Functional And Mechanistic Investigation Of MicroRNA-223 In Neutrophilic Inflammation And The Pathogenesis Of Nonalcoholic Steatohepatitis

Background Nonalcoholic fatty liver disease(NAFLD)is an obesity-related chronic liver disease.NAFLD has become a major liver disorder worldwide with the significant increased incidence of obesity and type 2 diabetes.NAFLD is divided into Nonalcoholic fatty liver(NAFL)and Nonalcoholic Steatohepatitis(NASH)according to clinical and pathological characteristic.NASH patients had markedly increased risk of progressing to fibrosis,cirrhosis and hepatocellular carcinoma compared with NAFL.However,the pathogenesis of NASH has not been fully elucidated,and there are no effective drugs for NASH.Liver inflammation is one of the typical histopathological features of NASH.Clinical and animal experiments have confirmed that increased neutrophil infiltration in liver is one of the typical histopathological features of NASH,but the molecular mechanism that mediates neutrophil liver infiltration in NASH is unclear.Micro RNA-223(miR-223)is a highly conserved and chemically stable endogenous non-coding RNA with high abundance in myeloid cells.In addition,clinical study revealed significant differences in the abundance of miR-223 in liver between NAFL and NASH group.Therefore,the investigation into pathophysiological functions of miR-223 in regulating neutrophilic inflammation and hepatic inflammation has vital scientific significance for further revealing the molecular mechanism of NASH occurrence and progression.Objectives 1)Evaluate the histopathological characteristics of the three method of NASH model respectively to establish the animal model basis for the study of the pathophysiological functions of NASH related target genes 2)To illuminate the tissue expression profile of miR-223 in NASH mouse model 3)To study the pathophysiological functions of miR-223 in liver metabolic inflammation and the progression of NASH through the strategy of loss-offunction 4)To demonstrate mechanism of miR-223 in regulating innate immune cell inflammatory response,mediating liver inflammation in NASH In conclusion,this study provides basic research data supporting the development of novel drugs for NASH including innovative traditional Chinese medicine with miR-223 as a potential targetMethods 1)MiR-223 level in various organs and tissues of C57 BL/6J wild-type mice were fed with standard chow to investigate the expression profile by q PCR.2)NASH models were induced by three methods,(1)ten week-old C57 BL/6J male mice were fed with Methionine-and-Choline-Deficient(MCD)diet for 5 weeks to induce NASH,and Methionine-and-Choline-Sufficient(MCS)diet as a control diet.(2)Four weeks old C57 BL/6J male mice were fed with High Fat High Cholesterol(HFHC)diet for 20 weeks with one time peritoneal injection of LPS to induce NASH,and standard chow as a control diet.(3)Four week-old Apo E knockout(Apo E KO)male mice were fed with HFHC diet for 20 weeks to induce NASH,and standard chow(SC)as a control diet.The NASH histopathological features were measured by H&E,Masson staining and detection of serum AST.In addition,the lipid metabolism and systemic metabolic phenotypes of mice were identified by body weight monitoring,fasting blood glucose and glucose tolerance tested through blood glucose test paper,and level of serum TG and TC.3)The investigation adopts the loss-of-function strategy to study the pathophysiology functions of miR-223 in NASH occurrence and progress.Week age matched miR-223 knockout(miR-223 KO)and wild type(WT)male mice induced NASH model through the above three means,and the NASH histopathological characteristics,lipid metabolism and systemic metabolic phenotypes were evaluated by the aforesaid methods.The expression of miR-223 in the liver of C57 BL/6J wild-type mice induced by MCD and HFHC+LPS was detected by q PCR.4)In studies of the function of miR-223 in mediating innate immune cells infiltration and inflammation in liver induced by NASH,innate immune cells such as macrophages and neutrophils infiltration and levels of MPO,PR3,NE and LCN2 in liver in the three models were detected through immunohistochemistry.The expression of MPO and LCN2 in hepatic nonparenchymal cell were measured by western blot.5)The investigation into molecular mechanism of miR-223 in mediating neutrophil inflammation,the expression of granular proteins in neutrophils and macrophages in miR-223 KO and C57 BL/6J male mice were detected by q PCR.Besides,the migration of neutrophils in miR-223 KO and C57 BL/6J male mice were tested through transwell.Results 1)Study on expression profile of miR-223 Systematic gene expression profile studies in C57 BL/6J wild-type mice fed with standard diet showed that the abundance of miR-223 in bone marrow cells and neutrophils was significantly increased compared with other organs/tissues,indicating significant myeloid cell specificity.2)The three NASH models were evaluated with the key clinical manifestations and histopathological characteristics of NASH(1)Methionine-and-Choline-Deficient Diet induced NASH model(MCD model): The typical liver histopathological features similar to human NASH,included liver steatosis,lobular inflammation and hepatocellular ballooning.Some systematic metabolic phenotypes were inconsistent with human NASH,such as body weight loss,increased insulin sensitivity,and no hyperlipidemia.