| BackgroundDiabetes mellitus is a group of metabolic diseases. Type 2 diabetes (T2D), accounting for the absolute proportion of diabetes, increases rapidly in the world. T2D has become the global health and economic burden. As estimated, the number of people living with diabetes would exceed 592 million worldwide till 2035. In China, with the arrival of the aging society, T2D and related complications would become more severe. According to the latest statistics, the incidence of diabetes in Chinese adults takes up 11.6%. However, the pathogenic mechanism of T2D has not been elucidated clearly. It is ergent to explore effective early intervention for diatetes. Diabetes and diabetes induced cardiovascular and cerebrovascular diseases or chronic kidney disease are becoming serious public health problems. Therefore, it is of great significance to investigate the mechanism of T2D, clarify the key virulence genes, find new targets for the early diagnosis, treatment of T2D and the development of new drugs.It is reported that the macrophage dysfunction and inflammasome activation play important roles in T2D pathogenesis and complications. In the process of T2D, high sugar, high fat environments can lead to fat cell hypertrophy, hypoxia and necrosis, which stimulate adipose tissue and vascular mesenchymal cells to release proinflammatory factors such as TNF-α or MCP-1. These factors recruit peripheral macrophages into adipose tissue. Activated macrophages secrete a number of inflammatory factors to promote the macrophages aggregate into the liver, pancreas and fat tissues. The vicious spiral of macrophage infiltration leads to the development of insulin resistance and T2D. In diabetic patients and animal models, monocyte derived macrophages secrete more IL-1β, accompanied by the activation of NLRP3 inflammasome. In T2D, IL-1β promotes insulin resistance by antagonizing insulin signaling, further inhibits glucose uptake and reduces glucose tolerance. At the same time, chronic low-grade inflammatory state and high fat and high sugar environments can induce the apoptosis of islet beta cell in an IL-1β dependent manner. Therefore, some scholars put forward that IL-1β is an important driving factor for T2D pathogenesis. In the development of T2D, the metabolic accumulation of "risk factors" activates NLRP3 inflammasome to induce IL-1β maturation and release. Mature IL-1β directly causes death and injury of islet beta cells. On the other hand, IL-1β leads to islet beta cell dysfunction by inducing the systemic inflammatory response and aggravating the infiltration of macrophages, and T2D is induced at last. Therefore, the immune molecules responsible for regulation of macrophages and inflammasome activation may play an important role in the occurrence and development of T2D.The T cell immunoglobulin- and mucin-domain-containing molecule (TIM-4) is a new type of immune regulatory molecule, which is selectively expressed on antigen-presenting cells, especially high expressed in macrophages and mature dendritic cells. TIM-4 plays an important role in the maintenance of immune tolerance. Evidences show that TIM-4, as the receptor of phosphatidylserine, involves in maintaining the stable internal environments by promoting the phagocytosis of apoptotic cells by phagocytes. TIM-4 blockade or knockout can induce the production of autoantibodies and autoimmune disease like symptoms, suggesting that TIM-4 may play a protective role in autoimmune diseases. Previous studies found that TIM-4 overexpression could down regulate the expression of CD80, CD86, MHC II molecule on macrophage cell line RAW264.7 and TNF-a generation, while blocking TIM-4 can promote LPS induced macrophage activation. These data indicate that TIM-4 negatively regulates the activity of macrophages. Intravenous transfusion of macrophages with Tim-4 overexpression can protect mice from ConA induced immunologic liver injury. In addition, our lab found enhanced TIM-4 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) compared with normal controls. Moreover, the expression level of TIM-4 was positively correlated with TNF-a in SLE patients, suggesting that the increased expression of TIM-4 may be a negative feedback mechanism of host. As reported in the literature, TIM-4 also closely related with rheumatoid arthritis, ankylosing spondylitis, allergic asthma and other immune diseases. However, the role of TIM-4 in the occurrence of T2D has not been reported in the literature. In this paper, the clinical study was performed to explore the correlation between TIM-4 and T2D, and further investigate the regulation of TIM-4 on NLRP3 inflammasome.AimsThis study was performed to clarify the correlation between TIM-4 and T2D and explore related mechanisms to provide new ideas for the diagnosis and treatment of T2D. This study aims to clarify the regulation of TIM-4 on the NLRP3 inflammasome to identify the novel biological functions of TIM-4, which would provide new targets for the intervention of NLRP3 inflammasome associated diseases. In addition, TIM-4 promoter reporter gene vector was constructed to detect its activity to lay the foundation for the study on the regulation of TIM-4 expression.3. Serum IL-1β was positively correlated with HbA1c in T2D patientsTo verify the important role of increased IL-1β in patients with T2D, we further analyzed the correlation of IL-1β and HbA1c, LDL, HDL and other clinical indicators. The results showed that IL-1β was positively correlated with HbA1c in T2D patients, but no correlation was found between IL-1β and other indexes, indicating that the high level of IL-1β may aggravate the T2D process.4. TIM-4 mRNA was negatively correlated with LDL and HbA1c in T2D patientsWe further analyzed the correlation between TIM-4 mRNA and metabolic indexes. Results showed that TIM-4 mRNA was negatively related to LDL. There was a negative correlation trend between TIM-4 and HbA1c. However, no correlation was found between TIM-4 and fasting blood glucose, total cholesterol, HDL cholesterol, triglycerides. These results suggest that TIM-4 may play a protective role in the process