AGEs/RAGE Induce Human Aortic Smooth Muscle Cell Apoptosis Through Activation Of P38MAPK And JNK Pathways

Objective: The role of vascular smooth muscle cell apoptosis in vascular calcification is more and more attention.Research shows that in the apoptosis of vascular smooth muscle cells in atherosclerotic plaques obviously increased;Because calcium phosphorus metabolism disorders in patients with chronic kidney disease, the large amount of calcium phosphate crystals was engulfed by vascular smooth muscle cells, again by cells lysosome degradation, causes a dramatic increase in intracellular ca lcium, phosphorus, lead to cell apoptosis, and then the formation of apo-ptotic bodies, these can be used as a vascular matrix mineralization of apoptosis body of nuclear point start calcification process.Advanced glyc-osylation end products(AGE) is a set of sugar in the body of the ald e-hyde group interact with protein amino generated a charged amino aci d residues, AGE expression in vivo is very low at physiological state.AGE through combined with corresponding receptors play a role of biol ogy, RAGE is he most studied receptors now.This study intends to expl ore the role of AGEs,which in human aortic smooth muscle cells(HSA SMC)apoptosis, whether through the AGE- RAGE binding way to play the molecular mechanism of biological effect and related signaling p athway.Methods:The 3-5 generation HSASMC used in the experiment.With diff-erent concentrations of AGE- BSA(50 mg/L, 100 mg/L, 200 mg/L) st imulate cell for 48 h, then flow cytometry instrument detection HASMC apoptosis and Western blot test cells promote apoptosis related protein p53, BAX, Caspase3, resistance to apoptosis related proteins p65, cox-2, Bcl2 and RAGE protein expression.In order to determine the role of R AGE in the AGE to promotecell apoptosis, respectively build people ex press and silent RAGE gene slow virus vector, its transfection to HAS MC respectively, Western blot and q- PCR detection RAGE protein e xpression efficiency.flow cytometry instrument testing apoptosis of HASM C with different levels RAGE protein expression,and Western blot test cells corresponding p53, BAX, promoting apoptosis related proteins in Caspase3, resistance to apoptosis related proteins p65, the expression of cox-2 and Bcl2 conditions.For clear p38 MAPK, JNK pathway are inv olved in cell apoptosis process,after AGE- BSA stimulate cells at diffe rent times(6 h, 12 h, 24 h, 48 h), Western blot detection activation o f phosphory-lated products of HASMC JNK and p38 MAPK.To contin ue to verify p38 MAPK, JNK pathway, respectively introduced sp600125 JNK inhibitors and p38 MAPK inhibitor SB203580 intervention cells, again using flow cytometry instrument detecting apoptosis and Western blot detectio n caspase3 protein expression in cells.Results: Successful culture human aortic smooth muscle cells, respectively to different concentrations(50 mg/L, 100 mg/L, 200 mg/L) AGE- BSA stimulation 48 h, Cells apoptosis increase concentration dependence, apoptosis rate are 8.43%, 12.49% and 8.43%;The promoting apoptosis related proteins p53, BAX, Caspase3 expression increase by concentration dependence,The resistance to apoptosis related proteins p65, cox-2, Bcl2 expression reduce by concentration dependence;RAGE protein expression in a concentration dependent increase in HASMC.Build a express/silent RAGE gene HASMC stable cell lines, each plant cell expression of green fluorescent protein success; RAGE protein expression levels were significantly higher than those control group in RAGE gene expression cells(0.97±0.02VS0.44±0.01,p<0.01),and RNA levels were significantly higher than those in control group(1588.55 vs1.26);RAGE protein expression levels in RAGE gene silencing cells ignificantly lower than the control group( 0.56±0.02,0.86±0.01VS1.25±0.02,p<0.01),and RNA level than the control group was significant interference, RAGE- sh RNA- 2 group at a rate of 99.55%;RAGE-sh RNA-3 groups of interference at a rate of 98.86%.RAGE- CDNA group of apoptosis rate was significantly higher(33.69% VS17.52 %), RAGE- sh RNAcell apoptosis rate was significantly lower in group three(8.41% VS17.52 %).RAGE- CDNA groups of cells and promote the expression of apoptosis protein p53, BAX and caspase3 than the control group significantly increased, the expression of antiapoptotic proteins cox-2、Bcl2、p65 than the control group significantly decreased.After 200 mg/L AGE- BSA stimulation for HASMC, then detection the protein expression of JNK and P- JNK, p38 MAPK and p-p38 MAPK at 6 h, 12 h, 24 h, 48 h.The results suggest that the level of p-JNK expression gradually increased, peaked at 12 h, the same as p-p38 MAPK and peaked at 48 h. while the total JNK and p38 MAPK have no obvious change.To verify P38 MAPK and JNK pathway in the role of cell apoptosis, SP600125 JNK inhibitors and P38 lightning SB203680 MAPK inhibitors were applied in the experiment. After s P600125, SB203680 intervention cells 24 hours, HASMC caspase3 protein expression were significantly suppressed,and the AGE-induced HASMC apoptosis rate was significantly lower(6. 25% VS 18.49%, 6.15% VS 18.49%)Conclusion:1.the AGE can promote apoptosis of human aortic smooth muscle cells.2.The RAGE play an important role in the process of AGE to promote human aortic smooth muscle cells apoptosis.3. AGEs/RAGE Induce Human Aortic Smooth Muscle Cell Apoptosis through Activation of p38 MAPK and JNK Pathways.