| BackgroundCardiovascular disease(CVD)and chronic kidney disease(CKD)are the main causes of disability and death worldwide.Vascular calcification(VC)refers to the abnormal deposition of calcium salt in the vascular wall,which is one of the cardiovascular complications of CKD patients.The incidence and severity of VC are increasing year by year,leading to the high mortality in CKD patients.Currently,there have been few effective treatments for VC.Hemodialysis cannot slow down the process of CKD-VC,and the effect of renal transplantation on VC remains controversial.Therefore,exploring the pathogenesis of VC and the pharmacological interventions,finding the effective molecular targets for VC diagnosis and prevention,are the key to improve the public health,which requires our research work urgently.The mechanisms of VC are complex.Many mechanisms and signaling pathways,such as autophagy and apoptosis,Wnt/?-catenin signaling pathway activation,endoplasmic reticulum stress,are involved in the regulation of VC.Recent studies have shown that VC is not only a passive deposition of calcium and phosphorus,but also a complex pathological process involving multiple mechanisms,which is similar to the process of bone formation.Among them,phenotype transformation of vascular smooth muscle cells(VSMC)plays an important role.In the process of VC,the expression of VSMC original contractile phenotypic markers,such as a-SMA,calponin,SM22a,is decreased,while the expression of osteogenic related transcription factors,such as Runx2,Msx2,osterix,is increased,thus promoting the downstream expression of a series of bone related proteins,such as bone morphogenetic protein-2(BMP2),osteocalcin,sclerostin,etc.This active differentiation of VSMCs into osteoblast like cells mediates the deposition of bone matrix in blood vessels.Therefore,inhibiting the phenotype transformation of VSMCs in the process of VC is the important step to prevent and treat VC.C1q/TNF related protein 9(CTRP9)is a new member of CTRPs family.CTRP9 is homologous and structurally similar to adiponectin.It was initially found in adipose tissue,and later proved to express in various cells including VSMCs.Recent studies have shown that CTRP9 is mainly derived from the heart and is involved in the regulation of multiple CVDs.CTRP9 inhibits the proliferation and migration of VSMCs,vascular inflammation and dilates blood vessels by regulating endothelial cells.In addition,clinical studies have shown that CTRP9 may be a marker of diabetic renal vascular injury,and the level of plasma CTRP9 is positively related to the degree of vascular stiffness in patients with type 2 diabetes.Recently,serum CTRP9 was reported to be negatively associated with ACAI(Aortic Calcification Area Index),indicating that CTRP9 might be involved in VC regulation.However,there is no study concerning about the mechanism of CTRP9 in VCAutophagy refers to the digestion and reuse of intracellular organelles and components by lysosomes.Autophagy is very important in maintaining cell homeostasis and stress response.Hyperphosphatemia is one of the significant clinical characteristics of CKD.Studies show that the autophagy level of VSMC increases significantly under the stimulation of high phosphorus,and in turn,the enhanced autophagy process could antagonize VSMC calcification induced by high phosphorus Autophagy is under the complicated regulation.MTOR is an upstream inhibitor of autophagy and was involved in VC regulation.At present,there is few researches on CTRP9 and autophagy.CTRP9 was reported to regulate autophagy in the early atherosclerosis plaque of apoE-/-mice.At present,there is no research on the link between CTRP9,autophagy,and VC.Ginsenosides are the main components of ginseng and Panax notoginseng,which can be divided into panaxadiols(PPD)and panaxatriols(PPT)according to chemical structure.Rbl is the main monomer of PPD,which plays a protective role in many CVDs and kidney injuries.Studies show that Wnt/?