| Research Background and Objective:Chronic kidney disease(CKD)is a global public health problem,which brings serious economic burden to the world.Cardiovascular diseases(CVD)is the most common complication and the main cause of death in CKD patients.With the progress of the disease,the incidence and mortality of CVD increase further.Vascular calcification(VC)is a serious and extremely common complication in CKD patients.It has been reported that vascular calcification is an independent risk factor for the occurrence and death of CVD in CKD patients,but the specific mechanism of vascular calcification in CKD is still unclear.Studies have found that TGF-β1induces the occurrence and development of vascular calcification,Our previous studies also found that COX-2 is involved in the process of vascular calcification.This study aims to investigate the effect and possible mechanism of TGF-β1 on vascular calcification,explores the role and potential mechanisms of COX-2 in the process of vascular calcification induced by TGF-β1,and evaluates the effect of COX-2 inhibitor-meloxicam on renal injury and vascular calcification in rats renal failure model,so as to provide a new target for the prevention and treatment of clinical vascular calcification,It is of great significance to reduce the morbidity and mortality of cardiovascular events and to improve the quality of life in patients with CKD.Methods:1.In vitro experiments(1)VSMCs were extracted from aorta of SD rats.(2)High phosphate-induced VSMCs calcification,Real-time PCR and Western Blot assay were used to detected the m RNA and protein exprssion of TGF-β1,The protein expression of COX-2、β-catenin、p-GSK3β、GSK3βwere tested by Western Blot.Luciferase reporter plasmid assay detected the effect of phosphate on TGF-β1 signaling.(3)VSMCs were infected with Adenovirus TGF-β1,Real-time PCR and Western Blot assay were used to detected the m RNA and protein exprssion of Osteogenic markers Runx2 、 OPN and VSMCs markersα-SMA 、 SM22α.Western Blot assay was used for the detection of COX-2、β-catenin、p-GSK3β、GSK3β、Sclerostion and c-myc protein expression.Immunofluorescent staining detected the protein expression ofβ-catenin.Alizarin Red S staining was used to test the effect of TGF-β1 on calcification in VSMCs.Aortic rings of SD rats was infected with Adenovirus TGF-β1,Von Kossa staining was used to test the effcet of TGF-β1 on calcification in aortic segments at day 14.(4)VSMCs were treated as follows: control,Ad TGF β1,NS398(COX-2 inhibitor),Ad TGF β1+NS398,Real-time PCR and Western Blot assay were used to detected the m RNA and protein exprssion of Runx2、OPN、α-SMA and SM22α.Western Blot assay was applied to detect the proteion expression of β-catenin、p-GSK3β、GSK3β and Sclerostion.The aortic rings of SD rats were treated in the same way,Von Kossa staining was used to test the effcet of TGF-β1 and/or NS398 on calcification in aortic segments.(5)VSMCs were infected with Ad TGF β1,Immunoprecipitaion(IP)assay detected the interaction between p-Smad2/3 and p-CREB in VSMCs.chromatin immunoprecipitation(CHIP)assay was used to detecte the recruitment of p-Smad2/3or p-CREB in the promoter regions of sclerostin and β-Catenin.2.In vivo experiments(1)Renal failure model was induced by 0.75% adenine diet.(2)Rats were randomly divided into 6 groups:Control group(normal diet 6W),low-dose prevention group(0.75% adenine diet + 1mg / kg meloxicam 6W),high-dose prevention group(0.75% adenine diet + 6mg /kg meloxicam 6W),model group(0.75% adenine diet 6W),low-dose treatment group((after treating with 0.75% adenine diet 6 w,intragastrically administrating 1 mg/kg meloxicam for 20 d),High dose treatment group(after treating with 0.75% adenine diet 6 w,intragastrically administrating 6 mg/kg meloxicam for 20 d)).(3)At the end of each time point,rats were sacrificed,body weight was measured,Orbital venous blood was collected,and detected blood biochemical indexes Calcium(Ca),phosphorus(Pi),urea nitrogen(BUN),creatinine(Scr)level.The expression of TGF-β1 and COX-2 in serum was detected by ELISA assay.The pathological changes of kidney were observed by HE staining,Western Blot assay was used to detect proteion expression of TGF-β1 and COX-2 on Rat thoracic aorta,at the same time,Von Kossa staining was used to test the calcification of thoracic aorta.3.Clinical sample collection(1)Blood samples were collected from CKD patients in the First Affiliated Hospital of Chongqing Medical University,Nephrology department.They were divided into two groups: Non-dialysis CKD patients and dialysis CKD patients.The inclusion and exclusion criteria for this study are as follows:Inclusion criteria: reached to The diagnosis criteria of CKD KDOQI stage 3 and above : Glomerular filtration(GFR)≤ 60 ml /(min·1.73m2).Exclusion criteria:1)Exist chronic basic diseases and infectious diseases,such as hepatitis B,syphilis and so on;2)Long term use of drugs affecting bone metabolism;3)Severe anemia or hemorrhagic diseases,such as disseminated intravascular coagulation,thrombotic thrombocytopenic purpura,etc;4)Exist Severe cardiovascular disease;5)coma;6)Without informed consent.(Note: the study was carried out only after informed consent and signature of all patients were obtained,and it has been reviewed by the ethics review committee of the First Affiliated Hospital of Chongqing Medical University.)