| Background and purposes: Vascular calcification(VC)is an active,highly regulated complex pathophysiological process similar to bone development and is prevalent in patients with coronary atherosclerosis,hypertension,diabetic vascular diseases,vascular injury,and chronic kidney diseases.And it is also an important risk factor for increased cardiovascular morbidity and mortality.Therefore,it is of great scientific and social value to actively conduct researches about the improvement of VC.The Klotho(KL)gene was discovered in 1997 and is extensively studied as an anti-aging gene.KL knockout mice exhibit a series of phenotypes similar to those in patients with premature aging syndrome,including VC.The in-vivo experiments confirm that the use of gene integration technology or exogenous supplementation of recombinant proteins to restore Klotho expression in Klotho-deficient mice can significantly ameliorate VC.And the in-vitro studies have shown that Klotho protein can significantly reduce high phosphorus-induced Osteogenic differentiation of mouse aorta smooth muscle cells(an important key point for VC).The anti-calcification mechanisms of Klotho may be partly attributed to two aspects.Firstly,the normal expression of Klotho in vivo can maintain the calcium-phosphorus balance of extracellular fluid.Secondly,it can inhibit intracellular insulin/IGF-1 and Wnt signaling pathways,and these factors are important pathogenic factors of VC.But whether there are other mechanisms involved remains unclear.Gap junction(GJ)is an important member of direct communication between cells.It is composed of connexin(Cx),which mediates the exchange of energy and substances between adjacent cells.In the Cx family,Cx43 is the most predominant Cx expressed in blood vessels,and studies have reported that osteogenic differentiation of vascular smooth muscles cells(VSMCs)is directly affected by GJ-mediated intercellular communication.Therefore,Cx43 can participate in the occurrence of VC by mediating osteogenic differentiation of VSMCs.Autophagy is a highly conserved process of degradation of intracellular proteins and organelles when stimulated,and is involved in the development of many diseases.Studies have confirmed that autophagy may affect VC,and autophagy can degrade Cx43.However,in the VC model,the relation between Klotho and autophagy is still unknown.Therefore,this study begins with in-vivo experiments to explore the changes in autophagy and VC in Klotho-deficient mice,and identify the effect of autophagy on VC by regulating autophagy.Then in-vitro experiments,experiments are employed to verify whether Klotho can inhibit VC through the inhibition of autophagy.Finally,experiments are utlilzed to explore whether Klotho can inhibit VC via autophagy-mediated Cx43 degradation.Methods: 2.1 The in-vivo VC detection Klotho heterozygous(HZ)mice were crossed to obtain young mice.After genotype identification of young mice,Klotho wild type(WT)and Klotho knockout(KO)mice were obtained.The 5-week-old male mice were used for experiments.The aorta of KL-deficient mice was obtained,and VC staining was detected by von kossa staining method.Calcium content in mouse aorta was detected by o-cresol oxime complex ketone(OCPC)method.The expression of Runx2 in mouse aorta was detected by immunohistochemistry assay.The q PCR assays were used to detect the m RNA expression of VC-related molecules-Pit1,Pit2,Runx2 and SM22.Western blot assay was used to detect the protein expression of Runx2 and α-SMA.2.2 The role of autophagy in VC caused by Klotho gene deletion 2.2.1 Detection of autophagy in the absence of Klotho The aorta samples of Klotho-deficient mice were obtained,total protein was extracted,and the expression of LC3-Ⅱ/Ⅰ and P62 was detected by western blot assays.After frozen sections of the aorta were performed,the difference of LC3 dots and P62 fluorescence in mice aorta between different groups were observed and counted through immunofluorescence assays.2.2.2 Observation of VC after regulation of autophagy WT and KO mice were intraperitoneally injected with appropriate amount of rapamycin(RA)and chloroquine(CQ).Two weeks later,these mice were sacrificed and the aorta samples were obtained.The protein expression of LC3-Ⅱ/Ⅰ,P62,Runx2 and α-SMA were detected by western blot assay.2.3 The