Study On The Mechanism Of Zinc Inhibiting Vascular Calcification By Regulating Autophagy Through ERK1/2 Pathway

Objective:The widespresd prevalence of chronic kidney disease(CKD)causes a significant health and economic burden.CKD–mineral bone disorders(CKD-MBD)is a broad and complex disorder caused by CKD,leading to vascular media calcification and abnormal bone metabolism,which play an important role in all-cause death and cardiovascular death among CKD patients.The phenotypic transition of vascular smooth muscle cells(VSMCs)in high phosphorus environment is an important pathophysiological basis for media calcification.Autophagy plays an important role in maintaining the stability of the internal environment and responding to external stimuli of VSMCs.In addition,autophagy can alleviate the calcification of VSMCs induced by high phosphorus.Zinc is a common trace element in the human body.It is essential for the structure and function of many macromolecules and enzymes,and is the basis for a variety of cell life activities.Zinc transporter,zinc receptor and zinc binding protein work together to maintain the intracellular zinc homeostasis.A large number of animal models and clinical studies have shown that abnormal zinc homeostasis can promote inflammation,oxidative stress,and interfere with apoptosis.Besides this,zinc is also involved in the growth and maintenance of healthy bones.Normal blood zinc levels and dietary zinc intake play an important role in preventing osteoporosis.Many in vitro experiments have shown that zinc can positively regulate autophagy and exert a protective effect in many types of cells.Among them,the extracellular regulatory kinase(ERK1/2)signaling pathway has been confirmed to be closely related to zinc-regulated autophagy.There is no animal study on whether zinc supplementation can relieve CKD associated vascular calcification and bone metabolism disorders.Moreover,the regulatory effects and mechanisms of zinc and zinc transporters on vascular calcification and autophagy have not been reported.The purpose of this study is to 1)Explore the effects of zinc supplementation on mineral metabolism,vascular calcification,bone metabolism,and autophagy levels in CKD rats through in vivo experiments;2)Explore the mechanism by which zinc affects autophagy and inhibits calcification by constructing VSMCs calcification model induced by high phosphorus in vitro;3)Explore changes in the levels of zinc transporters in different calcification stages of VSMCs and the effects of zinc transporters on calcification and autophagy.Methods:1.Study on the effect of zinc on CKD-MBD and autophagy in CKD rats:The CKD model was constructed by feeding Wistar rats with 0.25%adenine/low vitamin K.The rats were randomly divided into 6 groups:CTR(control group);CTR+ZH(zinc control group,60mg/kg Zn Cl₂);CKD(calcification group);CKD+ZL(calcification+low-dose zinc group,5mg/kg Zn Cl₂);CKD+ZM(calcification+medium-dose zinc group,30mg/kg Zn Cl₂);CKD+ZH(calcification+high-dose zinc group,60mg/kg Zn Cl₂).Zn Cl₂ was intragastrically administered according to the body weight every other day.Collect the urine and blood sample of the rats at baseline and every 2 weeks thereafter,and analyze the body weight,serum creatinine(SCr),serum calcium(SCa),serum phosphorus(SP),serum zinc(SZn),serum alkaline phosphatase(ALP),serum parathyroid hormone(PTH),urinary phosphorus(UP),and urinary calcium(UCa).Three months later,the rats were sacrificed.The aorta was collected for Alizarin Red staining and calcium quantification to detect vascular tissue calcification.Immunohistochemistry and Western Blot were performed to detect the expression levels of vascular smooth muscle marker?-SMA,osteogenic protein RUNX2,and autophagy-related proteins Beclin1 and LC3II/I;The bilateral femurs were collected for Micro-CT,Goldner staining,and TRAP staining to analyze the morphological parameters of bone.2.Study of ERK1/2 signal pathway in zinc regulating autophagy and calcification of VSMCs induced by high phosphorus:the CCK-8 was used to detect the effects of different zinc concentrations(10?80u M Zn Cl₂)on the viability of human aortic smooth muscle cells(h ASMCs).The?-glycerophosphate(?-GP)was used to construct a h ASMCs calcification model in vitro.Alizarin Red staining and Western Blot were used to explore the effects of different zinc concentrations on h ASMCs calcification and osteogenic transdifferentiation within a suitable range.The optimal zinc intervention concentration was selected and the expression of LC3 was detected by immunofluorescence,the expression of LC3II/LC3I,Beclin1 was detected by Western Blot,and the number of autophagosomes in VSMCs was observed by transmission electron microscope.Autophagy inhibitor 3-methyladenine(3-MA)was used to detect whether zinc can alleviate h ASMCs calcification by activating autophagy.ERK1/2pathway inhibitor(U0126)was used to detect whether zinc can promote autophagy and alleviate the calcification of h ASMCs induced by?-GP by activating the ERK1/2signaling pathway.3.Study of zinc transporter on?-GP induced calcification and autophagy of VSMCs:The q RT-PCR technique was used to detect the expression of zinc transporters(ZIP1?14)at different calcification stages(2d,7d,14d)of h ASMCs by zinc supplementation.The h ASMCs were transfected by liposome transfection.Alizarin Red staining,calcium quantitative analysis,Western Blot,and immunofluorescence were used to detect the effects of zinc and ZIP13 on?