The Study Of Jiangtang Sanhuang Tablet On TLR4/NF-κB Pathway Of Diabetic Nephropathy Rats

Literature research:Diabetic nephropathy is related to "Xia Xiao"、"Shen Xiao" which are complications of Xiaoke disease.The etiology are related to the deficiency of innate endowment,unrestrained diet,excessive sexual life,and emotional disorders and other factors.The pathogenesis is based on the deficiency of the spleen and stomach,the deficiency of the kidney,It is marked by dryness heat,phlegm,dampness,blood stasis,and phlegm poison.The main treatment is to improve the kidney and the spleen and to nourish yin and clear the heat,accompanied by reducing phlegm and water,promoting blood circulation and removing blood stasis,purging turbid phlegm.Modern medicine believes that the occurrence of diabetic nephropathy involves a variety of factors,such as the accumulation of advanced glycation end products,protein kinase C activation,polyol pathway activation,lipid metabolism disorders,hypertension,cytokines,etc.In recent years,more and more studies show that inflammation plays an important role in the pathogenesis of diabetic nephropathy.TLR4/NF-κB pathway is an important signaling pathway mediated inflammatory response.TLR4 can activate NF-κ B,increased expression of inflammatory cytokines,promote the progression of diabetic nephropathy.Jiangtang Sanhuang Tablet is a hospital preparation developed from Taohechengqi Decoction,with the effect of supplementing Qi and nourishing Yin,promoting blood circulation and removing blood stasis,purging heat and purging bowels.It has a certain effect in the prevention and treatment of diabetic nephropathy.Experimental StudyObjectiveTo observe the effect of Jiangtang Sanhuang Tablet(JTSH)on Toll like receptor 4(TLR4)/Nuclear factor-κB(NF-κB)pathway and the expression of Monocyte chemoattractant protein-1(MCP-1)of diabetic nephropathy(DN)rats,and investigate the protective effect and mechanism of JTSH on DN.MethodsAfter an adaptive feeding,80 SPF SD rats were randomly divided into normal group(n=10)and model group(n=70).Before modeling,fasting 12 hours,not stop drinking water,the model group were produced by intraperitoneal injection of streptozocin(STZ)at 45mg/kg,and the normal group was injected with the same amount of citric acid buffer.After modeling 72 hours,fasting blood glucose was measured in rats.If the blood glucose level was>16.7mmol/L,it was confirmed as a diabetic model.The normal group was fed with normal diet,the model group was fed with high sugar and high fat diet for three weeks.After three weeks,urine was measured.When urine volume>150%of the original urine volume,24 hours urine protein>30mg/24h,it was confirmed as diabetic nephropathy model.Diabetic nephropathy model rats were randomly divided into model group(n=12),low dose group of JTSH(n=11),middle dose group of JTSH(n=11),high dose group of JTSH(n=11),irbesartan group(n=11).The low dose group of JTSH was given 675mg/kg·d by intragastric administration.The middle dose group of JTSH was given 1350mg/kg · d by intragastric administration.The high dose group of JTSH was given 2700mg/kg·d by intragastric administration.The irbesartan group was given 13.5mg/kg·d by intragastric administration.Before intragastric administration,all the drugs were mixed with purified water to 2 ml,once a day for 8 weeks.Normal group and model group were given equal distilled water.At the end of the experiment,62 rats finished the study.Among them 10 rats were in the normal group,9 rats in model group,10 rats in low dose group of JTSH,11 rats in middle dose group of JTSH,11 rats in high dose group of JTSH,11 rats in irbesartan group.The fasting blood glucose was measured after 72 hours of molding and before the treatment.The urine protein was collected before treatment and after 8 weeks of treatment.After 8 weeks of treatment,blood samples were obtained from abdominal aorta by intraperitoneal injection of 10%chloral hydrate to narcotize.Fasting blood glucose,HbAlc,fasting insulin,total triglyceride,total cholesterol,high density lipoprotein cholesterol,low density lipoprotein cholesterol,serum creatinine,blood urea nitrogen and other indicators were detected.The expressions of TLR4,NF-κB(p65)and MCP-1 were detected by immunohistochemistry.Western blot method was used to detect the expression of TLR4,NF-κB(P-p65)and MCP-1 protein in renal tissue of each group.Results1.After 8 weeks of treatment,the fasting blood glucose of low,middle and high dose group of JTSH were significantly lower than that of model group(P<0.01).There was no significant difference in fasting blood glucose between the irbesartan group and the model group(P>0.05).HbAlc of low,middle and high dose group of JTSH were significantly lower than that of model group(P<0.01).There was no significant difference in HbAlc between the irbesartan group and the model group(P>0.05).TG,TC and LDL-C of low,middle and high dose group of JTSH were significantly lower than that of model group(P<0.01).HDL-C of low,middle and high dose group of JTSH were significantly higer than that of model group(P<0.01).There was no significant difference in TG,TC,LDL-C and HDL-C between the irbesartan group and the model group(P>0.05).The relative kidney weight of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).After 8 weeks of treatment,24h urine protein of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.05).After 8 weeks of treatment,there was no significant difference in 24h urine protein between high dose group of JTSH and irbesartan group(P>0.05).The serum creatinine of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.05).The serum creatinine of high dose group of JTSH were significantly lower than that of irbesartan group(P<0.01).The blood urea nitrogen of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).The blood urea nitrogen of high dose group of JTSH were significantly lower than that of irbesartan group(P<0.01).2.TLR4,NF-kappa B(p65)immunohistochemical staining showed that TLR4 average optical density of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.05).There was no significant difference in TLR4 average optical density between high dose group of JTSH and irbesartan group(P>0.05).NF-κB(p65)average optical density of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).There was no significant difference in NF-κB(p65)average optical density between high dose group of JTSH and irbesartan group(P>0.05).The expression of TLR4 and NF-κB(p65)protein was detected by Western blotting.Expression of TLR4 protein of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).There was no significant difference in the expression of TLR4 protein between high dose group of JTSH and irbesartan group(P>0.05).Expression of NF-κB(P-p65)protein of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).There was no significant difference in the expression of NF-κB(P-p65)protein between high dose group of JTSH and irbesartan group(P>0.05).3.MCP-1 immunohistochemical staining showed that MCP-1 average optical density of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.05).There was no significant difference in MCP-1 average optical density between high dose group of JTSH and irbesartan group(P>0.05).The expression of MCP-1 protein was detected by Western blotting.Expression of MCP-1 protein of low,middle,high dose group of JTSH and irbesartan group were significantly lower than that of model group(P<0.01).There was no significant difference in the expression of MCP-1 protein between high dose group of JTSH and irbesartan group(P>0.05).ConclusionJTSH can improve glucose and lipid metabolism,decrease the excretion of urinary protein,improve renal function.There was a dose-effect relationship between low,middle and high dose group of JTSH,and the effect of high dose group was the best.JTSH by inhibiting TLR4/NF-κB inflammatory signaling pathways,deduce the expression of MCP-1 cytokines,to reduce glomerular hypertrophy,reduce the excretion of urinary protein,protect renal function and delay the development of DN.