Mechanism Of Tripterygium Wilfordii Polyglycosides On Preventing And Treatment Of Type2Diabetic Nephropathy By Th Cells Balance

Objective Diabetic nephropathy (DN) is a common microvascular complications of diabetes mellitus and it has become the major cause of end stage renal disease (ESRD). Changes in glomerular hemodynamics, advanced glycation end products generation, anomalies of polyhydric alcohol metabolic pathway and other factors play a role in the development and progression of diabetic nephropathy. Recently, more and more studies have found that DN may be related to chronic inflammatory reaction which due to changes in immune mechanisms. Taking type2diabetes and diabetic nephropathy SD rats animal model which were built by sugary and fatty feedstuff and low dose STZ tail vein injection as our research objects. Taking Tripterygium Wilfordii Glycosides (TWP) as the treatment drug. We explored TWP effect and possible mechanism in changing rats blood Thl/Th2cells and the IFN-γ、 TNF-α、IL-4、IL-10levels in the blood and kidney tissue expression.Methods136clean grade male SD rats, were raised in cages in specific pathogen-free conditions. Select16of them randomly as prevention normal control (PNC groud, n=8) and treatment normal group (TNC groud, n=8) fed with regular feedstuff. Others with fatty and sugary feedstuff, and4weeks later, STZ was injected into caudal vein to induce type2diabetes mellitus. Successful model rats were randomly divided into prevention diabetes model group(PDM group, n=15), prevention TWP low dose group(PTL group,6mg/kg·d, n=15), prevention TWP middle dose group(PTM group,12mg/kg·d,n=15), prevent TWP high dose group(PTH group,24mg/kg·d n=15), treatment diabetes model group(TDM group, n=15), treatment TWP low dose group(TTL group,6mg/kg·d, n=15), treatment TWP middle dose group(TTM group,12mg/kg·d, n=15) and treatment TWP high dose group(TTH group,24mg/kg·d n=15). PNC, PDM, PTL, PTM, PTH group, began gavage4weeks after being DM model and was executed and removed organs in the4th weekend. While TNC, TDM、TTL、TTM、TTH group, began gavage4weeks after being DN model in the5th week and was executed and removed organs in the8th weekend. PNC, TNC, PDM and TDM group was used distilled water to fill the stomach. Body weight, blood glucose, hepatic and renal function, blood lipids and24h urinary albumin (UMA) were observed; Thl/Th2cells and IFN-γ、TNF-α、IL-4、 IL-10in peripheral blood were observed by flow cytometry and enzyme-linked immuno sorbent assay(ELISA); The expression of four cytokines:IFN-γ、TNF-α、 IL-4、IL-10were observed by RT-PCR and Western blotting personally from the genetic and protein levels in kidney tissues.Results1) Group of fatty and sugary feedstuff feeding rats before being DM model blood glucose is significantly higher than PNC, TNC group(P<0.05). After STZ injection, Group of fatty and sugary feedstuff feeding rats appeared performance of polydipsia, polyphagia and polyuria, the blood sugar and the weight is significantly higher and lower than before(P<0.05), meanwhile the24h urinary protein levels of the rats continued to rise from the4th week.2) PDM and TDM group significantly increased the levels of UMA and KW/BW than PNC and TNC group (P<0.05). Compared with the PDM and TDM group, TWP prevention and treatment of three dosage groups of UMA, Kidney Weight/Body Weight (KW/BW) were declined in different levels. PTM and TTM group produced the most obvious reduction of UMA and KW/BW than other administration groups (P<0.05). However, the levels of UMA and KW/BW in the PTH group was not changed significantly(P>0.05).3) General indicators:TWP prevention and treatment group no significantly changed alanine transaminase(ALT), creatinine(SCr), blood urea nitrogen(BUN), white Blood Count(WBC), Red Blood Cell(RBC), Hemoglobin(Hb), Platelets(PLT), Monocyte(MONO) and Neutrophile granulocyte(NEUT) than PNC and TNC group(P>0.05). In PTH group, aspartate aminotransferase(AST) was significantly raised as compared with other groups (P<0.05). TTL, TTM and TTH group obviously reduced the number of lymphocytes(LYM) than TNC Group (P<0.05), but no difference among the three groups(P>0.05). Group of fatty and sugary feedstuff feeding rats before being DM model triglyceride (TG) and total cholesterol (TC) level is significantly higher than PNC, TNC group(P<0.05). The level of TG and TC increased gradually in PDM and TDM group than PNC> TNC group (P<0.05), while TWP prevention and treatment three dosing groups were significantly declined TG and TC.4) FCM and ELISA:PDM and TDM significantly raised Thl cells, the ratio of Thl/Th2, IFN-γ and TNF-α levels and lowered Th2cells, IL-4and IL-10 levels in blood than PNC and TNC group (P<0.05). Compared with the PDM and TDM group, prevention and treatment three dosing groups had different degrees of reducing, PTM, TTM group declined significantly Thl cells, the ratio of Th1/Th2, IFN-γ and TNF-α levels (P<0.05), but TNF-α level in PTH group was not clearly reduced (P>0.05).5) RT-PCR and Western Blotting:compared with PNC and TNC group, there were more IFN-γ, TNF-αand less IL-10espressed in PDM and TDM group (P<0.05), but IL-4expressed no significantly reduced (P>0.05). In TWP prevention and treatment three dosing groups, there were different reduced IFN-γ and TNF-α expression and raised IL-4and IL-10expression than PDM and TDM group, while in PTH group, there were no obviously less TNF-α and more IL-4and IL-10expressed than PDM group(P>0.05).Conclusions1) We successfully prepared animal model of T2DM and DN rats, the24h urine protein features of the rats in line with the characteristics of the human type2diabetic nephropathy.2) The24h urinary protein levels and the kidney weight index of the group of fatty and sugary feedstuff feeding rats continued to rise from the4th week.Application of low and middle dose TWP can significantly lower the urinealbumin level of the rats to delay and treat occurrence and development of type2diabetic nephropathy and has a high security. While high doses Tripterygium glycosides may cause liver and kidney toxicity, and therefore should be strictly controlled indications and contraindications.3) T2DM model rats existed Th1/Th2cells and cytokines imbalance, and the increasing of Thl cells and pro-inflammtaory cytokines, the reducing of Th2cells and anti-inflammatory cytokines secretion can cause delayed immune inflammatory response may result in the occurrence and development of DN.4) Different doses of TWP can reduce the levels of UMA by adjusting Th1/Th2cells rato and ameliorating blood lipid. In the kidney, TWP may be a significant inhibition of pro-inflammatory cytokine production, increase the expression of anti-inflammatory cytokines, reduce local inflammation in the kidneys.