Expression And Role Of Notch1-RBP-Jk/Msx2 Signaling Pathway In The Aorta Of Different Vascular Calcification Rat Models

Part I ssion and role of Notch1-RBP-Jk/Msx2 signaling pathway n the aorta of diabetic nephropathy rats with vascular calcificationObjective: Vascular calcification,one of the major complication of tic nephropathy(DN),is the leading cause of death in patients with However,the precise mechanism of vascular calcification in DN nts is still not fully understood.So in order to explore the effect of h1-RBP-Jk/Msx2 signaling pathway on development of vascular ication occurred in DN,we observed the calcification of aorta in tic nephropathy rats with vascular calcification,and the expression Notch1-RBP-Jk/Msx2 signal on DN rats aorta.Methods: 42 male ats were randomly divided into normal group(Nor group,n=18),iabetic nephropathy with vascular calcification group(DN+VDN,n=24).DN+VDN group rats received intraperitoneal injection35 mg/kg streptozotocin(STZ)in citrate solution after 4 weeks of a fat diet to induce diabetes.Following this(after 3 days),blood se levels were detected utilizing One-Touch strip.Rats with blood se concentrations above 16.7 mmol/L were considered as diabetic ere used in the experiments followed.24 hours urinary protein of ling rats after 2 weeks was detected.If it were higher than 30 mg,N modeling succeed,otherwise it failed and would be given up.DN+VDN group rats were continued to treat with vitamin D3 and ne.The Nor group included age-matched rats that were only given a le buffer and a standard rat chow diet.Rats were sacrificed in each at 8 w,12 w and 16 w respectively after the success of modelingand 24-hour urine was reserved to test 24 hours urinary albumin(24-h Upro);blood was collected from abdominal aorta to test blood glucose,blood urea nitrogen(BUN),serum creatinine(Scr),calcium(Ca)and phosphorus(P);the characteristic to the aortic calcification was detected by Von Kossa staining,immunohistochemistry(IHC)was used to detect the protein expression of ?-SMA and Runx2.The protein expression of Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD in the aorta were tested by IHC.Real-time polymerase chain reaction(Real-time PCR)was applied to detect the gene expression levels of ?-SMA,SM22?,Runx2,ALP,Notch1,RBP-Jk,Msx2 and Jagged1 m RNA.Results: 1.General index(1)body weight: Compared with Nor group,model group decreased obviously(P<0.001).(2)Blood glucose: The level of blood glucose in Nor group fluctuated in the normal range,DN+VDN was higher than Nor group(P<0.01).(3)24-h Upro: The 24-h Upro in DN+VC group were gradually increased in 8th,12 th and 16 th weeks,and were higher than that group of Nor(P<0.001).(4)Renal function: BUN,Scr: The DN+VDN group rat BUN level were obviously higher than that of Nor group(P<0.001).Compared with Nor group,the level of Scr in DN+VDN group started increasing in 12 th week(P<0.001).(5)calcium(Ca),phosphorus(P),calcium-phosphorus product(Ca×P): The DN+VDN group rat serum Ca level were obviously higher than that of Nor group(P<0.05).Compared with the Nor,the level of serum P in DN+VDN group started increasing in 16 th week(P<0.05).The DN+VDN group rat Ca×P level were obviously higher than that of Nor group(P<0.05).2.The characteristics of the aortic calcification:(1)Von Kossa staining: The deposition of black granules in DN+VDN group was gradually increased in 8th,12 th and 16 th weeks,and were significantly higher than that group of Nor at each time point(P<0.001).(2)IHC tested the expression of ?-SMA and Runx2: As compared with Nor group,the expression of ?-SMA evaluated by IHC was relatively low,while the expression of Runx2 was relatively high in the DN+VDN rats at each time point(P<0.05).3.The level of Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD signal protein: The expression of Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD in the Nor group was weakly appeared in the aortic wall,while in DN+VDN group these signal protein expression increased significantly(P<0.05)and showed a continual increasing trend.4.?-SMA,SM22?,Runx2 and ALP m RNA expression: As compared with Nor group,the expression of ?