Effect Of Notch And Hh Signaling Pathways In Renal Tissue Of Rat With Diabetic Kidney Disease And The Intervention Effect Of Huayu Tongluo Herbs

Objective:This study was designed to investigate the intervention effects of Huayu Tongluo Chinese medicine on the blood stasis syndrome and renal function impairment in rats with diabetic kidney disease(DKD),and to explain whether the Notch and Hedgehog(Hh)pathways is participated.Methods:60 healthy male Sprague-Dawley rats were randomly divided into control group(Group C,n=10)and modelling group(n=50).To establish the diabetic models,rats in modelling group were fed with high-sugar,high-fat diet and with a single small dose(35 mg/kg)of streptozotocin(STZ)by intraperitoneal injection.The diabetic model rats were randomly divided into model group(Group M),Huayu Tongluo herbs group(Group Q)and irbesartan group(Group I).The rats in Group Q and Group I were given the corresponding drugs by gavage,and the rats in Group C and Group M were given the corresponding volume of purified water by gavage.All rats were intervened for 16 weeks.Random blood glucose,water intake,food intake,urine volume and 24h urinary total protein(24h UTP)were regularly determined.And the rats were weighed at the end of the 16~thh week.The blood and urine specimens of the rats were taken after anesthesia.Right kidneys were weighed.Renal tissues were fixed for pathological examination by light microscope and electron microscope.Immunohisto chemical(IHC)technique,Real-time PCR and Western Blot were utilized to detect the expression of protein and mRNA in Notch and Hh signaling pathways.Results:1 Interventional effects of Huayu Tongluo herbs on the indexes related to Blood Stasis in the rats with diabetic kidney disease1.1 General conditions of ratsThe rats in Group M,Group Q and Group I showed polydipsia,polyphagia,polyuria,loss of weight,fur with no lustre,dull response and cyanoderma of tongue and paw.In addition,some rats also appeared skin ulcer in the late period.The symptoms of rats in the treatment groups were improved compared with Group M from the 12~thh week.1.2 Random blood glucose and lipid testsCompared with Group C,the levels of triglyceride,low-density lipoprotein and very low density lipoprotein were increased greatly in Group M,Group Q and Group I(P<0.01).The levels of triglyceride,low-density lipoprotein and very low density lipoprotein in Group Q were significantly lower than Group M(P<0.01).There was no significant difference in random blood glucose among modelling groups(P>0.05).1.3 24h urinary total proteinFrom the 4~thh week,24h urinary total protein was significantly increased in modelling groups compared with Group C(P<0.01).At the end of the 12~thh and 16~thh week,24h UTP of rats in the treatment groups were significantly reduced compared with Group M(P<0.01).At the end of 16~thh week,24h UTP of rats in Group Q was significantly declined compared with Group I(P<0.01).1.4 Hemagglutination index testsCompared with Group C,prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT)and international standardization ratio(INR)were all reduced in modelling groups(P<0.05 or P<0.01),and the level of plasma fibrinogen(FIB)was increased in modelling groups(P<0.01).Compared with Group M,PT,APTT and INR were increased significantly in Group Q(P<0.05 or P<0.01),and there was no significant difference between Group Q and I(P>0.05).There was no significant difference in the TT and FIB among the modelling groups(P>0.05).2 Intervention effects of Huayu Tongluo herbs on kidney injury in rats with diabetic kidney disease2.1 Comparison of kidney hypertrophy indexCompared with Group C,the kidney hypertrophy index was significantly increased in modelling groups(P<0.01).Compared with Group M,the kidney hypertrophy index of rats in Group Q was significantly decreased(P<0.01).2.2 Observation by light microscopy and transmission electron microscopyPathological changes of kidney structure were observed in modelling groups.According to observation by light microscopy,it showed glomerular enlargement,narrow renal capsule,glomerular basement membrane thickening,increased mesangial matrix and swelling of tubular epithelial cells.According to observation by electron microscopy,it showed homogeneous thickening of glomerular basement membrane,and footsteps partly integrated and disappeared.The pathological changes of the treatment groups were improved in different degree.2.3 Renal function testCompared with Group C,the serum creatinine,urea nitrogen,and uric acid were significantly increased in