| Objective: The incidence and mortality of chronic kidney disease(CKD)is increasing year by year worldwide,and has brought a great economic and health burden.CKD vascular calcification can be actively regulated by a variety of cells,and the key event is the phenotypic transformation of vascular smooth muscle cells VSMCs to osteoblast/chondroid cells in a procalcified environment.Clock genes are a group of genes that regulate the diurnal fluctuation effect of biological functions.The BMAL1(brain and muscle ARNT-like 1)gene is a core member of the circadian feedback pathway,widely expressed in the human body,and involved in various cell biological processes.The expression of the BMAL1 gene in the human body is usually stable,but abnormal expression can lead to many diseases.Recent studies have shown that BMAL1 can further promote the phenotypic transformation of vascular smooth muscle cells by regulating calcium and phosphorus during the development of vascular calcification,thus exacerbating the progression of the disease.At present,there is no animal study on CKD on whether BMAL1 can inhibit vascular calcification,so this study by: 1)exploring the changes of mouse clock genes BMAL1 and NRF2/HO-1signaling pathways in the context of CKD through in vivo experiments,and exploring the possible mechanisms of the two in the process of vascular calcification;2)The changes of BMAL1 and NRF2/HO-1 signaling pathways were further observed by the construction of in vitro calcification models of VSMCs induced by high phosphorus,and the mechanism of action of the two in the calcification of VSMCs was explored.Research methods: 1.Study on the expression level of BMAL1,NRF2/HO-1 signaling pathway proteins in mice with chronic kidney disease under the background of CKDMBD: C57BL/6,0.25% adenine/low vitamin K feeding 12 W was used to construct a CKD mouse model.Mice are randomly divided into 2 groups: CTR(control group);CKD(Calcification Group);After 12 weeks,mice were collected and their body weight,SCr,BUN,SCa,SP,ALP and other blood biochemical indexes were analyzed.After the sacrifice of mice,the aorta was collected,alizarin red staining and calcium quantification experiments were performed to detect vascular tissue calcification,immunohistochemistry was performed to detect the expression levels of vascular tissue smooth muscle markers α-SMA and osteoblastin RUNX 2(Runt-related transcription factor 2)(Runt-related transcription factor 2)(Runt-related transcription factor 2),Western Blot was performed to detect vascular tissue osteoblastin MSX2(MSH Homeo Box 2),RUNX2,BMAL1,NRF2/ The expression level of the HO-1 signaling pathway protein.2.The mechanism of action of BMAL1 and NRF2/HO-1 signaling pathway changes in the in vitro calcification model of VSMCs in vascular smooth muscle cells: routine subculturation of rat vascular smooth muscle cells to detect the circadian expression rhythm of rat clock rhythm gene BMAL1,study the changes of biological clock protein BMAL1 and NRF2/HO-1 signaling pathway-related proteins induced by high phosphorus,and divide this part of the experiment into the following two groups:(1)control group(CTR);(2)High phosphorus-induced calcification group(HP).To investigate the role of BMAL1 in vascular calcification induced by high phosphorus,and the effect of BMAL1 alteration on VSMCs calcification and NRF2/HO-1 signaling.The effect of pathway-associated protein alteration,using inorganic phosphorus to construct the A7R5 calcification model,further divided the study into the following four groups: overexpression studies:(1)control group(CTR);(2)High phosphorus control group(HP);(3)high-phosphorus BMAL1 overexpression control group(Ad-NC+HP);(4)High phosphorus BMAL1 overexpression group(AdBMAL1+HP).Interference studies:(1)control group(CTR);(2)High phosphorus control group(HP);(3)High-phosphorus BMAL1 knockdown control group(SiNC+HP);(4)High-phosphorus BMAL1 knockdown group(Si-BMAL1+HP).Alizarin red staining was performed to observe the calcium salt deposition in cells;The calcium content kit was used to determine the intracellular calcium content of each group;PCR and Western blot detect gene and protein expression levels in RUNX2,MSX2,BMAL1,NRF2,and HO-1.Results: 1.Compared with the control group,the blood vessels of CKD mice showed obvious vascular calcification,the calcification level increased,and the expression level of clock gene BMAL1 in the vascular tissues was significantly down-regulated,along with the expression of NRF2/HO-1 signaling pathway protein.2.VSMCs alizarin red staining and calcium quantification showed that calcification was successfully induced in the high-phosphorus group.Western blot analysis showed that the expressions of osteoblast MSX-2 and RUNX-2 in the high-phosphorus group were significantly increased compared with those in the control group,which further indicated that the high-phosphorus group successfully induced cell calcification.3.The expressions of BMAL1,NRF2 and HO-1 genes and proteins in VSMCs in the high-phosphorus group were significantly lower than those in the control group.4.Overexpression of BMAL1 can increase the expression levels of NRF2 and HO-1 protein,decrease the expression levels of osteoblast proteins MSX-2 and RUNX-2,and improve calcification in the high-phosphorus BMAL1 overexpression group.BMAL1 knockdown results in the opposite effect.Conclusion: 1.In the context of CKD,vascular calcification may be related to the decreased expression level of the clock gene BMAL1 and the damaged NRF2/HO-1signaling pathway in vascular tissue,and its exact mechanism needs to be further studied.2.The decreased expression of BMAL1 and the damaged NRF2/HO-1signaling pathway in the cells under the condition of high phosphorus are the important mechanisms of vascular smooth muscle cell calcification.3.BMAL1 can positively regulate NRF2/HO-1 signaling pathway,which can improve the expression level of NRF2/HO-1 signaling pathway by promoting the expression of BMAL1 and reduce the calcification of vascular smooth muscle cells induced by high phosphorus. |
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