The Proliferation And Serection Effects Of IL-18 And PDTC On Mesangial Cells

[Background and aim]Diabetes mellitus (DM) is a chronic disease which is caused by many factors. At present it is the third death cause next to cardio vascular and cerebrovascular disease and carcinoma. The majority of diabetic patients were subjected to chronic complicants including the pathological changes of macro- and micro-vessels which lead to high fatality rate and high disability. Diabetic nephropathy (DN) is prevalent in diabetic patients, which is the main cause of end stage renal disease (ESRD)in the advanced country. The incident of DN increases year and year in our country recent years. Up to now, the pathogenesis of DN is still not clear to us. The main pathologic changes of DN are the thickening of basilar membrane, accumulating and producing of extracellular matrix too much. Recent studies demonstrated that inflammatory mechanism mediated by macrophages may play important roles in the pathogenesis of DN.Interleukin-18 (IL-18) is a proinflammatory cytokine derived from activated macrophages. Its structure is similar to IL-1βand function similar to IL-12. IL-18 can induce many kinds of cells to produce IFN-γand be named as IFN-γinducing factor (IGIF) previously. Then it is renamed as Interleukin-18 because of its different structure from others. IL-18 can also induce immunocells to produce many cytokines and express FasL and play important roles in the course of cytotoxic mediated via Fas. Besides, nuclear factor-κB (NF-κB) also takes part in the signal transduction to active IL-18. Recent studies suggest that IL-18 is related to primary renal glomerular disease, lupus nephritis, chronic renal failure, acute renal failure and so on. Recently, serum and urine IL-18 levels have been reported elevated in patients with T2DM. Its level increases with the progress of T2DM and renal lesions. There is high expression of IL-18 in glomerular nephritis. Serum IL-18 levels have been reported elevated in patients with DM and have direct correlation with AER, HbA1c, microglobulin and so on. Thus, IL-18 might be an important factor in the process of diabetic nephropathy. It is related to inflammatory reaction markedly.This study aims to investigate the proliferating and secrecting efects of IL-18 with different concentration to mesangial cell (MC) and learn the inhibiting effect of PDTC in the pathogenesis of DN.[Methods]1. Rat glomerular mesangial cells were isolated, cultured and passaged as previous description with different concentration IL-18 and/or PDTC. The proliferation effects of IL-18 and PDTC on cultured rat mesangial cells were evaluated by methylene blue assay (MTT method);2. ELISA method was used to examine the levels of type IV collagen (CO-IV) and laminin (LN) in the supernatant of cultured rat mesangial cells;3. The level of TNF-αmRNA in rat mesangial cells were detected by reverse transcription polymerase chain reaction (RT-PCR);4. Immunocytochemistry was used to detect the activity of NF-κB.[Results]1. Inverted phase contrast microscope showed the cultured cells appeared stellate or spindleshape with irregular cytoplasmic processus. Identification of mesangial cells was performed by immunocytochemistry staining techniques for positive staining with anti-desmin and alpha-smooth muscle actin and negative staining with anti-cytokeratin and anti-factor VIII-related antigen;2. The A value of the supernatant of cultured rat mesangial cells stimulated with different concentration IL-18 increased significantly compared with team A (P<0.01); The A value of the supernatant of cultured rat mesangial cells with 50μg/L IL-18 increased significantly compared with 25μg/L team (P<0.01); Team D was same to team A (P>0.05); Team E decreased significantly compared with team B (P<0.01); Team F decreased significantly compared with team C (PO.01);3. ELISA method showed that the level of CO-IV in the supernatant of cultured rat mesangial cells stimulated with IL-18 increased significantly in a dose dependent manner (compared with control, P<0.01). Also the level of LN in the supernatant of cultured rat mesangial cells stimulated with IL-18 increased significantly in 25μg/L and 50μg/L dose (compared with control, P<0.01). The level of CO-IV and LN in the supernatant of cultured rat mesangial cells inhibited with PDTC decreased significantly. Team E decreased significantly compared with team B (P<0.01); Team F decreased significantly compared with team C (P<0.01); Team D was same to team A (P>0.05);4. The expression of TNF-αmRNA in cultured rat mesangial cells stimulated with IL-18 increased significantly in a dose-dependent manner (compared with control, P<0.01). In the cultured rat mesangial cells inhibited with PDTC reduced the expression of TNF-αmRNA significantly (P<0.01).5. The stain of activiated NF-κB p65 appeared at the nucleus mainly in the MC cultured with different concentrate of IL-18, and the stain of NF-κB p65 at the cytoplasm mainly in the MC cultured with PDTC.[Conclusions]1. IL-18 could be able to induce MC proliferating significantly in a dose-dependent manner, and PDTC inhibits MC proliferating.2. IL-18 enhances the secreting of CO-IV and LN, which is inhibited by PDTC.3. Different concentrate of IL-18 can promotes the secreting and expressing of TNF-αand PDTC inhibits it.4. IL-18 activate NF-κB p65 and make it transfer from cytoplasm to nucleus, and PDTC reduce it.