Study on the mechanism of death related protein kinase 3 deficiency and inhibition of endoplasmic reticulum stress to alleviate vascular calcification / 中国综合临床

ABSTRACT

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 whether β-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 (Runχ 2, BMP2) was significantly decreased (Runχ 2 (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.