(2)High Fat High Cholesterol Diet with one time peritoneal injection of LPS induced NASH model(HFHC+LPS model): The part of liver histopathological features similar to human NASH,comprised liver steatosis,short lobular inflammation,no notable hepatocellular ballooning and fibrosis.In addition,the glycolipid metabolic was markedly impaired,such as body weight gain,damaged glucose tolerance and dyslipidemia.(3)High Fat High Cholesterol Diet induced Apo E KO mice NASH model(Apo E model): Partial liver histopathological features were analogous to human NASH,for instance,liver fat accumulation,mild or moderate liver inflammation,no obvious hepatocellular ballooning accompany with glycolipid metabolic dysfunction,such as body weight gain,impaired glucose tolerance and dyslipidemia.3)The pathophysiological function of miR-223 in NASH(1)Mice phenotypes were concurrence of the three model,liver inflammation and hepatocellular injury were significantly enhanced in miR-223 KO mice compared with wild-type mice.Liver histopathological features(liver inflammation,hepatocellular ballooning and histopathological CRN Score)were markedly augmented in miR-223 mice compared with wild-type mice as well as the level of AST in serum,which is a biochemical index of liver function,was significantly upregulated in miR-223 KO mice(n=6/7,P<0.05),This result demonstrated that miR-223 deficiency enhanced mice experimental NASH.(2)There were no significant differences in liver lipid metabolism and metabolic phenotypes between the miR-223 KO group and the wild-type control group.Metabolic phenotypes show that no marked difference in fast blood glucose and glucose tolerance between miR-223 KO group and wild-type group in HFHC+LPS model.Moreover,there were no significant differences in body weight and serum TG,TC level between miR-223 and Apo E double KO group and Apo E KO group in Apo E model.In addition,there were no marked differences in body weight and serum TG,TC level between miR-223 KO group and wild-type group in MCD model.Suggesting that miR-223 absence has no notable effect on liver lipid metabolism and metabolic phenotypes.4)Studies of the function of miR-223 in mediating innate immune cells infiltration and inflammation in liver induced by NASH.(1)Immunohistochemistry of macrophage marker F4/80 and neutrophil marker MPO data shows that macrophages and neutrophils infiltration were obviously increased in liver in miR-223 KO mice compared with wild-type mice fed with MCD diet.(2)In MCD model,immunohistochemistry data shows that the level of neutrophil granular proteins(MPO,PR3,NE and LCN2)were significantly increased in live in miR-223 KO mice compared with wild-type mice fed with MCD diet.(3)In HFHC+LPS model,immunohistochemistry data shows that the level of neutrophil granular proteins(MPO,PR3,NE and LCN2)were markedly upregulated in liver in miR-223 KO mice compared with wild-type mice fed with HFHC diet.(4)In Apo E model,immunohistochemistry data shows that the level of neutrophil granular proteins(MPO,PR3 and LCN2)were obviously augment in live in miR-223 KO mice compared with wild-type mice fed with HFHC diet.(5)In MCD and HFHC+LPS model,the level of MPO and LCN2 in hepatic nonparenchymal cell was significantly increased in miR-223 KO mice with NASH.5)Molecular mechanism of miR-223 in mediating neutrophil inflammation(1)QPCR data shows that the level of MPO and LCN2 in neutrophil were significantly increased in miR-223 KO mice compared with wild-type mice.(2)QPCR data demonstrates that the level of MPO,PR3,NE and LCN2 were obviously upregulated in bone marrow cell in miR-223 KO mice compared with wild-type mice.(3)Transwell data reveals that neutrophils migration were markedly enhanced in miR-223 KO mice compared with wild-type mice.Conclusions(1)Three NASH mice models(MCD,HFHC+LPS,Apo E)in this study all show different degrees in clinical NASH key histopathological features and specific metabolic phenotypes.Analysis by synthesis,these models had their own characteristics and limitations.Consequently,in the study of NASH related gene function or the preclinical evaluation of new drugs,single model or combined application should be selected according to the research purpose and the key characteristic of NASH.(2)MiR-223 deletion markedly accelerated diet induced liver metabolic inflammation,and significantly augment the susceptibility to experimental mice of NASH.(3)MiR-223 absence significantly enhanced the liver recruitment of innate immune cells such as macrophages and neutrophils(4)MiR-223 deficiency obviously upregulated the m RNA and protein level of granular proteins MPO,PR3,NE and LCN2.(4)MiR-223 deletion markedly enhanced neutrophil migration.In conclusion,the study results revealed that miR-223 significantly dampened neutrophil-macrophage crosses talk,abated the liver metabolic inflammation and its induced hepatocyte injury,improve mice NASH through inhibiting the expression of several granular proteins to suppress neutrophil inflammation.Thus,miR-223 and its regulated granular proteins are new potential target for the development of novel drugs for NASH including innovative traditional Chinese medicines.