identification of mouse aorta vascular smooth muscle(MOVAS)cells Cellular immunofluorescence assay was employed to clearly observe the expression of Alexa-488 labeled α-SMA in MOVAS cells.2.4 Calcification induction and identification of MOVAS cells MOVAS cells were inoculated into 6-well plates.The experimental group was treated with calcification medium(CM)for 8-12 days,and the control group was treated with common culture solution,and solution was changed every 2 days.Calcium deposition was observed by alizarin red S staining assay,and the calcium content in MOVAS cells was detected by OCPC method 2.5 The effect of Klotho recombinant protein on autophagy and VC in vitro 2.5.1 The effect of Klotho recombinant protein on autophagy MOVAS cells were treated with Klotho recombinant protein with or without CQ(autophagy inhibitor).The protein expression of LC3-Ⅱ/Ⅰ and P62 were detected by western blot assays.After the double fluorescent LC3 adenovirus was transfected into MOVAS cells,the red and yellow dots between different groups were detected to indicate autophagy flux.2.5.2 The mechanism of Klotho recombinant protein on VC MOVAS cells were treated with Klotho recombinant protein with or without CQ(autophagy inhibitor).The expression of Runx2 and α-SMA protein was gauginged by western blot assays.Calcium deposition was observed by alizarin red S staining.The calcium content of MOVAS cells were detected by OCPC method.2.6 The effect of Klotho on Cx43 2.6.1 The change of Cx43 expression in Klotho-deficient mice The expression of Cx43 protein in aorta of Klotho-deficient mice was detected by western blot assay.2.6.2 The effect of KL on Cx43 through the regulation of autophagy in Klotho-deficient mice Western blot assay was conducted to detect the expression of Cx43 protein after exogenous intraperitoneal administration of KL recombinant protein with/without CQ.2.7 The effect of Klotho recombinant protein on Cx43 through the regulation of autophagy in vitro In vitro experiments,on the basis of the presence of Klotho recombinant protein,western blot assay was used to detect Runx2 and α-SMA protein expression,alizarin red S staining was used to observe calcium deposition,and OCPC method was employed to detect the calcium content of MOVAS cells after Cx43 si RNA was used to interfere Cx43 expression.Results: 3.1 Klotho-deficient mice phenotype and genotype identification Morphologically,KO mice were significantly smaller than WT and HZ mice,which exhibited hunchback and poor skin gloss.Then we extracted rat tail DNA,ran electrophoresis and developed after PCR experiment.WT mice had only wild alleles(458 bp),KO mice had only mutant alleles(920 bp),and HZ mice had both alleles.3.2 The aorta of Klotho-deficient mice showed obvious VC The results of the Von kossa experiment showed that Compared with WT mice,the calcified area began to appear in the aorta of HZ mice,and more and more obvious calcification areas were observed in the aorta of KO mice.The OCPC method was used to detect the calcium content of aorta.The results showed that the calcium content gradually increased with the gradually deficiency of the Klotho gene(WT vs.HZ vs.KO;p < 0.05,n=6).The relative expression of Runx2 in the aorta was detected by immunohistochemistry assay.The results showed that the relative expression of Runx2 in HZ group and KO group was higher than that in WT group(p < 0.05,n=6),and the relative expression of Runx2 in KO group was higher than that in HZ group(p < 0.05,n=6).The q PCR assays were used to detect the m RNA expression of VC-related factor.The results showed that the relative expression of Pit1,Pit2 and Runx2 m RNA increased after Klotho gene was deficient(WT vs.HZ vs.KO;p < 0.05,n=6),but the relative expression of SM22 m RNA decreased(WT vs.HZ vs.KO;p < 0.05,n=6).The protein expression of VC-related factor was detected by Western blot assays.The results showed that the relative expression of Runx2 protein increased after Klotho gene was deficient(WT vs.HZ vs.KO;p < 0.05,n=6),but the relative expression of α-SMA protein reduced(WT vs.KO;p < 0.05,n=6).3.3 Autophagy increased after the Klotho gene was knocked out Western blot analysis was used to detect the autophagy in mouse aorta after Klotho gene was deficient.The results showed that the relative expression of LC3-II/I