The Role And Mechanism Of Adiponectin In Endoplasmic Reticulum Stress Induced Vascular Calcification

Adiponectin is secreted by fat cells, a specific bioactive peptides, is an important metabolic regulator, adiponectin has anti-diabetic, anti-inflammatory, anti-atherosclerotic plaque formation and vascular protective the physiological effects. Recent studies indicate that adiponectin may play a protective role by inhibiting the activation of the vascular endothelial cells, inhibition of endoplasmic reticulum stress-induced apoptosis, promote endothelial cell repair pathways, but the exact mechanism remains to be studied. In this study, through the establishment of apoptosis in vitro, in vivo calcification model,combined with pathological analysis, molecular biology and cell biology techniques,explained adiponectin alleviate endoplasmic reticulum stress-induced vascular calcification and thus alleviate the mechanism.PartⅠ Adiponectin attenuates vascular calcification in ratsObjective:An in vivo rat vascular calcification model was adopted to investigate the inhibitory effect of adiponectin.Methods:Healthy male SD rats were randomly divided into three groups.control group:Were given saline administered intramuscularly and pure peanut oil. VDN group: Rats(n=10)were intramuscularly injected with vitamin D3(300,000 IU/kg) and given nicotine(25mg/kg, dis-solved in peanut oil) intragastrically. Rats were additionally administered nicotine 12 h later. VDN+g Ad group: Model making with calcification group, while thetongue intravenous adiponectin(1 μg ? kg-1 / day). Removal of the thoracic aorta after all rats were fed for 42 d. Combined with pathological analysis(Von Kossa staining was observed in vascular calcification), thoracic aortic calcium content and ALP activity assay was observed degree of calcification, we assessed vascular calcification markers OPG,Runx2 by quantitative real-time PCR and western blot.Results:(1)Von Kossa staining showed no calcium deposition in the sham group.In the VDN group, vascular calcium deposition visible black particles in the membrane fraction of elastic fibers disorders, fracture. In the VDN + g Ad group, only a small amount of black particles of calcium deposition; Compared with the sham group, the VDN group calcium content and activity of ALP were significantly increased(P <0.01), the VDN + g Ad group compared with the VDN group, calcium content and activity of ALP was significantly decreased(P <0.05).(2) Compared with the sham group, the VDN group OPG m RNA expression was significantly decreased, Runx2 m RNA expression was significantly increased(P <0.01); The VDN + g Ad group compared with the VDN group, OPG m RNA expression was significantly increased, Runx2 m RNA expression was significantly decreased(P <0.01).(3) Compared with the sham group, in the VDN group, OPG protein expression was significantly decreased, Runx2 protein expression was significantly increased(P <0.01); The VDN + g Ad group compared with the VDN group, OPG protein expression was significantly increased, Runx2 protein expression was significantly decreased(P <0.01).Conclusion:g Ad inhibits vascular calcification in rats.PartⅡ Endoplasmic reticulum stress-mediated apoptosis promotes vascular calcificationObjective:An in vitro primary cultured rat aortic smooth muscle cells calcification model was adopted to investigate the endoplasmic reticulum stress-induced apoptosis of vascular calcification.Methods:Primary cultured rat aortic VSMCs after established calcification model were randomly divided into four groups.(1) control group;(2) β-GP group: VSMCs were treated with 10 mmol/L β-glycerophosphate;(3)TG group: VSMCs were treated with 3μmol/L TG and 10 mmol/L β-glycerophosphate;(4) 4-PBA group: VSMCs were treated with 1μg/ml4-PBA and 10 mmol/L β-glycerophosphate. Von Kossa staining was observed in cells calcification. Calcium content and ALP activity of the cells were observed to assess degree of calcification was measured.TUNEL method and caspase-3 activity was measured to assess smooth muscle cell apoptosis. We assessed endoplasmic reticulum stress,apoptosis,calcification index(GRP78, caspase12, CHOP, OPG, Runx2) by quantitative real-time PCR and western blot.Results:(1) Compared with the control group, in the β-GP group and TG group, calcium content and activity of ALP were significantly increased(P < 0.01), in the 4-PBA group,calcium content and activity of ALP was obviously increased(P < 0.05); The TG group compared with the β-GP group, calcium content and activity of ALP were significantly increased(P < 0.05);The 4-PBA group compared with the β-GP group, calcium content and activity of ALP were significantly decreased(P < 0.05).