| Objective To investigate the effect of GDF11 regulation on arterial calcification in C57BL-6 mice;To observe the relationship between GDF11,Smad2/3 and calcification degree in calcified aorta induced by GDF11 up-regulation and down-regulation;To observe the relationship between GDF11,Smad2/3 and calcification degree in calcified aorta induced by Smad2/3 inhibition.Methods1.The arterial calcification model of C57BL-6 mice was established: The 12-week-old C57BL-6 male mice were used in this experiment.Adaptive feed after 1 week,vitamin D3(Vit D3)·anhydrous ethanol solution was inject by intramuscular,nicotine·peanut oil suspension was lavaged,after 3,5,7,9 weeks respectively,through the Von Kossa-hematein-eosin(Von Kossa-HE)staining to observe and compare the four groups of elastic fiber fracture,extent of vascular intima and the media calcification,immunohistochemical observe calcification marker protein BMP2,RUNX2 distribution,so as to establish the mature and stable and time-dependent C57BL-6 mouse calcification gradient model;2.Exogenous mouse source r-GDF11 was given by intraperitoneal injection,and the effect of GDF11 on arterial calcification process in C57BL-6 mice was observed: Take 5weeks old of C57BL-6 mice 30,randomly divided into 5 groups,after adaptive feed 1week,saline solution,source buffer solvent,low concentration of exogenous r-GDF11,medium concentration of r-GDF11,high concentration of r-GDF11 was daily intraperitoneal injection respectively,after pretreatment of seven weeks,then induce calcification 5 weeks,eyeball blood before separation of the aorta.We observed the calcium deposition and elastic fiber breakage by Von kossa-HE staining and compared among the five groups.Western Blot was used to detect the contents of calcification marker proteins BMP2,RUNX2 and GDF11 in arterial blood vessels,and the distribution of immunohistochemical calcification marker proteins BMP2,RUNX2 and GDF11 were also compared.3.Down-regulated GDF11 expression by AAV transfection in aorta,observe the arterial calcification process in C57BL-6 mice : Take 11 weeks of C57BL-6 mice 18,they were divided into 3 groups randomly,adaptive feed after 1 week,blank group tail intravenous saline,empty virus group tail intravenous injection,experimental group tail intravenous packaged AAV,1 week after PCR method to observe the AAV transfection success,then induce aortic calcification 3 weeks.Eyeball blood the samples,isolated the aorta,and the calcium salt deposition and elastic fiber fracture of Von kossa-HE staining were observed and compared among the three groups.Western Blot was used to detect the contents of calcification marker proteins BMP2,RUNX2 and GDF11 in the arterial blood vessels,and the distribution of calcification marker proteins BMP2,RUNX2 and GDF11 were also detected by immunohistochemical.4.In the first part of the experiment,aortic proteins up-regulated and down-regulated GDF11 were melted at low temperature,and Western Blot analysis was performed to observe the expression of calcification marker proteins BMP2 and RUNX2,as well as GDF11,p-Smad2/3 and Smad2/3.5.Take 11 weeks of C57BL-6 mice,18 were divided into 3 groups randomly,the adaptability of feeding 1 week,the blank group,control group injected with normal saline,the experimental group injected Smad2/3 inhibitor,after 1 week,3 weeks to induce calcification,then eyeball blood,separation of the aorta,to observe and compare the three groups between Von kossa-HE calcium salt deposition rate of the HE staining and elastic fiber fracture,Western Blot detection calcification marker protein BMP2,RUNX2,GDF11,Smad2/3,p-Smad2/3 content in arteries.Results1.C57BL-6 mice calcification model had been established: We observed the calcification by Von kossa-HE,3-week group blood vessels are almost no calcification,5-week of the dark brown calcium salt deposits in the membrane,but the membrane elastic fiber continuity was complete,in 7-week and 9-week group dark brown calcium salt deposits increased significantly compared with the first two groups,and the membrane elastic fiber fracture had no continuity.In immunohistochemistry,calcification marker proteins BMP2,RUNX2 were gradually concentrated in the3-week group,the 5-week group the 7-week and 9-week group,and there was no significant difference in the distribution of BMP2,RUNX2 and GDF11 between the7-week group and the 9-week group in the light microscope.2.Calcification was induced after intraperitoneal injection of r-GDF11: In Von kossa-HE staining,calcium salt deposition in the low,medium and high concentration r-GDF11 group was reduced compared with the blank group and the solvent group,and the elastic fibers were more complete,the higher the concentration of r-GDF11,the less calcium salt deposition and the more complete the elastic fibers were.In Western Blot,the expression levels of the calcification marker proteins RUNX2 and BMP2 in the group with low,medium and high r-GDF11 concentration were lower than those in the blank group and the solvent group,the higher the concentration of r-GDF11 was,the lower the expression levels of the calcification marker proteins were(p < 0.05).In immunohistochemical analysis,the higher the concentration of r-GDF11 was,the denser the GDF11 was in the arterial tissue,and the thinner the pale RUNX2 and BMP2 calcification markers were.3.In Von kossa-HE,the arterial intima of the blank group and control group were smooth,and the elastic fibers of the middle membrane were complete,without calcium salt deposition.However,the inner membrane of the experimental group was not smooth,the elastic fibers of the middle membrane were broken,and dark brown calcium salt deposition was observed in the middle membrane.In Western Blot,the expression of calcification marker proteins BMP2 and RUNX2 in the experimental group increased significantly compared with the blank group and the control group,while the expression of GDF11 decreased significantly(compared with the blank group,p < 0.05).In immunohistochemistry,calcification marker proteins BMP2 and RUNX2 were more densely distributed in the experimental group than in the blank group and control group,while GDF11 was more sparse4.In the first part of the experiment,aortic proteins up-regulated and down-regulated GDF11 were melted at low temperature,and Western Blot analysis was performed to observe the expression of calcification marker proteins BMP2 and RUNX2,as well as GDF11,p-Smad2/3 and Smad2/3.5.Take 11 weeks of C57BL-6 mice,18 were divided into 3 groups randomly,the adaptability of feeding 1 week,the blank group,control group injected with normal saline,the experimental group injected Smad2/3 inhibitor,after 1 week,3 weeks to induce calcification,then eyeball blood,separation of the aorta,to observe and compare the three groups between Von kossa-HE calcium salt deposition rate of the HE staining and elastic fiber fracture,Western Blot detection calcification marker protein BMP2,RUNX2,GDF11,Smad2/3,p-Smad2/3 content in arteries.Conclusion GDF11 could affect arterial calcification in C57BL-6 mice: Up-regulation of GDF11 could reduce the degree of arterial calcification;Downregulation of GDF11 can aggravate the degree of arterial calcification;GDF11 inhibited arterial calcification in C57BL-6 mice by phosphorylating Smad2/3. |
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