-catenin pathway is an important signaling pathway in the process of osteogenesis and VC.Activation of pathway promotes the expression of calcifying factors,upregulate the activity of alkaline phosphatase(ALP),and eventually start and promote the occurrence and development of VC;on the other hand,inhibiting Wnt/?-catenin pathway has been demonstrated to ameliorate VC.PPAR-y belongs to the nuclear receptor superfamily,and the mutual antagonistic effect of PPAR-y and Wnt/?-catenin pathway is crucial in the process of osteogenesis and VC.Ginsenosides belong to the steroidal saponin family which share similar structures with steroids.It has been reported that Rb1 can regulate downstream genes by activating various nuclear receptors,including PPAR-y.Furthermore,the crosstalk between Wnt/?-catenin pathway and mTOR/autophagy pathway play a vital role in VC.For example,atorvastatin inhibits the ?-catenin pathway,induces the autophagy,and thereby inhibiting VC.Until this study,there has been no study on the effect of ginsenoside Rbl on VC.We speculate that Rbl may inhibit the Wnt/?-catenin pathway through activating PPAR-y,thus enhancing the mTOR-dependent autophagy and protecting VC.In conclusion,to elucidate the role of CTRP9 and ginsenoside Rbl in CKD-VC and the underlying molecular mechanisms,we collected clinical samples,established the VC model of CKD rats by adenine gavage,extracted and cultured the primary VSMC of rats,established the VSMC calcification model by ?-GP induction,and studied the related scientific issues.This paper will be divided into the following two parts for research and discussion:PART ?:the effect and mechanisms of CTRP9 in VC;PART ?:the effect and mechanisms of Rbl in VC;PART ?:The Effect and Mechanisms of CTRP9 in VC1.Purpose(1)To study and clarify the expression and role of CTRP9 in VC:to clarify the role of CTRP9 in calcium deposition and VSMC osteogenic phenotype transformation in VC.(2)To study the regulatory effect of CTRP9 on autophagy and mTOR pathway:to clarify the link between CTRP9 and autophagy,mTOR,and whether CTRP9 regulates VC through the mTOR-dependent autophagy pathway.(3)To study the direct molecular interaction of CTRP9 on mTOR:to determine whether there is a direct interaction between CTRP9 and mTOR.If so,whether this interaction is mediated by direct binding between DNA,mRNA or protein levels.2.Methods(1)Clinical specimen collection:the patient(n=3)and the donor(n=3)were diagnosed as CKD G5 in Qilu Hospital of Shandong University.The vascular tissue samples of the discarded iliac/renal artery were collected.(2)CKD-VC rat model establishment:after two weeks of tail vein injection of lentivirus,the CKD-VC rat models were established by intragastric administration of adenine for 8 weeks;the control group was given saline by gavage.(3)Extraction and culture of primary rat VSMCs:the method of "sticking to the wall" was used to extract:aseptic dissection and separation of thoracic aorta segment,stripping of vascular adventitia and curettage of intima.The vascular medial tissue mass was evenly attached to the bottom wall of the culture flask,then the complete culture medium was added.After about 5-7 days,VSMC migrated from the edge of tissue.(4)VSMCs verification and VSMC calcification establishment:The passage 3 to 8 of VSMCs were used in vitro after VSMCs were confirmed by ?-SMA immunofluorescence staining.The calcification model of VSMC was established by stimulating with 10mM ?-GP for 3-12 days.(5)Heart rate(HR)and blood pressure(BP):at the end of the experiment,the HR and BP of rats were measured by non-invasive tail blood pressure meter,and the systolic blood pressure(SBP),diastolic blood pressure(DBP),pulse pressure(PP)and HR of rats were recorded,which were measured three times continuously and the average value was taken.(6)Echocardiography:at the end of the experiment,small animal echocardiography was used to measure and calculate the following parameters:ventricular septal thickness(IVS),left ventricular internal diameter(LVID),left ventricular posterior wall thickness(LVPW),left ventricular volume(LVvol),etc.Five cardiac cycles were measured continuously and the average value was taken.