of T2D.5. Vector constructionpcDNA3.0 was used as matrix vector and pcDNA3.0-hTIM-4 was used as the template, pcDNA3-mTim-4-HA containing full length of mouse Tim-4 CDS fused with HA tag was constructed. pcDNA3-hTIM-4-HA as a template, pcDNA3-hTIM-4 (△mucin)-HA with mucin domain deletion was constructed using a mutagenesis Kit. The correctness of the recombinant was tested by enzyme digestion and sequencing. Western blot was performed to detect the expression of TIM-4 fusion protein. By blast analysis, the sequence of target gene was consistent with that provided by Genbank. Western blot detected the expression of label protein HA.6. The establishment of cell model of NLRP3 inflammasome activation7. The inhibition of TIM-4 on NLRP3 inflammasome in vivo by peritonitis modelThe 6-8 weeks of age, male C57BL/6 mice were administered LPS (20 mg/kg) by intraperitoneal injection.2 h after intraperitoneal injection,800μg AL (OH)3 was given by intraperitoneal injection.4 h later, peritoneal fluid was collected to detect of IL-1β levels by ELISA. The results showed that higher levels of IL-1β was detected in the peritoneal fluid of mice from LPS/AL (OH)3 injection group, indicating that the peritonitis model was constructed successfully.60μg pcDNA3-mTim-4-HA or empty vector pcDNA3 plasmid DNA were mixed with transfection reagent PEI according to the ratio of nitrogen to phosphorus at 10:1.200 μl mixture was prepared and incubated at room temperature for 15 min. C57BL/6 mice were randomly divided into two groups, and 3 mice in each group. Mice in one group were injected with pcDNA3-mTim-4-HA plasmid, and mice of the other group were injected with empty vector plasmid pcDNA3.24 h later, two groups of mice were injected with 25 mg/kg LPS and 800μg AL (OH) 3. ELISA was performed to detect IL-1β in peritoneal fluid, and RT-PCR was used to detect Tim-4 mRNA expression. The results showed that IL-1β of Tim-4 plasmid DNA injection group was significantly lower than that of the empty vector control group. The expression of Tim-4 mRNA in Tim-4 plasmid injection group was higher than that of the control group.Tim-4 siRNA lentivirus or control virus (4×107 U/0.1 ml/) was intravenously injected into C57BL/6 mice.7 d later, the peritonitis model was constructed as above. IL-1β level in peritoneal fluid, Tim-4 mRNA and protein expression were assayed. The results showed that IL-1β levels in the peritoneal fluid of Tim-4 siRNA group were significantly higher than those in the control group. The expression of Tim-4 mRNA in spleen and liver mononuclear cells from siRNA injection group was significantly lower than the control group. Flow cytometry results showed that Tim-4 expression decreased in the spleen cells and liver mononuclear cells.8. Effects of TIM-4 on expression of NLRP3 inflammasome components in macrophagesTIM-4 siRNA and control NC siRNA were transfected into THP-1 cells using polyplus.48 h after transfection, RT-PCR was used to detect TIM-4, NLRP3, ASC, caspase-1 and IL-1β mRNA expression respectively. The results showed that the expression of TIM-4 in siRNA transfection group was decreased, while the expression level of ASC was increased in TIM-4 siRNA group6% starch solution were administered by intraperitoneal injection to induce macrophages.72 h later, peritoneal macrophages were isolated regularly. Tim-4 siRNA or control were transfected into macrophages.48 h after transfection, expression of Tim-4, NLRP3 inflammasome components was tested. The results showed that the expression level of Tim-4 mRNA in Tim-4 siRNA transfection group decreased, and the expression level of IL-1β mRNA was increased in Tim-4 siRNA group.9.Construction of TIM-4 promoter and detection of TIM-4 promoter activity.The peripheral blood genomic DNA was used as template and PCR was performed to amplify TIM-4 promoter fragment. The human TIM-4 promoter pGL3-hTIM-4(-1241～+49) was constructed, and identified by digestion, sequencing. HEK293 cells were seeded at 1.5×105/ml in a 48-well culture plate,500 ng promoter plasmid DNA and 10 ng pGL-TK were transfected into HEK293 cells using Lipo2000.48 h after transfection, luciferase activity of promoter and reference was analyzed by the fluorescence detection system counter. Sequencing analysis results indicated that the recombinant pGL3-hTIM-4(-1241～+49) was constructed successfully. In addition, luciferase reporter gene assay showed that the promoter had good activity.Conclusion1. The TIM-4 mRNA expression level in peripheral blood mononuclear cells from T2D patients was significantly higher than that of healthy controls. In T2D patients, TIM-4 mRNA was positive correlated with the level of hsCRP, but was negatively correlated with IL-1β and LDL. There were negative correlation trend between TIM-4 mRNA and IL-18, HbA1c in T2D patients. IL-1β was positive correlated with HbA1c. These results showed that TIM-4 may be involved in T2D process by influencing the production of IL-1β.2. TIM-4 can inhibit the activation of NLRP3 inflammasome, but the key domain of TIM-4 involved in this process and the related molecular mechanism remain unclear. In addition, TIM-4 might inhibit the expression of ASC, IL-1β mRNA. Howevere, the specific mechanism needs further study.3. Get human TIM-4 promoter with good activity.Innovation and significanceIn this study, the in vivo experiments showed that TIM-4 can inhibit the NLRP3 inflammasome activation. We also found that TIM-4 mRNA in peripheral blood mononuclear cells from T2D patients was significantly increased. In T2D patients, TIM-4 mRNA was positive correlated with the level of hsCRP, but was negatively correlated with IL-1β and LDL. There were negative correlation trend between TIM-4 mRNA and IL-18, HbA1c in T2D patients. Our results suggest that TIM-4 may participate in T2D process through inhibiting NLRP3 inflammasome activation. This study clarifies the novel biological function of TIM-4, The results of this paper would provide the new thought for the study of T2D pathogenesis, and provide new candidate target for diagnosis and treatment of T2D. |
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