| Background Nonalcoholic fatty liver disease(NAFLD)represents a wide spectrum including simple steatosis,nonalcoholic steatohepatitis(NASH),and liver fibrosis and cirrhosis that progress from NASH.Due to the rapidly increasing incidence of obesity and metabolic syndrome,NAFLD has become the leading chronic liver disease worldwide.Relative to simple steatosis,the patients with NASH have significantly increased risk of disease progression to end-stage liver diseases.However,the molecular mechanism underlying the initiation and progression of NASH to liver fibrosis has not been fully elucidated.There are no effective drugs for the treatment of NASH and its related fibrosis.Clinical and animal histopathological results have well documented that enhanced infiltration of neutrophils in the liver is one of the key histopathological features of NASH.Neutrophil granule proteins function as the crucial modulator of immunity and inflammatory response in neutrophils.Mi R-223 is highly expressed in neutrophils.It regulates the activation and function of neutrophils in various inflammatory diseases.Therefore,the investigation of the function and molecular mechanism of miR-223/neutrophil granule protein axis regulating NASH-induced liver fibrosis can provide an important basis for the development of new drug for NASH therapy.Objectives This project primarily aims to study the function and molecular mechanism of miR-223 / neutrophil granule protein axis in regulating NASH-induced liver fibrosis,so as to provide support for the research and development of new drugs for prevention and treatment of NASH.Specifically,this study aims: 1.To establish a mouse model with good capacity in recapitulating the key histological features of human NASH and fibrosis by a low methionine and choline deficiency and L-amino acid-defined high-fat diet(CDAHFD);2.To investigate the pathophysiological role of miR-223 in the pathogenesis of NASH and its related liver fibrosis by employing loss-of-function research strategy;3.To identify the key transcription factors mediating the regulatory effect of miR-223 on neutrophil granule proteins by transcriptome sequencing 4.With bone morrow transplantation,to validate whether or not myeloid cells and it-derived neutrophils functions as the key contributor to the regulatory role of miR-223 in inflammatory response in neutrophils and the development of NASH to liver fibrosis.Methods 1.NASH associated liver fibrosis was induced in 10-week-old male C57 BL/6J mice by the feeding with CDAHFD diet for 10 weeks.The histopathological features,liver fibrosis and liver injury degree of NASH were analyzed by H&E staining,Masson staining,Sirius red staining,immunohistochemical staining of ?-SMA,m RNA level detection of liver fibrosis-related genes,and serum ALT and AST biochemical detection.The obesity metabolic phenotype of mice was identified by the detection of body weight,instant blood glucose,as well as oxygen consumption and body fat rate.2.The expression levels of miR-223 in the liver tissue of wild type(WT)male mice fed normal standard diet and CDAHFD diet for 10 weeks were measured by quantitative real time PCR.3.Mi R-223 knockout(miR-223 KO)and wild type mice in C57 BL/6J genetic background were fed with CDAHFD diet for10 weeks to induce NASH and its related liver fibrosis.NASH histopathology,liver fibrosis,liver injury,and metabolic phenotype were detected using the above experimental methods.Immunohistochemistry was used to detect neutrophil liver infiltration and migration in LPS-induced inflammation,and expression of liver neutrophil granule protein.Meanwhile,the expression of neutrophil granule proteins in liver tissue were detected by western blotting analysis.After traditional Chinese medicine intervention,m RNA levels of TNF-?,IL-1?,IL-10,IL-6,MPO,LCN2,PR3,and NE in LPS-activated macrophages were detected.4.Transcriptome sequencing of miR-223 was performed to screen out possible key transcription factors that mediates the regulatory role of miR-223 on neutrophil granulocyte proteins.The m RNA levels in neutrophils and liver non-parenchymal cells of wild-type and miR-223 KO mice were verified and further screened for transcription factors.The dual luciferase reporter gene was used to detect whether miR-223 directly binds to transcription factors and exerts its effect.RT-PCR was used to detect the m RNA levels of MPO,LCN2,PR3,and NE in bone marrow cells and neutrophils of wild-type and miR-223 KO mice.5.Bone marrow transplantation was employed to selectively change the expression of miR-223 in myeloid cells.Liver histology,including lobular inflammation,hepatocyte ballooning,and liver fibrosis,together with metabolic phenotypes were assessed as described above.Results 1.Mouse model induced by CDAHFD feeding recapitulated the key histological features of NASH-induced liver fibrosis and metabolic disorders in humans CDAHFD-fed mice exhibited typical histopathological features of NASH,including steatosis,balloon-like changes and liver inflammation in combination with evident liver fibrosis.In addition,the model showed several metabolic disorders,such as increased body fat rate and decreased metabolic oxygen consumption(n=5/group).2.Mi R-223 abundance was significantly enriched in the liver from CDAHFD-fed mice The data of quantitative real time PCR demonstrated that miR-223 abundance in the liver from CDAHFD-fed mice was significantly enriched(n=5/group).3.Pathophysiological role of miR-223 in the pathogenesis of NASHinduced liver fibrosis 1)Mi R-223 deficiency significantly aggravated experimental NASH in mice Under the condition of CDAHFD diet feeding,miR-223 