(2)In the morning,when the patients were on an empty stomach,blood samples were collected before dialysis;(3)Carefully checked the patient’s name,gender,age,bed number and hospitalization number before blood collection.(4)After blood collection,it was allowed to stand at room temperature for 2 hours and centrifuged at low temperature(4 ℃)and3500rmp for 5 minute.Serum samples were collected and stored at-80 ℃.(5)Detection of blood biochemical parameters Calcium(Ca),phosphorus(Pi),urea nitrogen(BUN),creatinine(Scr)level.(6)The expressions of TGF-β1,COX-2 and sclerostin in serum samples were detected by ELISA assay.(7)At the beginning of our study,a total of 175 patients participated in the study.After screening by exclusion criteria,96 patients were selected.Results:1.In vitro resluts(1)Phosphate up-regulated the m RNA and protein levels of TGF-β1in VSMCs,and the signal transcription activity of TGF-β1was enhanced.Meanwhile,the protein levels of COX-2,β-Catenin,p-gsk3β were up-regulated,but the protein level of GSK3 β was not changed.(2)TGF-β1 up-regulated the m RNA and protein levels of Runx2 and OPN in VSMCs,down regulated the m RNA and protein levels of α-SMA and SM22αTGF-β1 can induce calcification of VSMCs and rat aortic rings.TGF-β1 up-regulated the expression of COX-2,β-Catenin,p-GSK3βand down regulated the expression of sclerostin in VSMCs during the process of calcification.(3)NS398,a COX 2 inhibitor,can decrease the up regulation of Runx2 and OPN expression induced by TGF-β1 and increase the expression of α-SMA and SM22 reduceed by TGF-β1.NS398 can reduce the calcification of VSMCs and aortic rings induced by TGF-β1.NS398 decreased the m RNA and protein levels of β-Catenin and p-GSK3β,which induced by TGF-β1,and increased the m RNA and protein levels of sclerostin decreased by TGF-β1 in VSMCs,but the protein level of GSK3βwas not changed.(4)IP results showed that the interaction between p-Smad2/3 and p-CREB in VSMCs.CHIP assay results showed that the recruitment of p-Smad2/3 or p-CREB at the promoter region of sclerostin and β-Catenin in VSMCs.2.In vivo resluts(1)Body weight measurement results: compared with the control group,the body weight of the model group was significantly decreased,while the body weight of the treatment group was significantly improved.(2)Serum biochemical results: the levels of Ca、Pi、BUN and Scr in the model group were significantly higher than those in the control group.The levels of Ca、Pi、BUN and Scr in the prevention group were higher than those in the treatment group,but lower than those in the model group.(3)The results of ELISA: the serum levels of COX-2 and TGF-β1in the model group were higher than those in the control group.(4)The results of HE staining:the renal structure of the control group was normal,and there were no abnormal changes in glomeruli,renal tubules and interstitium;the renal pathological changes of the model group were obvious: the number of glomeruli was significantly reduced,the renal tubules were dilated,there were brown crystal material deposition in the tubules,the interstitial area was widened,the interstitial fibrous tissue proliferation and fibrosis were obvious;the renal pathological changes of the prevention group were more obvious than those of the model group In the treatment group,the renal pathology was partly improved.(5)Results of rat thoracic aorta tissue detection: WB results showed that compared with the control group,the protein levels of COX-2 and TGF-β1 in the model group were significantly higher than those in the control group;silver nitrate staining results showed that the vascular calcification was obvious in the model group,the improvement of vascular calcification was not obvious in the prevention group,but was obvious in the treatment group.3.Clinical samples results(1)Results of serum biochemical analysis: BUN and Scr levels of all patients in this study reached CKD KDOQI stagⅤ,The serum phosphorus level increased significantly,while the level of serum calcium decreased。(2)ELISA results: the serum levels of COX2 and TGF-β1 in hemodialysis patients were significantly lower than those in non dialysis patients,and the serum levels of sclerostin in non dialysis patients were significantly lower than those in hemodialysis CKD patients.Conclusions:1.In the process of vascular calcification induced by high phosphorus,it promotes the expression of TGF-β1 and activates TGF-β1 signal in VSMCs.2.TGF-β1 can induce osteogenic differentiation of VSMCs and arterial ring Calcification,and also promote high phosphorus-induced Calcification in VSMCs.3.High phosphorus and TGF-β1 activate Wnt/β-catenin signaling pathway in the process of promoting VSMCs calcification.4.The expression of COX2 was up-regulated in the process of promoting calcification of VSMCs by high phosphorus and TGF-β1,and inhibition of COX2 could significantly inhibit the calcification of VSMCs induced by high phosphorus and TGF-β1.5.COX 2 was involved in regulating the activation of Wnt/β-catenin signaling pathway by high phosphorus and TGF-β1.Inhibition of COX 2significantly attenuates the activation of Wnt/β-catenin signaling pathway by high phosphorus and TGF-β1.6.In vivo rat renal failure model,meloxicam,a COX 2 inhibitor,can significantly reduce vascular calcification and partly improve renal injury. |
PDF Full Text Request