A Study On Tangshenheji Treating Diabetic Nephropathy By Regulating Macrophage Polarization In Kidney Of Diabetic Rats

Background:The occurrence and development of diabetic nephropathy(DN) involves many factors. Current researches suggest that The role of inflammatory response in diabetic nephropathy can not be ignored. Macrophage plays an important role in the inflammatory response. The infiltration of macrophages is closely related to the elevation of blood glucose, the deterioration of renal function and renal fibrosis. M2 and M1 are two different phenotypes of macrophages.M1 macrophages and their secretion of inflammatory factors in DN increase significantly, so that the local inflammatory response to enhance, and ultimately cause kidney damage. M2 macrophages can secrete anti-inflammatory cytokines, and inhibit the activation of M1, thereby promoting tissue repair.Modern medicine for the treatment of DN has not achieved the desired results, and the traditional Chinese medicine in this area shows the advantage. A variety of traditional Chinese medicine and compound has been proven to reduce the inflammatory medium and slow down the rate of DN progress. However, there is no study show whether the single herb or compound can impact macrophage phenotype.Tangshenheji is an empirical prescription of Professor Yao’s treatment of DN, which has achieved good therapeutic effect in clinical therapy. Previous studies showed traditional Chinese medicines of improving kidney play a role in anti-fibrosis, detoxification can inhibit inflammatory signaling pathways. However, the mechanism of Tangshenheji for the treatment of DN is not yet clear, whether the impact of the changes in the macrophage need to be further explored.Objective:Observe the changes of M1, M2 macrophages and inflammatory mediators tumor necrosis factor alpha in the kidney of diabetic rats. After the intervention of DN rat model by using the Tangshenheji, we then observe the effect of Tangshenheji on the different phenotypes of macrophages in renal tissue, and discuss the mechanism of the therapeutic effect of DN.Method:High fat diet combined with low dose streptozotocin intraperitoneal injection to establish DN rat model. Successful model rats are randomly divided into 9 group:model group(MX), high dose Tangshenheji group(TG), low dose Tangshenheji group(TD), high dose detoxification group(DG), low dose detoxification group(DD), high dose huangqi group(HG), low dose huangqi group(HD), atorvastatin group(AT), every group 8 rats. Normal and model groups are treated with normal saline at 15mL/(kg·d).Other groups rats are treated with corresponding drugs at 15mL/(kg-d). Record weight and blood sugar when model is successful and after drug intervention; detect 24-hour urinary protein excretion, blood serum creatinine, blood urea nitrogen, albumin and total cholesterol. Western blot (WB) is used to detect the expression expression of Ml marker (iNOS), TNF-α and M2 marker (CD163). Light microscope is used to observe pathological changes in renal tissue after stained with hematoxylin-eosin (HE). Immunohistochemistry measure the expression of iNOS, CD 163 in kidney. Experimental data are analyzed by SPSS 17.0.Result:(1)24-hour urinary protein excretion:model group, high dose detoxification group, high dose huangqi group, low dose huangqi group, atorvastatin group are higher than the normal group; the model group is significantly higher than that of high dose tangshenheji group,low dose tangshenheji group,high dose detoxification group,low dose detoxification group,high dose huangqi group,low dose huangqi group and atorvastatin group (P<0.01); high dose tangshenheji group,low dose tangshenheji group are lower than that of huangqi groups and detoxification groups (P<0.05); high dose tangshenheji group,low dose tangshenheji group,high dose detoxification group and high dose huangqi group are lower than that of atorvastatin group (P<0.05)(2) Serum albumin:compared with the normal group, the model group serum albumin level is significantly lower (P<0.01); the serum albumin of high dose tangshenheji group, low dose tangshenheji group,high dose detoxification group, low dose detoxification group, high dose huangqi group, low dose huangqi group and atorvastatin group are improved when compared with model group. The albumin of high dose tangshenheji group and low dose tangshenheji group are higher than the Atorvastatin group (P< 0.05).(3) Renal function:serum creatinine and blood urea nitrogen of model group is higher than that of the normal group (P<0.01); high dose tangshenheji group and low dose tangshenheji group are lower than model group (P<0.05); high dose tangshenheji group and low dose tangshenheji group are lower than atorvastatin group,but the difference has no statistical significance(P> 0.05).