(2)It is rare TUNEL positivecells in the control group. Compared with the control group, in the β-GP group and the TG group, TUNEL positive cells and activity of caspase-3 was significantly increased(P<0.01).In the 4-PBA group, TUNEL positive cells and caspase-3 activity was significantly increased(P<0.05).Apoptotic cells and caspase-3 activity increased significantly in the TG group(P < 0.05). The 4-PBA group compared with the β-GP group,TUNEL positive cells and caspase-3 activity were significantly decreased(P < 0.05).(3)Compared with the control group, in the β-GP group and the TG group, m RNA and protein expression of GRP78, CHOP,caspase-12 and Runx2 were significantly increased, m RNA and protein expression of OPG was significantly decreased(P < 0.01);The TG group compared with the β-GP group, m RNA and protein expression of GRP78,CHOP,caspase-12 and Runx2 were significantly increased, m RNA and protein expression of OPG was significantly decreased(P < 0.05). The 4-PBA group compared with the β-GP group, m RNA and protein expression of GRP78, CHOP,caspase-12 and Runx2 were significantly inhibited, m RNA and protein expression of OPG was significantly increased(P < 0.05).Conclusion:Endoplasmic reticulum stress-mediated apoptosis promotes vascular calcification.Part Ⅲ Adiponectin Attenuates Vascular Calcification by Inhibits Endoplasmic Reticulum Stress-mediated ApoptosisSection 1 Adiponectin Attenuates Vascular Calcification by Inhibits Endoplasmic Reticulum Stress-mediated Apoptosis in VivoObjective:An in vivo rat vascular calcification model was adopted to investigate the role of adiponectin in vascular calcification of endoplasmic reticulum stress-mediated apoptosis.Methods:The experiment group are consistent with PartⅠ.Results:(1) Compared with the sham group, calcium content and ALP activity of the VDN group were increased significantly(P < 0.01), The VDN+g Ad group compared with the VDN group, its calcium content and ALP activity was significantly decreased(P < 0.05).(2) There are rare TUNEL positive cells in the sham group. Compared with the sham group,in the VDN group and VDN+g Ad group,TUNEL positive cells and caspase-3 activity were increased significantly(P < 0.01). Compared with the VDN group, TUNEL positive cells and caspase-3 activity of the VDN+g Ad group were decreased significantly(P < 0.05).(3)Compared with the sham group, m RNA and protein expression of GRP78,CHOP,caspase-12 and Runx2 of the VDN group were significantly increased, m RNA and protein expression of OPG were significantly decreased(P < 0.01). m RNA and protein expression of GRP78, CHOP,caspase-12 and Runx2 of the VDN+g Ad group were significantly inhibited, m RNA and protein expression of OPG were significantly increased(P < 0.05).Conclusion:g Ad attenuated rat vascular calcification by suppress endoplasmic reticulum stress-mediated apoptosis.Section 2 Adiponectin Attenuates Smooth Muscle Cells Calcification by Inhibit Endoplasmic Reticulum Stress-mediated Apoptosis in VitroObjective:An in vitro smooth muscle cells calcification model was adopted to investigate therole of adiponectin in vascular calcification of endoplasmic reticulum stress-mediated apoptosis.Methods:Primary cultured rat aortic VSMCs after established calcification model were randomly divided into 6 groups.(1) control group:(2) g Ad group: VSMCs were treated with 2μg/ml g Ad;(3)β-GP group: VSMCs were treated with 10 mmol/Lβ-glycerophosphate;(4)β-GP + g Ad(0.5μg/ml)group: VSMCs were treated with 0.5μg/ml g Ad and 10 mmol/L β-glycerophosphate;(5)β-GP + g Ad(1μg/ml)group: VSMCs were treated with 1μg/ml g Ad and 10 mmol/L β-glycerophosphate;(6) β-GP + g Ad(2μg/ml)group: VSMCs were treated with 2μg/ml g Ad and 10 mmol/L β-glycerophosphate. To further investigate the mechanisms responsible for the protective effect of g Ad, observation indicators in line with PartⅡ.Results:(1) Compared with the control group, calcium content and ALP activity of the β-GP group were difference significantly(P < 0.01), compared with the β-GP group, calcium content and ALP activity of the β-GP+1μg/ml g Ad group and the β-GP+2μg/ml g Ad group were significantly decreased(P < 0.05).(2) TUNEL positive cells and caspase-3 activity of the β-GP group were significantly increased.Compared with the β-GP group, TUNEL positive cells and caspase-3 activity of the β-GP+1μg/ml g Ad group and the β-GP+2μg/ml g Ad group were decreased significantly(P < 0.05).(3) Compared with the control group,m RNA and protein expression of GRP78, CHOP,caspase-12 and Runx2 of the β-GP group and the β-GP+0.5μg/ml g Ad group were significantly increased, m RNA and protein expression of OPG were significantly decreased(P < 0.01);Compared with the β-GP group,m RNA and protein expression of GRP78, CHOP,caspase-12 and Runx2 of theβ-GP+1μg/ml g Ad group and the β-GP+2μg/ml g Ad group were significantly decreased,m RNA and protein expression of OPG were significantly increased(P < 0.05).Conclusion:g Ad attenuated rat smooth muscle cells calcification by suppress endoplasmic reticulum stress-mediated apoptosis in a dose-dependent manner.