(7)Serum biochemical parameters:at the end of the experiment,rats were euthanized with pentobarbital and serum was collected.Serum creatinine(Cr),urea nitrogen(BUN),alkaline phosphatase(ALP),calcium(Ca),phosphorus(P)were measured.(8)Evaluation of the calcification of arteries/VSMC:alizarin red S staining,von Kossa staining were used to evaluate the calcification of the arteries and the formation of calcium nodules in VSMCs.(9)The calcium concentration of vascular tissue/VSMCs homogenate were detected by kits.(10)Detection of the phenotype transformation of VSMCs:select the contraction phenotype markers:a-SMA,calponin,SM22a;osteogenic phenotype markers:RUNX2,BMP2.Immunohistochemical staining and immunofluorescence staining were used to detect the location and expression of the markers in the vascular wall/VSMCs,and Western blot was used to detect the protein expression of the markers.(11)Detection of the expression of CTRP9:the circulating CTRP9 in serum was detected by ELISA;the location and expression of CTRP9 in arterial wall was detected by immunohistochemistry;the protein expression of CTRP9 was detected by Wester-n blot.(12)The autophagy of VSMCs was detected by transmission electron microscope(TEM):the number of autophagosomes and autolysosomes.(13)Detection of the expression of mTOR/autophagy pathway:mTOR,S6K,ATG5,p62,LC3 were detected.The localization and expression of mTOR and LC3 in the arterial wall/VSMCs,and the number of LC3 fluorescent puncta were detected by immunofluorescence staining;the mRNA and protein expression of related proteins were detected by RT-qPCR and Western blot.(14)Detection of protein-protein direct binding:double immunofluorescence staining and laser confocal microscopy observation of subcellular co-location;Co-IP.(15)Liquid Chromatography-tandem Mass Spectrometry(LC-MS/MS):Primary antibody was used to immunoprecipitated target protein,followed by electrophoresis.When protein is about to separate during electrophoresis,Coomassie brilliant blue staining was performed.The positive gel bands were taken,enzymolysed and desalted,then identified by LC-MS/MS.(16)RNA immunoprecipitation(RIP):Primary antibody was used to precipitate the target protein bound RNA complex.After dissolving and removing proteins,RNA was amplified by RT-qPCR and the amplified product was detected by agarose gel electrophoresis.(17)Statistical analysis.3.Results(1)The arteries of CKD patients and rats developed significant medial VC,and the expression of CTRP9 was upregulated in the heart and arterial tissues of CKD rats;there was no significant change of the serum circulating CTRP9 level in CKD rats.(2)Overexpression of CTRP9 promoted the formation of VSMC calcium nodules,the calcium concentration of VSMCs homogenate,and the osteogenic phenotype transformation of VSMCs;gene silencing of CTRP9 inhibited the above-mentioned calcification indexes of VSMC;in vivo silencing of CTRP9 reduced the serum level of ALP,HR,SBP,PP,IVS,LVvol.LVID.etc.in CKD rats,and reduced the calcification and phenotype transformation of the arteries.(3)In the process of calcification,VSMC and vascular autophagy were increased;gene silencing CTRP9 further enhanced autophagy;3-mA inhibited autophagy and reversed the protective effect of silencing CTRP9 on VSMC phenotype transformation.(4)During the process of calcification,the expression of mTOR was upregulated;overexpression of CTRP9 was upregulated,while silencing of CTRP9 downregulated the mRNA and protein expression of mTOR and its substrate S6K;mTOR agonist(leucine)inhibited the pro-autophagic effect of CTRP9 gene silencing.(5)MTOR gene silencing or mTOR inhibitor(rapamycin)inhibited the effect of overexpression of CTRP9 on the formation of calcium nodules and the expression of RUNX2;mTOR agonist(leucine)reversed the above-mentioned calcification protective effect of CTRP9 silencing.(6)In VSMC,CTRP9 protein could not directly bind to mTOR protein.(7)In rat VSMCs,99 proteins were identified by Co-IP and LC-MS/MS.After ranking by peptide-spectrum matches(PSMs),most of the top ten proteins belong to the heterogeneous nuclear ribonucleoprotein(hnRNP)family.HnRNP A2/B1 is the 3rd of the list.