(4) Serum cholesterol:model group is higher than the normal group (P<0.05); atorvastatin group,high dose tangshenheji group,low dose tangshenheji group and high dose detoxification group are lower than that of the model group (P<0.05); There is no difference between atorvastatin group and other traditional Chinese medicine groups(P> 0.05).(5) Renal pathology:HE staining shows that the glomerular volume of the model group increased,glomerular balloon thickening, part of the glomerulus and renal capsule adhesion, capillary plexus with lobulation,mesangial matrix hypertrophy,partly moderate to severe, basement membrane thickening, some areas of glomerulus focal sclerosis; tubular epithelial cell degeneration, loss, focal tubular dilation, interstitial fibrosis, and scattered inflammatory cells.The change of high dose tangshenheji group is slight, no glomerulus significant sclerosis, mesangial matrix mild hyperplasia; a small amount of renal tubular mild dilation and atrophy, focal inflammatory cell infiltration in partial area, no fibrosis formation. The low dose tangshenheji group shows that mesangial mild to moderate hyperplasia, focal renal capsule mild adhesion, no glomerulus sclerosis; tubular dilatation, protein casts, scattered inflammatory cell mild infiltration, pathological change compared with the model group reduced. High and low dose huangqi and detoxify groups, and atorvastatin statin group show mesangial matrix with moderate to severe hypertrophy. some areas sclerosis, tubular epithelial cell vacuolar degeneration, scattered inflammatory cell infiltration. Compared with model group,pathological changes are reduced.But the efficacy is inferior to high and low dose tangshenheji groups. Compared with atorvastatin statin group, mesangial hypertrophy and glomerular sclerosis are less in high and low dose huangqi and detoxify groups.(6) Immunohistochemistry:Normal group is hardly detected iNOS (M1 macrophages) and CD 163 (M2 macrophages) in glomerulus and interstitium. Model group has a large number of M1 macrophages and a little M2 macrophages. All treatment groups have less M1 macrophages and more M2 macrophages than the model group (P<0.05). Compared with atorvastatin statin group,there are less M1 macrophages and more M2 macrophages in high and low dose tangshenheji groups. Pathological changes of high and low dose huangqi and detoxify groups are similar to statins atorvastatin groups,and no statistical difference between them (P>0.05)(7) Western Blot:iNOS and TNF-a are hardly detected in normal renal tissue and only a small amount of CD 163 is observed. Model group has a large number of iNOS, TNF-a and a little CD 163.There are iNOS、TNF-a and CD 163 in high dose tangshenheji group, low dose tangshenheji group,high dose detoxification group, low dose detoxification group, high dose huangqi group, low dose huangqi group and atorvastatin group. All treated groups have less iNOS、TNF-a and more CD163 than the model group (P<0.01);high dose tangshenheji group,low dose tangshenheji group, high dose detoxification group and high dose huangqi group have less iNOS、TNF-a than atorvastatin group (P<0.05); all high dose groups of traditional Chinese medicine have less iNOS、TNF-a than all low dose groups (P<0.05); high dose tangshenheji group and low dose tangshenheji group have less iNOS、TNF-a than detoxification groups and huangqi groups (P<0.05); TNF-a is positively correlated with the expression of iNOS. All traditional Chinese medicine groups have more CD 163 than atorvastatin group(P<0.01); high dose tangshenheji group and low dose tangshenheji group have moe CD 163 than detoxification groups and huangqi groups (P<0.05)Conclusion:(1) M1, M2 macrophages in DN rats increase, especially the number of M1 macrophages.The number of TNF-a increases with the increase of M1 macrophages. This change suggests different phenotypes of macrophages are involved in the inflammation of DN.(2) Tangshenheji can reduce the 24-hour urine protein, serum creatinine, urea nitrogen, cholesterol, elevate serum albumin and reduce the pathological changes.Tangshenheji has the protective effect on the kidney of diabetic rats by inhibiting M1 macrophages infiltration, reducing the expression of TNF-a and promoting M2 macrophages activation.(3)Huangqi and detoxification dampness Chinese herbs also can affect macrophages.But the effect of macrophage activation, proteinuria reduction, elevated serum albumin is not as good as Tangshenheji. Huangqi and detoxification dampness Chinese herbs fail to improve renal function. Detoxification dampness Chinese herbs can reduce serum total cholesterol.