-SMA and SM22? m RNA in the DN+VDN group were relatively low,yet the expression of Runx2 and ALP m RNA were relatively higher in the DN+VDN rats at each time point(P<0.05).5.Transcription level of Notch1,RBP-Jk,Msx2 and Jagged1: The Real-time PCR analysis results showed the level of Notch1,RBP-Jk,Msx2 and Jagged1 m RNA in the DN+VDN group at each time point were significantly up-regulated compared to the Nor group(P<0.05).Conclusion: 1.There has existed phenotypic changes in smooth muscle cells and activation of osteogenic factors and Notch1-RBP-Jk/Msx2 signaling pathway in diabetic nephropathy with vascular calcification;2.Notch1-RBP-Jk/Msx2 signaling pathway may be involved in the pathogenesis of vascular calcification in diabetic nephropathy,and may be one of the important signal transduction pathways.Part II The expression and role of Notch1-RBP-Jk/Msx2 signaling pathway in the aorta of chronic renal failure rats with vascular calcificationObjective: Vascular calcification(VC)is the main manifestation of cardiovascular complications in patients with CKD,is also the main cause of cardiovascular events in dialysis patients(stage CKD 5D).Understanding the mechanism of vascular calcification is the key to prevent and block the pathological process of VC.So in order to explore the effect of Notch1-RBP-Jk/Msx2 signaling pathway on development of vascular calcification occurred in chronic renal failure(CRF),we observed the calcification of aorta in chronic renal failure rats with vascular calcification,the activation of vascular smooth muscle cell phenotypic marker ?-SMA,SM22?,osteogenic transcription factor ALP and Runx2.In addition,we observed Notch1-RBP-Jk/Msx2 signal pathway activation on CRF+VC rats aorta.Methods: 40 male SD rats were randomly divided into normal group(Nor group,n=15),and chronic renal failure with vascular calcification group(CRF+VC group,n=25).CRF+VC group were placed on a high phosphate(1.8%)diet.In addition,the CRF+VC group were given adenine gavage with 250mg/kg/d for four weeks.After four weeks,adenine gavage were given every other day.The Nor group included age-matched rats that were only given a vehicle buffer and a standard rat chow diet.Rats were sacrificed in each group at 4 w,6 w and 8 w respectively after the success of modeling and 24-hour urine was reserved to test 24-h Upro;blood was collected from abdominal aorta to test BUN,Scr,Ca and P;the characteristic to the aortic calcification were detected by alizarin red Sstaining,IHC was used to test the protein expression of ?-SMA,Runx2,Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD in the aorta.Real-time PCR was applied to detect the gene expression levels of ?-SMA,SM22?,Runx2,ALP,Notch1,RBP-Jk,Msx2 and Jagged1 m RNA.Results: 1.General index(1)body weight: compared with Nor group,CRF+VC group decreased obviously(P<0.01).(2)24-h Upro: The 24-h Upro in group of CRF+VC were significantly increased in 4th,6th and 8th weeks,and were higher than that group of Nor(P<0.05).(3)Renal function:BUN,Scr: compared with Nor group,the level of BUN and Scr in the CRF+VC group were increased in 4th,6th and 8th weeks(P <0.01).(4)Ca,P,Ca×P: The CRF+VC group rat serum Ca level was obviously higher than that of Nor group in 4th and 8th weeks(P<0.01),while serum Ca showed no difference between 2 groups in 6th week.The level of serum P and Ca×P in the CRF+VC group were increased significantly at each time point compared with Nor group(P<0.01).2.The characteristics of the rat aortic calcification:(1)Alizarin red S staining: Numerous continuous calcified nodules were detected in the vascular wall of the CRF+VC group.(2)IHC tested the expression of ?-SMA and Runx2: As compared with Nor group,the expression of ?-SMA evaluated by IHC was relatively low,while the expression of Runx2 was relatively high in the CRF+VC rats at each time point(P<0.05).3.The level of Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD signal protein: The expression of Notch1,RBP-Jk,Msx2,Jagged1 and N1-ICD in the Nor group was weakly appeared in the aortic wall,while in CRF+VC group these signal protein expression increased significantly during the experimental period(P<0.05).4.?-SMA,SM22?,Runx2 and ALP m RNA expression: As compared with Nor group,the expression of ?-SMA and SM22? m RNA in the CRF+VC group were relatively low,yet the expression of Runx2 and ALP m RNA were relatively higher in the CRF+VC rats at each timepoint(P<0.01).5.Transcription level of Notch1,RBP-Jk,Msx2 and Jagged1: The Real-time PCR analysis results showed the level of Notch1,RBP-Jk,Msx2 and Jagged1 m RNA in the CRF+VC group at each time point were significantly up-regulated compared to the Nor group(P<0.05).Conclusions: 1.There has existed phenotypic changes in smooth muscle cells and activation of osteogenic factors and Notch1-RBP-Jk/Msx2 signaling pathway in chronic renal failure with vascular calcification;2.Notch1-RBP-Jk/Msx2 signaling pathway may be involved in the pathogenesis of vascular calcification in chronic renal failure,and may be one of the important signal transduction pathways.