ratio increased with the deletion of Klotho gene(WT vs.HZ vs.KO;p < 0.05,n=6),while the relative expression of P62 protein decreased(WT vs.HZ vs.KO;p < 0.05,n=6).The expression of LC3 and P62 were detected by immunofluorescence assays.The results showed that when Klotho gene was deficient,the relative amount of LC3 puncta/cell increased(WT vs.HZ vs.KO;p < 0.05,n=6),while the relative fluorescence intensity of P62 gradually decreased(WT vs.HZ vs.KO;p < 0.05,n=6).3.4 Detection of VC after the regulation of autophagy in Klotho-deficient mice Western blot analysis was employed to detect VC after RA was used to promote autophagy,and the results showed that after exogenous administration of RA,the relative ratio of LC3-II/I increased(KO vs.KO+RA;p < 0.05,n=6)but the relative expression of P62 protein decreased(KO vs.KO+RA;p < 0.05,n=6),and the relative protein expression of VC-related transcription factor Runx2 decreased(KO vs.KO+RA;p < 0.05,n=6),while the relative expression of the skeleton protein α-SMA increased(KO vs.KO+RA;p < 0.05,n=6).Western blot analysis was used to detect VC after CQ was used to inhibit autophagy,and the results showed that after exogenous administration of CQ,the relative ratio of LC3-II/I and the relative expression of P62 protein both increased(KO vs.KO+CQ;p < 0.05,n=6),and the relative protein expression of the VC-related transcription factor Runx2 increased(KO vs.KO+CQ;p < 0.05,n=6),while the relative expression of α-SMA reduced(KO vs.KO+CQ;p < 0.05,n=6).The aortic calcium content was detected by OCPC method.The result showed that after the administration of RA to promote autophagy,the aortic calcium content decreased(KO vs.KO+RA;p < 0.05,n=6).And after the administration of CQ to inhibit autophagy,the aortic calcium content increased(KO vs.KO+RA;p < 0.05,n=6).3.5 Detection of calcification and autophagy after the administration of Klotho recombinant protein in vitro Firstly,the obtained cells were identified as MOVAS cell lines by cell immunofluorescence assay,and the calcification induction was successfully confirmed by alizarin red S staining and calcium content detection experiments.Western blot analysis was used to detect autophagy after treatment with Klotho recombinant protein.The results showed that the relative ratio of LC3-II/I increased after calcification induction(WT vs.CM;p < 0.05,n=3),but the relative protein expression of P62 reduced(WT vs.CM;p < 0.05,n=3).After successful calcification induction,cells were treated with Klotho recombinant protein.The results showed that the relative ratio of LC3-II/I continued to increase(CM vs.CM+KL;p < 0.05,n=3),while the relative protein expression of P62 continued to decrease(CM vs.CM+KL;p < 0.05,n=3).After the employment of Klotho recombinant protein and CQ,the relative ratio of LC3-II/I further increased(CM+KL vs.CM+KL+CQ;p < 0.05,n=3),while the relative protein expression of P62 increased(CM+KL vs.WT+KL+CQ;p < 0.05,n=3).Autophagy flux was detected using a dual-fluorescence LC3 adenovirus transfection assay.The results showed that after calcification induction,the number of autophagosomes(Control vs.CM;p < 0.01,n=3)and autolysosomes(Control vs.CM;p < 0.01,n=3)increased,which indicated the up-regulation of autophagy.After calcification induction,the employment of KL recombinant protein could augment the number of autophagosomes(CM vs.CM+KL;p < 0.01,n=3)and autolysosomes(CM vs.CM+KL;p < 0.01,n=3),indicating that autophagy continues to increase.After calcification induction,the simultaneously empoloyment of Klotho recombinant protein and CQ augmented the number of autophagesomes(CM+KL vs.CM+KL+CQ;p < 0.01,n=3),while the number of autolysosomes decreased(CM+KL vs.CM+KL+CQ;p < 0.01,n=3),indicating that the binding of autophagosomes and autolysosomes was blocked,and autophagy was inhibited.3.6 Klotho could inhibit calcification through promoting autophagy in vitro The effect of Klotho on the MOVAS cell calcification was detected by Western blot assay.The results showed that the relative expression of Runx2 protein increased after calcification induction(Control vs.CM;p < 0.05,n=3),while the relative expression of α-SMA protein decreased(Control vs.in CM;p < 0.05,n=3).After treatment with Klotho recombinant protein,the relative expression of Runx2 protein decreased(CM vs.CM+KL;p < 