Roles And Mechanisms Of RAI16 In Mouse Models Of Ulcerative Colitis And Non-alcoholic Fatty Liver Disease

Retinoic acid induced（RAI）is a family of proteins induced by retinoic acid involved in intracellular signal transduction and transcriptional regulation.RAI is conserved in human and many other animals by homology analysis,which indicated that RAI might play an important role in basic cell function.Retinoid-induced protein 16（RAI16,also known as FAM160B2）is a member of the RAI family.The roles of RAI16 on cell growth and differentiation were mentioned by some researches,but there have been relatively few reports on the structure and function of RAI16 proteins.Our group has been focusing on and working on the possible pathophysiological functions of RAI16.Previously,our group firstly reported that RAI16 enhanced tumorigenesis in hepatocellular carcinoma（HCC）due to the resistance to apoptosis and could serve as biomarker for HCC diagnosis.However,the functional study on RAI16 is still limited.Considering that inflammation is closely related to the development of tumors,while the role of RAI16 in inflammatory diseases has not been reported.Thus,we generated the RAI16 knockout（RAI16-/-）mouse model by CRISPR/Cas9 strategy,in order to further evaluate the function of RAI16 by comparing RAI16-/-mice with wild type mice in various inflammatory disease models.In this study,RAI16-/-knockout mice were used to establish some mouse sterile inflammatory disease models including dextran sodium sulfate（DSS）-induced acute colitis and high fat diet induced non-alcoholic fatty liver disease（NAFLD）models in order to identify the specific function and the precise molecular machinery of RAI16.This reaearch may help us comprehensively explore the biological functions of RAI16 and will provide theoretical and experimental basis for better understanding of the pathogenesis and prevention and treatment of ulcerative colitis and non-alcoholic fatty liver disease.Part 1:Roles and mechanisms of RAI16 in DSS-induced colitis miceUlcerative colitis（UC）is one of inflammatory bowel disease involving colon and rectum.Patients with UC are at a higher risk of developing colitis-associated colorectal cancer（CAC）.The etiology of ulcerative colitis is still unknown and may be related to a variety of factors.In this part,RAI16^-/-mice were given 3%DSS to establish the acute colitis models to determine the contribution of RAI16 in the pathogenesis of the inflammatory diseases.We found that in 3%DSS-induced colitis model,RAI16-/-mice showed significantly enhanced colitis symptoms,including increased weight loss,colon shortening and colonic mucosa damage and severity,together with enlarged spleens and much more white blood cells.However,The m RNA expression of several cytokines（IL-1β,IL-6 and TNF-α）in colonic tissues showed no significance between WT and RAI16-/-mice.Furthermore,it was found that m RNA expressions of several repair-associated cytokines including cyclooxygenase-2（Cox2）,epithelial growth factor receptor ligand Epiregulin（Ereg）and matrix metalloproteinase 10（MMP10）were siginificant decreased in RAI16-/-mice.The results above showed that RAI16 deficiency could exacerbate the severity of colitis and RAI16-/-mice exhibited an impaired tissue repair in response to DSS.Part 2: Roles and mechanisms of RAI16 in high fat diet induced NAFLD miceA growing number of studies have found that obesity is a chronic inflammatory disease of the body.With the improvement of people’s living standards,the prevalence of non-alcoholic fatty liver disease（NAFLD）,diabetes,coronary heart disease and other obesity-related metabolic diseases increases gradually,which seriously affects human health and life.NAFLD is a metabolic stress liver injury closely related to insulin resistance and other factors.It is mainly characterized by hepatic fat accumulation and metabolic disorders.Much more researches have conducted the mechanisms of NAFLD,however,the exact pathological procession and mechanisms are still unclear and the specific therapeutic strategies against NAFLD are needed.In the present study,RAI16-/-mice were fed with high fat diet（HFD）for up to 12 months to induce NAFLD models.And we aimed to explore the possible role and the molecular mechanism of RAI16 in NAFLD.We found that RAI16-/-mice fed on HFD gained much more body weight and body fat content within 4 months than WT mice on HFD.RAI16-/-mice on HFD also showed increased serum ALT and AST levels compared with WT mice on HFD.Histopathology results revealed significant lipid droplet accumulation in the liver of RAI16-/-mice.Moreover,the expressions of fatty acid synthesis associated molecules Ppar-γ,Srebp-1c and Fas were further increased in RAI16-/-mice compared with WT mice on HFD.The expression levels of inflammatory cytokines TNF-alpha and IL-6 in the liver of RAI16-/-mice were significantly increased.In addition,macrophage infiltration related molecules Mcp-1 and F4/80 and proinflammatory factor Lcn2 were significantly increased in RAI16-/-mice compared with WT mice on HFD.Fasting blood glucose and insulin were higher in RAI16-/-mice compared with WT mice on HFD.Furthermore,GTT and ITT results indicated that RAI16-/-mice were more glucose intolerant and insulin-resistant than WT mice on HFD.Taken together,RAI16-/-mice fed on HFD showed severer hepatic lipogenesis,macrophage infiltration and insulin resistance compared with wild-type mice.Thus,we simulated the progress of NAFLD with long-term high-fat diet and demonstrated for the first time that RAI16 deficiency exacerbates hepatosteatosis and insulin resistance in HFD induced NAFLD.Our study complemented the biological functions of RAI16 and provided new insights into the pathogenesis and targeted therapy of NAFLD.