KO mice exhibited more severe NASH histopathological features,including steatosis,hepatocellular ballooning,and lobular inflammation,when compared with wild type controls(n=5/group).2)Mi R-223 deficiency notably promoted NASH-induced liver fibrosis in mice In comparison to wild type controls,CDAHFD diet feeding-evoked liver fibrosis in miR-223 KO mice were notably potentiated,evidenced by the significantly enhanced collagen deposition and up-regulated m RNA levels of several genes involving hepatic fibrogenesis(n=4-5/group).3)Mi R-223 knockout markedly potentiated NASH-induced hepatocellular injury Circulating levels of two most commonly used biochemical markers of hepatocellular injury,including ALT and AST,were markedly higher in miR-223 KO mice than those of wild type controls,indicating the significantly enhanced hepatocellular injure in mice resulted from miR-223 knockout(n=4-7/group).4)Mi R-223 deficiency did not affect CDAHFD-induced metabolic phenotypes Upon the feeding with CDAHFD,body weight,blood glucose level,body fat rate and oxygen consumption,were similar between miR-223 KO and wild type control mice(n=4-12/group).4.The role of miR-223 in the regulation of hepatic infiltration of neutrophils and inflammatory response in neutrophils 1)Mi R-223 deficiency significantly boosted the infiltration of neutrophils in the liver in CDAHFD-fed mice CDAHFD feeding-evoked hepatic infiltration of neutrophils was significantly boosted in miR-223 KO mice when compared with wild type control mice(n=5/group).2)Mi R-223 deficiency significantly enhanced the migration capacity of neutrophils In response to the stimulation with LPS,the number of neutrophils in the liver of miR-223 KO mice was significantly higher than that of wild-type mice(n=5/group).3)Mi R-223 deficiency significantly increased the expression of neutrophil granule proteins in liver of mice fed with CDAHFD In the case of CDAHFD induction,the protein levels of MPO,LCN2,and PR3 in the liver of miR-223 KO mice were higher than those of wildtype mice(n=5/group).4)Turbidity resolved TCM inhibits the expression of proinflammatory factors and granulosa proteins in immune cells 5.Transcriptome sequencing identified the key transcription factors mediating the regulatory effect of miR-223 on neutrophil granule proteins 1)Sequencing and screening results of key transcription factors that miR-223 regulated the expression of neutrophil granule proteins Transcriptome sequencing data demonstrated two transcription factors with significantly up-regulation in miR-223 knockout neutrophils,namely Fosl2,Plagl1.2)The validation of mediating effects of key transcription factors on miR-223 regulated expression of neutrophil granule proteins Compared with WT mice,m RNA levels of Fosl2 and Plagl1 in nonparenchymal cells and primary neutrophils from miR-223 KO mice were significantly increased,indicating that Fosl2 and Plagl1 were key transcription factors mediating the regulatory effects of miR-223 on neutrophil granulocyte proteins.3)Double-luciferase reporter method was used to detect the interaction between miR-223 and identified transcription factors Mi R-223 overexpression resulted in the expression of firefly luciferase decreased when transfected with recombinant plasmids containing the 3'-UTR sequence of the Fosl2 gene,suggesting that miR-223 directly acted on the 3'-UTR sequence of Fosl2 m RNA and significantly inhibited Fosl2 expression.4)Mi R-223 modulates the expression of neutrophil granule protein by regulating transcription factors Mi R-223 inhibited the expression of granule protein in bone marrow cells and neutrophils.Based on the above results,miR-223 inhibited the expression of neutrophil granulocyte protein by inhibiting the expression of transcription factor Fosl2.6.Pathophysiological function of bone marrow-derived miR-223 in NASH-induced liver fibrosis Mi R-223 chimerism in bone marrow-derived cells significantly determined phenotypes of NASH and NASH-related liver fibrosis in mice upon CHAHFD diet feeding.Nonetheless,Mi R-223 chimerism in bone marrow-derived cells did not affect the metabolic phenotype of obesity induced by CDAHFD in miR-223 KO mice.Conclusions 1.CDAHFD diet-induced mouse model recapitulated the key histological characteristics of NASH-related liver fibrosis in humans.This model can sever as a suitable animal model in the investigation of mechanisms underlying the transition from NASH to liver fibrosis and preclinical test of new drugs.2.Mi R-223 deficiency significantly aggravated liver inflammation,liver injury and liver fibrosis in mice,but did not affect the metabolic phenotypes.3.Mi R-223 deficiency significantly increased the infiltration and migration capacity of neutrophils in the liver,and resulted in the increased expression of neutrophil granule proteins in the liver.4.Mi R-223 inhibited the expression of neutrophil granule proteins by directly inhibiting the expression of the transcription factor Fosl2.5.Myeloid cells and it-derived neutrophils functions were the key contributor to the regulatory role of miR-223 in inflammatory response in neutrophils and the development of NASH to liver fibrosis.In summary,the data in this study revealed that miR-223 significantly reduced NASH liver inflammation,liver injury and associated liver fibrosis by directly inhibiting the expression of transcription factor Fosl2,thereby inhibiting the expression of neutrophil granule proteins.Thus,miR-223,Fosl2 and neutrophil granule proteins may represent the key targets for the development of new drugs for the treatment of NASH and liver fibrosis. |
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