死亡相关蛋白激酶3缺乏与抑制内质网应激减轻血管钙化的机制研究

Study on the mechanism of death related protein kinase 3 deficiency and inhibition of endoplasmic reticulum stress to alleviate vascular calcification

目的探究死亡相关蛋白激酶3(death-associated protein kinases 3,DAPK3)缺乏是否通过抑制内质网应激而减轻血管钙化。方法采用前瞻性队列研究方法,通过体外细胞培养实验进行观察分析。人主动脉血管平滑肌细胞(vascular smooth muscle cell,VSMC)采用F10K Kaighn′s改良培养基培养,根据培养基中是否添加β磷酸甘油(10 mmol/L)分为钙化组和非钙化组。钙化组细胞和非钙化组细胞又分别分为DAPK3抑制组及其对照组、内质网应激抑制组及其对照组、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)激活组及其对照组、DAPK3抑制+腺苷酸活化蛋白激酶(AMP-activated protein Kinase,AMPK)抑制以及空白对照组。测定各组的VSMC中DAPK3 mRNA以及蛋白浓度、钙含量、碱性磷酸酶、VSMC分化标志基因(SM22α、α-SMA)蛋白浓度、成骨分化转录因子[Runχ2、骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)]蛋白表达浓度、内质网应激标志蛋白(AFT4、GRP78、GRP94以及CHOP)表达水平以及p-PAMK蛋白表达。结果钙化细胞DAPK3 mRNA(第14天达最高值15.24±0.72)以及蛋白(第14天达最高值11.31±0.38)显著高于非钙化细胞(5.63±0.62、2.59±0.33),差异有统计学意义(P<0.001)。钙化细胞中,DAPK3缺乏组钙含量(86.54±8.21)mmol/g较对照组(194.63±8.54)mmol/g显著降低(t=22.35,P<0.001)、碱性磷酸酶活性显著降低[(96.27±10.28)IU/g蛋白与(224.67±10.94)mmol/g蛋白,t=20.951,P<0.001]、VSMC分化标志基因(SM22α、α-SMA)蛋白的表达显著上升[SM22α:(0.82±0.14)与(0.44±0.13),t=4.872,P=0.001;α-SMA:(0.95±0.18)与(0.56±0.13),t=4.303,P=0.002]、而骨分化转录因子(Runχ2、BMP2)蛋白表达显著降低[Runχ2:(1.12±0.28)与(2.21±0.35),t=5.957,P<0.001;BMP2:(0.82±0.12)与(1.26±0.16),t=5.39,P<0.001]、MAPK水平上调(DAPK3抑制组钙化细胞0.74±0.12、非钙化细胞1.04±0.14高于对照组钙化细胞0.44±0.10、非钙化细胞0.78±0.12,t=4.704,P=0.001;t=3.454,P=0.006);抑制ESR钙化细胞的钙含量显著降低(细胞经AMPK通路抑制后转染shRNA组150.21±11.98、细胞转染shRNA组83.21±12.12低于转染空白对照组164.82±12.34,P<0.001);碱性磷酸酶活性显著降低(细胞经MAPK通路抑制后转染shRNA组226.54±16.57、细胞转染shRNA组112.34±15.96低于转染空白对照组242.32±16.32,P<0.001);激活MAPK钙化细胞的钙含量显著降低(P<0.001)、碱性磷酸酶活性显著降低(P<0.001)。结论DAPK3缺乏通过AMPK信号通路抑制内质网应激达到减缓VSMC钙化的作用。

Objective To investigate whether the deficiency of death associated protein kinases(DAPK)3 can reduce vascular calcification by inhibiting endoplasmic reticulum stress.Methods The method of prospective cohort study was used to observe and analyze the cell culture in vitro.Human aortic vascular smooth muscle cells(VSMC)were cultured in F10K Kaighn′s modified medium,and divided into calcified group and non-calcified group according to whetherβ-phosphoglycerin(10 mmol/L)was added into the medium.The cells in calcified group and non calcified group were divided into DAPK3 inhibition group and its control group,endoplasmic reticulum stress inhibition group and its control group,mitogen-activated protein kinase(MAPK)activation group and its control group,DAPK3 inhibition+AMP-activated protein kinase(AMPK)inhibition and blank control group,respectively.DAPK3 mRNA and protein concentration,calcium content,alkaline phosphatase,protein concentration of VSMC differentiation marker genes(SM22α,α-SMA),osteogenic differentiation transcription factor(Runχ2,bone morphogenetic protein-2,BMP-2),endoplasmic reticulum stress markers(AFT4,GRP78,GRP94 and CHOP)and p-PAMK protein expression were measured.Results The mRNA level(highest value was 15.24±0.72 on the 14th day)and protein level(highest value was 11.31±0.38 on the 14th day)of DAPK3 were significantly higher than those in non calcified cells(5.63±0.62,2.59±0.33,respectively).The difference was statistically significant(P<0.001).In the calcified cells,calcium content(86.54±8.21)mmol/g in dapk3 deficient group was significantly lower than that in control group(194.63±8.54)mmol/g(t=22.35,P<0.001),alkaline phosphatase activity was significantly decreased((96.27±10.28)IU/g vs.(224.67±10.94)IU/g,t=20.951,P<0.001),the expression of the VSMC differentiation marker genes(SM22α,α-SMA)were upregulated significantly(SM22α:(0.82±0.14)vs.(0.44±0.13),t=4.872,P=0.001;α-SMA:(0.95±0.18)vs.(0.56±0.13),t=4.303,P=0.002),the level of bone differentiation transcription factor(Runχ2,BMP2)was significantly decreased(Runχ2:(1.12±0.28)vs.(2.21±0.35),t=5.957,P<0.001;BMP2:(0.82±0.12)vs.(1.26±0.16),t=5.39,P<0.001),MAPK level was up-regulated(DAPK3 inhibited group 0.74±0.12 of calcified cells,1.04±0.14 of non calcified cells,higher than the control group 0.44±0.10 of calcified cells,0.78±0.12 of non calcified cells,t=4.704,P=0.001;t=3.454,P=0.006),and the inhibited calcium content of ESR calcified cells significantly reduced(after inhibition of AMPK pathway,cells transfected with shRNA group 150.21±11.98,cells transfected with shRNA group 83.21±12.12 were lower than those transfection blank control group 164.82±12.34,P<0.001).The activity of alkaline phosphatase was significantly reduced(226.54±16.57)IU/g protein in the shRNA group and(112.34±15.96)IU/g protein in the shRNA group were significantly lower than 242.32±16.32 in the blank control group,P<0.001);calcium content and ALP activity in the calcified MAPK cells were significantly reduced(P<0.001).Conclusion DAPK3 deficiency can inhibit endoplasmic reticulum stress through AMPK signaling pathway to slow down VSMC calcification.