| Part one Protective effect of Huayu Tongluo Herbs on renal in diabetic nephropathy ratsObjective: to observe the effect of Huayu Tongluo Herbs on physical and chemical indexes and renal histopathological changes in rats with diabetic nephropathy,and to verify the protective effect of Huayu Tongluo Herbs on renal.Methods: 10 clean male SD rats were randomly selected as normal group(C group),and the other 45 rats were fed with high fat diet combined with intraperitoneal injection of streptozotocin(STZ,40 mg/kg)to establish diabetic model.The successful rats were randomly divided into model group(M group),Huayu Tongluo Herbs group(Z group)and Irbesartan group(I group).The corresponding drugs were gavaged respectively.At the end of 0th,4th,8th,12 th and 16 th week,fasting blood glucose(FBG)was measured,24-hour drinking water and food intake of each rat were collected and recorded,24-hour urine was collected and recorded,and part of the urine was retained for 24-hour urine total protein(24h UTP).At the end of 16 th week,blood samples were taken to detect serum creatinine(Scr),blood urea nitrogen(BUN),uric acid(UA),total cholesterol(TC)and triglyceride(TG).Renal tissue was taken for PAS and Masson staining to detect the degree of pathological injury and fibrosis.Results:1.General condition of rats in each groupWithin 16 weeks of intervention,there was no death in C group,4 rats were death in M group,3 rats were death in Z group and 4 rats were death in I group.At the end of the experiment,the final number of rats used for data statistics was as follows: C group(n = 10),M group(n = 10),Z group(n = 11)and I group(n = 10).In C group,the mental state of rats was good,the activity was sensitive,the hair was smooth and shiny,and the posture was normal;The rats in M group were dispirited,slow,dishevelled,dull and thin;Compared with M group,the mental state,activity and coat color of rats in Z group and I group were alleviated in varying degrees.2.Comparison of FBG,body weight,water intake,food intake and urine volume of rats in each group at different time points2.1 Comparison of FBG of rats in each group at different time pointsAt the same time(at the end of 0th,4th,8th,12 th and 16 th week of administration),compared with C group,the level of FBG was significantly higher in M group(P < 0.01),but compared with M group,there was no significant change in Z group and I group(P > 0.05).2.2 Comparison of body weight of rats in each group at different time pointsAt the 0th week of administration,there was no significant difference in body weight among the three groups(P > 0.05).At the same time(at the end of the 4th,8th,12 th and 16 th week of administration),compared with C group,the body weight was significantly lower in M group(P < 0.01),but compared with M group,there was no significant change in Z group and I group(P >0.05).2.3 Comparison of water intake of rats in each group at different time pointsAt the same time(at the end of 0th,4th,8th,12 th and 16 th week of administration),compared with C group,the level of water intake was significantly higher in M group(P < 0.01),but compared with M group,there was no significant change in Z group and I group(P > 0.05).2.4 Comparison of food intake of rats in each group at different time pointsAt the 0th week of administration,there was no significant difference in food intake among the three groups(P > 0.05).At the same time(at the end of the 4th,8th,12 th and 16 th week of administration),compared with C group,the food intake was significantly higher in M group(P < 0.01),but compared with M group,there was no significant change in Z group and I group(P >0.05).2.5 Comparison of urine wolume of rats in each group at different time pointsAt the same time(at the end of 0th,4th,8th,12 th and 16 th week of administration),compared with C group,the level of urine wolume was significantly higher in M group(P < 0.01),but compared with M group,there was no significant change in Z group and I group(P > 0.05).3.Comparison of 24 h UTP of rats in each group at different time pointsAt the 0th week of administration,there was no significant difference in24 h UTP among the three groups(P > 0.05).As time went by,the level of 24 h UTP increased gradually in M group,Z group and I group.At the same time(at the end of the 4th,8th,12 th and 16 th week of administration),compared with C group,the level of 24 h UTP