0.05,n=3),while the relative expression of α-SMA protein restored(in CM vs.CM+KL;p < 0.05,n=3),indicating the relief of calcification.However,when inhibition of autophagy with CQ based on Klotho recombinant protein,the relative expression of Runx2 protein increased again(CM+KL vs.in CM+KL+CQ;p < 0.05,n=3),and the relative expression of α-SMA protein decreased again(CM+KL vs.in CM+KL+CQ;p < 0.05,n=3),indicating that the calcification deteriorated again.The effect of Klotho on the MOVAS cell calcification could be detected through alizarin red S staining and calcium content detection assays.In terms of morphology,the Klotho group exhibited less calcium deposition compared with CM group,and after additionally use of CQ to inhibit autophagy,calcium deposition increased again compared with KL group.And result of the calcium content assay showed that after treatment with Klotho recombinant protein,the intracellular calcium content of MOVAS cells decreased(CM vs.CM+KL;p < 0.05,n=3),and after autophagy was inhibited with CQ,the calcium content increased again(CM+KL vs.CM+KL+CQ;p < 0.05,n=3).3.7 The effect of Klotho on Cx43 through autophagy The expression of Cx43 in the aorta of Klotho-deficient mice was detected by Western blot analysis.The result showed that the relative expression of Cx43 protein in aorta of KO mice was higher than that in aorta of WT mice(p < 0.01,n=6),and the relative expression of Cx43 protein in aorta of KO mice was also higher than that in aorta of HZ mice(p < 0.01,n=6).The effect of Klotho recombinant protein on the expression of Cx43 protein was detected by Western blot assay.The result showed that the relative expression of Cx43 protein in aorta increased after Klotho gene was knocked out(WT vs.in KO;p < 0.01,n=6),and the relative expression of aortic Cx43 protein decreased after exogenously supplementation of Klotho recombinant protein(KO vs.KO+KL;p < 0.05,n=6).After additional administration of CQ,the relative expression of aortic Cx43 protein increased(KO+KL vs.KO+KL+CQ;p < 0.05,n=6).3.8 Effect of Klotho on autophagy-mediated Cx43 degradation on calcification Western blot assays were employed to detect the effect of Klotho on MOVAS cell calcification induced by autophagy-mediated Cx43 degradation.The results showed that after calcification induction,the relative expression of Runx2 protein increased(Control vs.CM;p < 0.01,n=3)and the relative expression of α-SMA protein decreased(Control vs.CM;p < 0.01,n=3).After treatment with Klotho recombinant protein,the relative expression of Runx2 protein decreased(CM vs.CM+KL;p < 0.01,n=3),and the relative expression of α-SMA protein restored(CM vs.CM+KL);p < 0.05,n=3).Based on the Klotho recombinant protein,Cx43 si RNA was used to interfere the expression of Cx43,and the relative expression of Runx2 protein further decreased(CM+KL vs.CM+KL+Si Cx43;p < 0.05,n=3),while the relative expression of α-SMA protein further restored(CM+KL vs.CM+KL+Si Cx43;p < 0.05,n=3)Alizarin red S staining and calcium content detection assays were used to detect the effect of Klotho induced autophagy-mediated Cx43 degradation on MOVAS cell calcification.The result of alizarin red S staining assay showed that calcium deposition increased after calcification induction.Treating with Klotho recombinant protein inhibited intracellular calcium deposition.And simultaneous treatment with Cx43 si RNA and Klotho recombinant protein contributed to further decrease of calcium deposition In the calcium content detection experiment,calcium content in MOVAS cells significantly increased after calcification induction(Cotrol vs.CM;p < 0.01,n=3),and after the administration of Klotho recombinant protein,intracellular calcium content decreased(CM vs.CM+KL;p < 0.01,n=3).After the Cx43 si RNA was employed on basis of Klotho recombinant protein,the intracellular calcium content further decreased(CM+KL vs.CM+KL+Si Cx43;p < 0.01,n=3).Conclusions: 4.1 Apparent VC can be detected in five-week-old Klotho-deficient mice.4.2 Autophagy increases in the aorta of Klotho-deficient mice,and this up-regulation is a protective change.4.3 Klotho can inhibit the progression of VC by promoting autophagy.4.4 Klotho can inhibit the progression of VC by promoting autophagy-mediated Cx43 degradation. |
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