The Role Of TRIF-NF-?B Inflammatory Signal Pathway In Islet ? Cell Injury Induced By TLR3

Objective: Diabetes and its complications,as a kind of common chronic diseases,bring great burden to patients and society.There are many studies on the pathogenesis of diabetes.Insulin resistance,islet ?-cell damage and dysfunction are considered to play important roles in the pathogenesis of diabetes,while systemic chronic inflammation is considered to be an important factor leading to islet resistance and absolute / relative deficiency of insulin secretion.As an important receptor of the innate immune system,Toll-like receptors(TLRs)play important roles in inducing the release of inflammatory factors and promoting the occurrence of related diseases.As a highly expressed receptor in the pancreas,TLR3 and related signaling pathway are involved in the regulation of immune inflammation and glucose homeostasis,but the mechanism needs to be further studied.In this study,Poly(I:C)(PIC),as a specific agonist of TLR3,was used to stimulate the mouse islet ? cell line(NIT-1 cell line).The purpose of this study was to explore the effect of activating TLR3 signal pathway on the proliferation and apoptosis of islet ? cells and its possible mechanism.Methods: NIT-1 cell line was used as the research object.The cells were cultured inDMEM medium containing antibodies,and the cell culture medium was changed regularly.Subculture was carried out according to cell growth,and experimental grouping was carried out when the proportion of cells in the proliferative phase reached 80%-90%.They were then divided into blank control group and experimental group,and were inoculated on 96-well plate.After 24 hours,the adhesion and growth of cells in each group were observed,and then NIT-1 cells in the experimental group were stimulated with different concentrations of PIC(30 ?g/ml,60 ?g/ml,90 ?g/ml).After 48 hours of intervention,the following indexes were detected: 1.Cell proliferation was detected by flow cytometry;2.The expression of Toll/IL-1 receptor junction protein(TRIF)and nuclear factor-kappa B(NF-?B)m RNA were quantitatively detected by real-time fluorescence quantitative PCR(real-time PCR,q PCR),and the expression of cell cycle-related protein Cyclin-D1(CCND1),apoptosis related protein Caspase-3,inflammation related TRIF and NF-?B protein were detected by Western blot.Results: 1.The cell proliferation was detected by flow cytometry.The results showed that compared with the blank control group,the proportion of NIT-1 cells stuck in G0/G1 phase increased significantly with the increase of PIC concentration(P < 0.0001),while the proportion of cells in S phase and G2/M phase decreased significantly(P < 0.0001).2.Compared with the blank control group,the m RNA expression of TRIF and NF-?B in islet ? cells increased with the increase of PIC concentration(P < 0.05).The expression of m RNA showed an upward trend at low concentration of PIC(30 ?g/ml)(P > 0.05),but the expression of m RNA only increased significantly at middle and high concentration(P < 0.01).3.By comparing the gray values of CCND1 and GAPDH of Western blot bands stimulated by different concentrations of PIC,it was found that compared with the control group,the expression of CCND1 decreased significantly under different concentrations of PIC stimulation,and decreased significantly with the increase of PIC concentration,and the decrease was the highest at 90 ?g/ml(P < 0.0001).4.After treatment with different concentrations of PIC,the expression of apoptosis-related protein Caspase3 changed significantly.The expression of pro-CASP3 in low and middle concentration PIC stimulation was different from that in the blank control group(P < 0.05),but the degree of increase was significantly lower than that in high concentration stimulation,and the expression of cleaved-CASP3 protein in each intervention group was also significantly higher than that in the blank control group,and the increase was concentration-dependent(P < 0.0001).5.After the intervention of different concentrations of PIC,the expression of TRIF protein in NIT-1 cells were significantly higher than that in the blank control group(P < 0.0001).The increase of TRIF protein at 60 ?g/ml and 90 ?g/ml were significantly different from that at low concentration(30 ?g/ml), and showed a concentration-dependent trend(P < 0.0001).6.Compared with the blank control group,the expression of NF-? B protein in NIT-1 cells increased significantly after the intervention of different concentrations of PIC,and increased in a concentration-dependent manner(P < 0.0001).Under the stimulation of high concentration of PIC(90 ?g/ml),the expression of NF-?B was significantly higher than that of the control group(P < 0.0001),and higher than that of the low and middle concentration stimulation groups.Conclusion: 1.The activation of TLR3 could down-regulate the expression of cyclin D1 in islet ? cells through TRIF-NF-?B signal pathway,and then inhibit the proliferation of islet ? cells in G0-G1 phase.2.The activation of TLR3 signal pathway up-regulates the expression of caspase 3,an important protein that induces apoptosis,suggesting that it is involved in islet ?-cell apoptosis.3.The regulation of TLR3-TRIF-NF-?B inflammatory signal pathway on islet ? cell proliferation and apoptosis provides a new theoretical basis for the treatment of diabetes.