was significantly higher in M group(P < 0.01);Compared with M group,the level of 24 h UTP in Z group and I group was significantly lower(P < 0.05).At the end of the 16 th week,compared with I group,the level of 24 h UTP in Z group was significantly lower(P < 0.05).4.Comparison of BUN,Scr,UA,TC and TG of rats in each groupAt the end of the 16 th week,compared with C group,the levels of BUN,Scr and UA in M group were significantly higher(P < 0.01);Compared with M group,the levels of BUN and Scr in Z group and I group were significantly lower(P < 0.01),while the level of UA had no significant change(P > 0.05);Compared with I group,the levels of BUN and Scr in Z group had no significant change(P > 0.05).At the end of the 16 th week,compared with C group,the levels of TC and TG in M group were significantly higher(P < 0.01);Compared with M group,the levels of TC and TG in Z group were significantly lower(P < 0.01),while the level of TC and TG in I group had no significant change(P > 0.05);Compared with I group,the levels of TC and TG in Z group was significantly lower(P < 0.05).5.Renal pathological changes of rats in each group under Optical MicroscopePAS staining showed thickening of capillary basement membranes and hyperplasia of extracellular matrix in M group rats,which was significantly restored by Huayu Tongluo Herbs.Additionally,Masson’s staining showed a large number of blue collagen fiber in glomerular basement membranes,mesangial matrix and renal interstitium,however,smaller blue stained area was observed when treated with Huayu Tongluo Herbs.Part two Protective effect of Huayu Tongluo Herbs on diabetic nephropathy rats by reducing inflammation and then relieving renal fibrosisObjective: to observe the effect of Huayu Tongluo Herbs on inflammation and renal fibrosis related proteins in diabetic nephropathy rats,and to explore the intervention effect of Huayu Tongluo Herbs on inflammation and renal fibrosis.Methods: 10 clean male SD rats were randomly selected as normal group(C group),and the other 30 rats were fed with high fat diet combined with intraperitoneal injection of STZ(40 mg/kg)to establish diabetic model.The successful rats were randomly divided into model group(M group)and Huayu Tongluo Herbs group(Z group).The rats of Z group were gavaged with Huayu Tongluo Herbs.At the end of 16 th week,blood samples were taken from femoral artery to detect the levels of serum interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein-1(MCP-1).The expression of CD68 and inducible nitric oxide synthase(i NOS)was detected by immunofluorescence,the expression of collagen III(Col-III),collagen IV(Col-IV)and fibronectin(FN)was detected by immunohistochemical,and the levels of IL-1β,TNF-α and MCP-1 m RNA were detected by fluorescence quantitative PCR.Results:1.General condition of rats in each groupWithin 16 weeks of intervention,there was no death in C group,4 rats were death in M group and 3 rats were death in Z group.At the end of the experiment,the final number of rats used for data statistics was as follows: C group(n = 10),M group(n = 10)and Z group(n = 11).2.Compariton of serum IL-1β,TNF-α and MCP-1 of rats in each groupCompared with C group,the levels of IL-1β,TNF-α and MCP-1 in M group were significantly higher(P < 0.01),while the levels of IL-1β,TNF-αand MCP-1 in Z group were significantly lower than those in M group(P <0.01).3.Expression of CD68 and i NOS proteins in kidney tissue of rats in each groupImmunofluorescence results showed that there was only a small amount of CD68 and i NOS proteins expression in renal interstitium in C group,but a large amount of CD68 and i NOS proteins expression in renal interstitium in M group,while the expression of CD68 and i NOS proteins in Z group was significantly lower than those in M group.4.Expression of Col-Ⅲ,Col-IV and FN proteins in kidney tissue of rats in each groupThe immunohistochemistry results showed that there was a small amount of Col-III and FN protein expression in the renal interstitium in C group and a large amount of Col-III and FN protein expression in the renal tubulointerstitium in M group,whereas these changes were significantly inhibited by Huayu Tongluo Herbs.The expression of Col-IV protein in glomerular capillary wall and renal tubule basement membrane was linear in C group,the expression of Col-IV protein in glomerular capillary wall and tubular