| ObjectiveDiabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus (DM) and the most important reason of end stage renal disease (ESRD). Now, the etiology of DN has not yet been fully clarified, and recent studies have confirmed that the immune inflammatory injury plays an important role in the progression of DN.Macrophage and its related inflammatory factors may lead to kidney injury by proinflammatory effect.Tripterygium wilfordii polyglucoside(TWP), as an immuno-suppressant, has a strong anti-inflammatory and anti-immunity effect. Taking streptozotocin (STZ)-mediated type2diabetes mellitus (T2DM) and DN rats as our research objects, we explored the effection of TWP on the macrophages and interleukin-12(IL-12), signal transducers and activators of transcription4(STAT4), gamma interferon(IFN-y) in DN and the possible mechanism.Methods136clean grade male SD rats were adopted as the research objects and select16rats of them randomly as prevention normal control group (PNC group) and treatment normal control groups (TNC group), with8rats in each group, fed with regular diet.The other120rats were taken to establish the rat model of T2DM with the method of four weeks’high-sucrose-fat diet in combination with low dose STZ (30mg/kg) injected from tail vein. The DM model rats were randomly divided into prevention model control group (PDM group), prevention TWP low-dose group (PTL group,6mg/kg), prevention TWP middle-dose group (PTM group,12mg/kg) and prevention TWP high-dose group (PTH group,24mg/kg); treatment model control group (TDM group), treatment TWP low-dose group (TTL group,6mg/kg), treatment TWP middle-dose group (TTM group,12mg/kg) and treatment TWP high-dose group (TTH group,24mg/kg), with15rats in each group. The rats of TWP prevention group began intragastric administration immediately after being into DM model and were killed after4weeks, while the TWP treatment group began intragastric administration in the5th week after being into DM model and were killed at the end of the8th week. The PNC, TNC, PDM and TDM group were treated with equal amount of distilled water. The changes of body weight (BW), blood glucose,24-hour urine microalbumin (UMA) were recorded during the experiment. Blood lipids were tested by the means of inner canthus before STZ injection.When the experiment was at the end:①Blood specimens were kept for liver and kidney function,blood lipids determination,blood routine examination and ELISA method to detect IL-12and IFN-y,then BW and kidney weight (KW) were also recorded.②Kidney tissue samples were kept for RT-PCR assay to detect the expression of CD68, IL-12, STAT4and IFN-γ, Western Blotting assay to detect the expression of IL-12, STAT4, phospho-STAT4and IFN-y, and immunohistochemical assay to detect the expression of CD68.Results1. The results of model:the basic blood glucose, triglyceride (TG), total cholesterol (TC) in rats with4weeks’high-sucrose-fat were significantly higher than normal control group; after STZ injection,the diabetic model rats had high blood glucose,polydipsia, polyuria, polyphagia and weight loss, and UMA was significantly higher than normal control group since the4th week.2. General indicators:①BW and blood glucose:compared with PNC and TNC group, the blood glucose in TWP prevention and treatment group was increased while the body weight was decreased(P<0.05);②Liver and kidney function:the aspartate aminotransferase (AST) in the PTH group was higher than PNC, PDM, TWP prevention group (P<0.05); the AST in TWP treatment group had no significant difference(P>0.05); there were no significant differences in alamine aminotransferase (ALT), serum creatinine (SCr) and blood urea nitrogen (BUN) in each group(P>0.05).③Blood lipids:the TG and TC in PDM and TDM group were higher than PNC and TNC group(P<0.05),while the TG and TC were lower in TWP prevention and treatment group than PDM and TDM group(P<0.05),and there was no significant differences in each group(P>0.05).④Blood routine examination:compared with TNC group, the lymphocyte in the TWP treatment administered group was significantly reduced(P<0.05); the lymphocyte in TWP prevention group had no significant difference(P>0.05); there were no significant differences in white blood cell,red blood cell, platelet,hemoglobin, monocyte and neutrophile granulocyte in each group(P>0.05). 3. UMA and index of kidney weight (KW/BW):compared with PNC and TNC group, the UMA and KW/BW was significantly higher in the PDM and TDM group(P>0.05); compared with the PDM group, the UMA and KW/BW in the TWP prevention group was reduced to varying degrees, with the most significant decline in PTM group(P<0.05), followed by PTL group (P<0.05), and the PTH group had no significant change(P>0.05);compared with the TDM group, the UMA and KW/BW in the TWP treatment group was significantly reduced with the most significant decline in TTM group.4. ELISA:compared with PNC and TNC group, there were more IL-12and IFN-y expressed in PDM and TDM group (P<0.05); compared with PDM and TDM group, there were less IL-12and IFN-y expressed in the TWP prevention and treatment group (P<0.05) with the most significant decline in PTM and TTM group.5. RT-PCR:compared with PNC and TNC group, PDM and TDM group expressed more CD68, IL-12, STAT4and IFN-y (P<0.05); compared with PDM and TDM group, there were less CD68, IL-12, STAT4and IFN-y expressed TWP prevention and treatment group (P<0.05) with the most significant decline in PTM and TTM group.6. Western Blotting:compared with PNC and TNC group, PDM and TDM group expressed more IL-12, phospho-STAT4and IFN-γ (P<0.05); compared with PDM and TDM group, there were less IL-12, phospho-STAT4and IFN-y expressed in the TWP prevention and treatment group (P<0.05) with the most significant decline in PTM and TTM group.7. Immunohistochemical assay:there were more CD68labeled cells infiltrating in the renal glomerular mesangial areas and tubules in PDM and TDM group than PNC and TNC group; while TWP prevention and treatment group expressed less CD68than PDM and TDM group(P<0.05) with the most significant decline in PTM and TTM group.Conclusions1. The rat model of T2DM and DN were successfully prepared, and the characteristics of high blood sugar, high cholesterol and UMA were in accordance with human T2DM and DN. 2. The expression of CD68, IL-12, phospho-STAT4and IFN-y in the kidney and the expression of IL-12and IFN-y in the serum of DN rats were significantly increased, which may involve in the development of DN by proinflammatory effect.3. The prophylactic and therapeutic applications of low-dose (6mg/kg) and middle-dose (12mg/kg) of TWP for DN could reduce the UMA with the most significant decline in the middle-dose group.4. The prophylactic and therapeutic applications of three doses of TWP for DN could all reduce the inflammatory injury thereby delaying the occurrence and development of DN by inhibiting macrophage infiltration and down-regulating the expression of IL-12, phospho-STAT4and IFN-y, with the most significant decline in the middle-dose group.5. The low-dose and middle-dose of TWP had better security, while the high-dose of TWP (24mg/kg) may lead to liver injury.Thus the clinical application of TWP should pay more attention to the dose and closely watched the side effects. |
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