Co and Se decreased significantly after 9 weeks of MCD diet.(3)The contents of Fe,Cu,Zn,Mn,Se,Cd,Mo,Co and V in liver were significantly correlated with the contents of TG and TC,and the contents of Cu,Zn,Se,Mo,Co in liver were significantly correlated with the activities of MDA,T-AOC and SOD. |
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