basement membrane was significantly increased in M group,while the expression of Col-IV was significantly decreased in Z group.5.Compariton of serum IL-1β,TNF-α and MCP-1 m RNA of rats in each groupThe q PCR results showed that,compared with C group,the levels of IL-1β,TNF-α and MCP-1 m RNA in M group were significantly higher(P <0.01),while the levels of IL-1β,TNF-α and MCP-1 m RNA in Z group were significantly lower than those in M group(P < 0.05).Part three Effect of Huayu Tongluo Herbs on reducing inflammation and renal fibrosis in diabetic nephropathy rats by inhibiting NF-κB and TGF-β1/MAPK pathwayObjective: to observe the effect of Huayu Tongluo Herbs on NF-κB and TGF-β 1/MAPK signal pathway in diabetic nephropathy rats,and to explore the molecular mechanism of Huayu Tongluo Herbs in alleviating inflammation and renal fibrosis.Methods: the methods of modeling,grouping and administration of SD rats were the same as those of the second part.At the end of 16 th week,the renal tissue was taken and the expression of transforming growth factor-β1(TGF-β1)was detected by immunohistochemistry,and the expression of TGF-β1 m RNA was detected by q PCR.The expression of NF-κB and TGF-β1/MAPK signal pathway related proteins(NF-κB p65,nuclear NF-κB p65,c-Raf,p-c-Raf,ERK,p-ERK,TAK1,p-TAK1,JNK,p-JNK,P38 MAPK and p-P38MAPK)were detected by Western blot.Results:1.General condition of rats in each groupWithin 16 weeks of intervention,there was no death in C group,4 rats were death in M group and 3 rats were death in Z group.At the end of the experiment,the final number of rats used for data statistics was as follows: C group(n = 10),M group(n = 10)and Z group(n = 11).2.Expression of TGF-β1 protein in kidney tissue of rats in each groupThe immunohistochemical results showed there was a small amount of TGF-β1 protein expression in glomeruli and tubulointerstitium in C group,and the expression of TGF-β1 protein in glomeruli and tubulointerstitium was significantly increased in M group,but Huayu Tongluo Herbs downregulated the expression of TGF-β1.3.Compariton of TGF-β1 m RNA in kidney tissue of rats in each groupThe q PCR results showed that,compared with C group,the levels of TGF-β1 m RNA in M group were significantly higher(P < 0.05),while the levels of TGF-β1 m RNA in Z group were significantly lower than those in M group(P < 0.05).4.Comparison of NF-κB p65,nuclear NF-κB p65,c-Raf,p-c-Raf,ERK,p-ERK,TAK1,p-TAK1,JNK,p-JNK,P38 MAPK and p-P38 MAPK proteins in renal tissue of rats in each groupThe western blot results showed that the protein expression of nuclear NF-κB p65,p-c-Raf,p-ERK1/2,p-TAK1,p-JNK and p-P38 MAPK in M group rats were obviously higher than those in C group rats(P < 0.01),Huayu Tongluo Herbs significantly decreased the protein expression of nuclear NF-κB p65,p-c-Raf,p-ERK1/2,p-TAK1,p-JNK and p-P38MAPK(P < 0.01,P < 0.05).Whereas,the expression of NF-κB p65,c-Raf,ERK1/2,TAK1,JNK and P38 MAPK in each group had no difference(P > 0.05).Conclusions:1.Huayu Tongluo Herbs can relieve proteinuria and renal injury in DN rats,and regulate lipid metabolism.Compared with the positive control drug irbesartan,the effect is more lasting and the overall regulation is better.2.Huayu Tongluo Herbs can obviously reduce the infiltration and activation of macrophages in DN rats,reduce the levels of inflammatory factors such as IL-1β,TNF-α and MCP-1,and then reduce the inflammatory reaction.3.Huayu Tongluo Herbs can significantly reduce the expression of fibrosis-related proteins such as Col-III,Col-IV and FN in renal tissue of DN rats,thus relieve renal fibrosis.4.Huayu Tongluo Herbs can inhibit the excessive activation of NF-κB and TGF-β1//MAPK signaling pathways,which may be one of the mechanisms of renal protection,reduction of inflammatory reaction and renal fibrosis.The limitations of this experiment are:(1)Huayu Tongluo Herbs consists multiple herbs,and most of bioactive components in each herb and their interactions are unclear until now.Therefore,We need to deeply study the bioactive components of each herb and carry out strict quality control analysis,in order to achieve high level of efficacy and safety.(2)The sample size is small,and future research needs to increase the sample size to provide more rigorous and high-level evidence for the treatment of DN with Huayu Tongluo Herbs. |
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