-GP induced calcification and autophagy of h ASMCs.Results:1.Effects of zinc on body weight,serum biochemistry and urine biochemistry in CKD rats:Compared with the CTR and CTR+ZH groups,the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups showed decreased body weight,decreased levels of SCa and UP,while increased levels of SCr,SP,ALP,PTH,and UCa;Compared with the CKD group,the levels of SCa,ALP,and UP in CKD+ZL,CKD+ZM,and CKD+ZH groups increased,while the levels of SCr,SP,PTH,UCa decreased.There was no significant difference in SZn levels among the groups.2.Effect of zinc on aortic calcification in CKD rats:Compared with the CTR and CTR+ZH groups,the aortic wall of the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups showed different degrees of orange-red calcium deposits;Compared with the CKD group,the CKD+ZL,CKD+ZM,and CKD+ZH groups showed decreased calcium deposition with a dose-dependent manner.Aortic calcium quantification,immunohistochemical staining and Western Blot showed that compared with the CTR and CTR+ZH groups,the aortic wall of the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups showed increased calcium deposition,decreased?-SMA expression,and increased RUNX2 expression.Compared with the CKD group,the CKD+ZL,CKD+ZM,and CKD+ZH group showed decreased calcium deposition,increased expression of?-SMA,and decreased expression of RUNX2 with a dose-dependent manner.3.The effect of zinc on aortic autophagy in CKD rats:The immunohistochemical staining and Western Blot showed that compared with the CTR and CTR+ZH groups,the expression of LC3II/LC3I and Beclin1 in aorta of the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups were increased;compared with the CKD group,CKD+ZL,CKD+ZM,and CKD+ZH group showed increased expression of LC3II/LC3I and Beclin1 with a dose-dependent manner.4.Effect of zinc on bone metabolism in CKD rats:Bone Micro-CT showed that compared with the CTR and CTR+ZH groups,the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups showed decreased bone volume ratio(BV/TV),bone area ratio(BS/TV),number of trabecular bone(Tb.N),trabecular bone density(Tb.BMD),total cortical bone area(Tt.Ar),cortical bone area(Ct.Ar),cortical bone area ratio(Ct.Ar/Tt.Ar)),and cortical bone thickness(Ct.Th).There was no significant difference in trabecular bone separation(Tb.Sp),cortical bone porosity(Ct.Po),bone trabecular thickness(Tb.Th),and bone cortical density(Ct.BMD).Compared with CKD group,CKD+ZL,CKD+ZM,and CKD+ZH groups showed increased BV/TV,BS/TV,Tb.N,Tb.BMD,Tt.Ar,Ct.Ar,and Ct.Ar/Tt.Ar and decreased Tb.Sp.In addition,Tb.Th,Ct.Th,Ct.BMD showed no significant difference.Goldner staining and TRAP staining of femur showed that compared with the CTR and CTR+ZH groups,the CKD,CKD+ZL,CKD+ZM,and CKD+ZH groups showed increased osteoid area/bone area(OS/BS)),osteoid width(Ow),and osteoclast area/bone area(OC.S/BS),and decreased mineralization volume(MLV).Compared with CKD group,CKD+ZL,CKD+ZM,and CKD+ZH groups showed increased MLV,OS/BS and Ow,and decreased OC.S/BS.5.Effect of zinc on?-GP induced calcification of h ASMCs:CCK-8 showed that10-40u M zinc has no effect on the viability of h ASMCs.Within this concentration range,zinc can reduce the calcium nodules,calcium content,and RUNX2 expression of h ASMCs in high-phosphorus environment,and increase the expression of?-SMA.In addition,30u M zinc showed the best inhibitory effect on calcification.6.Effect of zinc on?-GP induced autophagy and calcifaication of h ASMCs:30u M zinc can further promote the expression of autophagy protein of VSMCs in high phosphorus environment.After treatment with 3-MA,h ASMCs in in high-phosphorus environment showed decreased expression of LC3II/I and Beclin1,fluorescence accumulation of LC3in the cytoplasm,and number of autophagosomes.At the same time,zinc cannot reduce the calcium nodules and calcium content of h ASMCs treated with?-GP,with increased expression of RUNX2 and decreased expression of?-SMA.7.The effect of ERK1/2 signaling pathway in the regulation of autophagy by zinc in?-GP induced calcification of h ASMCs:30u M zinc can further promote the expression of phosphorylated ERK1/2 protein in VSMCs treated with?-GP.After treatment with U0126,zinc cannot reduce the calcium nodules and calcium content of VSMCs in high-phosphorus environment.The expression of phosphorylated ERK1/2,?-SMA,LC3II/I,and Beclin1 decreased,while the expression of RUNX2 increased.In addition,the fluorescence accumulation of LC3 in the cytoplasm decreased,and the number of autophagosomes decreased.8.The effect of zinc transporter on?-GP induced calcification and autophagy of h ASMCs:High phosphorus environment reduced the expression of ZIP13 in h ASMCs,while 30u M zinc can promote the expression of ZIP13 in h ASMCs under high phosphorus environment.The ZIP13-si RNA group showed decreased calcium nodules,calcium content,and RUNX2 expression,and increased?-SMA expression in h ASMCs treated with?-GP.In addition,ZIP13-si RNA group showed decreased protein expression of LC3II/I,Beclin1,and fluorescence accumulation of LC3 in the cytoplasm.Conclusions:1.Zinc can improve CKD-MBD.While inhibiting vascular calcification,it can also improve bone metabolism disorders.2.Zinc can activate autophagy and inhibit vascular calcification by activating autophagy.3.ERK1/2 signaling pathway is involved in the regulation of autophagy of h ASMCs by zinc in?-GP induced calcification of h ASMCs.Zinc can enhance autophagy and inhibit calcification by activating the ERK1/2 signaling pathway.4.ZIP13 gene silencing could inhibit calcification,but reduced autophagy induced by?-GP and zinc in h ASMCs.