modelling groups(P<0.01).The serum creatinine and serum uric acid in Group Q were significantly lower Group M(P<0.01).3 The intervention effect of Huayu Tongluo herbs on expression of Notch pathway in renal tissue of rats with diabetic kidney disease3.1 Detection of Notch1,Jagged1,HES1 and Hey1 mRNA expression in rats'kidney by Real-Time PCRCompared with group C,the mRNA expression of Notch1,Jagged1,HES1 and Hey1 was significantly increased in modelling groups(P<0.01).Compared with group M,the mRNA expression of Notch1,Jagged1,HES1and Hey1 was significantly decreased in group Q and I(P<0.01).The mRNA expression of HES1 was significantly declined in group Q compared with group I(P<0.01).3.2 Detection of Notch1,Jagged1,HES1 and Hey1 protein expression in rats'kidney by IHCNotch1 proteins with yellow or brown color were observed by IHC in cytoplasm of renal glomeruli and tubular epithelial cells.Hey1 proteins were observed in the nuclear of glomeruli and renal tubular epithelial cells,and also found in cytoplasm of intrinsic glomerular cells.While HES1 protein were found in nuclear of glomeruli and renal tubular epithelial cells.Notch1,HES1and Hey1 proteins were weakly expressed in group C.Compared with group C,Notch1,HES1 and Hey1 protein expression was significantly increased in modelling groups(group M is higher than group Q and I).Jagged1 protein was mainly expressed in both kidney tubular basement membrane and epithelial cells.The expression of Jagged1 in group M was increased compared with group C,but decreased compared with group Q and I.3.3 Detection of Notch1,Jagged1,HES1 and Hey1 protein expression in rats'kidney by Western BlotCompared with group C,the protein expression of Notch1,Jagged1,HES1 and Hey1 was significantly increased in modelling groups(P<0.01).Compared with Group M,the protein expression of Notch1,Jagged1,HES1and Hey1 was significantly decreased in group Q and I(P<0.01);Compared with Group I,the protein expression of HES1 and Hey1 was significantly reduced in Group Q(P<0.01).4 The intervention effect of Huayu Tongluo herbs on Hh pathway in tubular interstitium of rats with diabetic kidney disease4.1 Detection of GLI1,SHH,FSP1 and LN protein expression in rats'kidney by IHCGLI1,SHH,FSP1 and LN proteins with yellow or brown color were observed in cytoplasm of tubular epithelial cells.The four proteins(GLI1,SHH,Fsp1,and LN)were weakly expressed in group C.The expression of the four proteins in group M was much increased compared with group C,and also higher expressed than group Q and I.4.2 Detection of GLI1,SHHand FSP1 mRNA expression in rats'kidney by Real-Time PCRCompared with group C,the mRNA expression of GLI1,SHH and FSP1was significantly increased in modelling groups(P<0.01);Compared with group M,the mRNA expression of SHH mRNA was significantly decreased in group Q and I(P<0.05),and there was no significant difference between Group Q and I(P>0.05).Compared with group M,the mRNA expression of GLI1 and FSP1 mRNA was significantly decreased in group Q and I(P<0.01),and compared with group Q,it was increased in group I(P<0.05 or P<0.01)4.3 Detection of GLI1,SHH and FSP1 protein expression in rats'kidney by Western blotCompared with group C,the protein expression of GLI1,SHH,and FSP1was significantly increased in modelling groups(P<0.01).Compared with Group M,the protein expression of GLI1,SHH,and FSP1 was significantly decreased(P<0.01).Compared with Group I,the protein expression of GLI1,SHH,and FSP1 was significantly reduced in Group Q(P<0.01).Conclusion:1.The proteinuria and DKD pathological changes of kidney appeared gradually after the blood glucose of the model rats increased,suggesting that the DKD model was successfully prepared.In addition,there was a disorder of glucose and lipid metabolism,blood hypercoagulability and typical symptoms of blood stasis in the model rats,which were consistent with the changes of blood stasis syndrome in the model rats in traditional Chinese medicine.2.Huayu Tongluo herbs can significantly improve the blood stasis symptoms,relieveproteinuria in DKD rats and renal pathological damageto protect the kidney of rats.3.Huayu Tongluo herbs can inhibit the protein expression of Notch1,Jagged1,HES1 and Hey1 in Notch pathway.And that may be one of the reasons of relieving proteinuria and reducing urine protein and kidney injury.4.Huayu Tongluo herbs can partially inhibit activation of Hh pathway,decreased the expression of FSP1 and LN protein.And that may be one of the important ways to reduce renal tubular injury and renal interstitial fibrosis.