(8)HnRNP A2/B1 protein can directly bind to CTRP9 protein,and hnRNP A2/B1 protein can directly bind to mTOR mRNA.(9)Overexpression of hnRNP A2/B1 reversed the inhibition effect of CTRP9 silencing on mTOR and S6K mRNA.4.Conclusion(1)The expression of CTRP9 in VSMCs was upregulated in CKD-VC;(2)CTRP9 promotes medial VC.while gene silencing of CTRP9 inhibits VC;(3)CTRP9 regulates vascular calcification through the mTOR-dependent autophagy pathway;(4)HnRNP A2/B1 directly binds to CTRP9 protein and mTOR mRNA,which mediates the mRNA regulation of CTRP9 on mTOR pathway.Part ?:The Effect and Mechanisms of Rbl in VC1.Purpose(1)To study and clarify the role of Rbl in VC:to clarify the role of Rbl in calcium deposition,ALP activity and osteogenic phenotype transformation of VSMCs.(2)To study and clarify the regulatory effect of Rb1 on PPAR-? and Wnt/?-catenin pathway:to clarify the link between Rbl and PPAR-?,Wnt/?-catenin pathway;whether Rb1 inhibits the Wnt/?-catenin pathway through PPAR-y,thereby inhibiting VC.;the upstream and downstream relationships of PPAR-y and Wnt/?-catenin pathway.(3)To explore the role of Rbl in the mTOR/autophagy pathway:to discuss whether Rb1 can regulate the mTOR/autophagy pathway through pathway cross-talking.2.Methods(1)CKD-VC rat model establishment:the animal model was established by intragastric administration of adenine for 6 weeks;the control group was given saline by gavage;Rb1(40 mg/kg/d)or saline were given by intraperitoneal injection.(2)Extraction and culture of primary VSMCs:the same as above.(3)VSMCs verification and VSMC calcification establishment:the same as above.(4)Serum biochemical parameters:the same as above.(5)Evaluation of vascular/VSMC calcification:the same as before.(6)ALP activity and calcium concentration of vascular tissue/VSMCs homogenate were detected by kits.(7)Detection of the phenotype transformation of VSMCs:selected contraction phenotype markers:?-SMA,calponin;osteogenic phenotype markers:RUNX2.Immunohistochemical staining was used to detect the location and expression in the vascular wall/cell,and Western blot was used to detect the protein expression of the markers.(8)Detection of the expression of PPAR-y and Wnt/?-catenin pathway:PPAR-?,GSK3 ?,?-catenin.Immunofluorescence staining combined with confocal microscopy was used to observe the expression and the nuclear translocation of?-catenin;cytoplasm and nucleus proteins were extracted separately and Western blot was used to detect the expression and the nuclear translocation of ?-catenin;Western blot was used to detect the expression of other related proteins.(9)Statistical analysis.3.Results(1)Intraperitoneal injection of Rbl in CKD rats reduced the serum phosphorus levels,reduced the VC significantly,ALP activity and calcium concentration of vascular homogenates;in vitro,Rb1(40mM)reduced the calcium nodules formation and calcium concentration of VSMCs.(2)Intraperitoneal injection of Rb1 in CKD rats reduced the expression of RUNX2,and increased the expression of ?-SMA and calponin in calcified arteries;In vitro,Rbl increased the expression of ?-SMA and calponin,and decreased the expression of RUNX2 during VSMC calcification in a dose dependent manner.(3)During the process of vascular calcification,Wnt/?-catenin pathway was activated.The nuclear translocation of ?-catenin was increased.PPAR-? expression was decreased;Rb1 upregulated the expression of PPAR-? and inhibited the expression of ?-catenin in vivo and in vitro;Rbl inhibited the nuclear translocation of?-catenin in VSMCs.(4)Wnt/?-catenin pathway agonist(SKL2001)reversed the protective effect of Rb1 on VSMC calcium deposition and phenotype transformation.(5)GW9662,an inhibitor of PPAR-?,reversed the inhibition effect of Rb1 on?-catenin.4.Conclusion(1)Rb1 inhibits the calcification of CKD-VC in vivo and in vitro;(2)Rbl regulates VC by activating PPAR-y and inhibiting Wnt/?-catenin pathway. |
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