The Effect Of NNMR Activation On Regulating Rat Aortic Endothelial Cell Apoptosis Induced By Ox-LDL And Hyperhomocystein And Its Mechanisms

Stimulation of non-neuronal muscarinic receptor (NNMR) can depress the endothelial cells damage caused by ox-LDL and homocysteine, and it also has beneficial effects on anti-atherosclerosis. The apoptosis of endothelial cells and the dysfunction of endothelium play a crucial role in the development of atherosclerosis and other vascular disease.The current study examined the effect of hyperlipemia on the NNMR mRNA expression of rabbit aortic endothelium. The results showed that the mRNA expression of muscarinic receptor subtypes M3, M2 and Ml decreased under hyperlipemia damage.The effect of NNMR stimulation on the apoptosis of rat aortic endothelial cells (RAEC) damaged by ox-LDL and homocysteine were investigated. The results showed that arecoline, carbachol and pivanampeta (PPVP) can depress the apoptosis of RAEC induced by Hcy tested by hoechst33342 staining method. Atropine can reverse the protection of arecoline. It shows that arecoline can decrease the apoptosis rate with concentration dependant (0.1 to 10 mM) tested by flow cytometry method with Annexin V-FITC and propidium iodide staining. Atropine can also increase the apoptosis decreased by arecoline.The effect of NNMR stimulation on the apoptosis related gene expression was tested by reverse transcription-polymerase chain reaction (RT-PCR). The protein of Bcl-2, Bax and caspase-3 were tested by western blot. The results showed that stimulation of NNMR by arecoline, carbachol and PPVP can increase the mRNA expression of Bcl-2, Bcl-x attenuated by Hcy. Arecoline, carbachol and PPVP also inhibited the mRNA expression of Bax, Fas, Fas-L, casepase8, casepase3, caspase-6 and p53 stimulated by Hcy. Atropine can reverse the effect of arecoline. The effects of arecoline, carbachol and PPVP on the expression of Bcl-2, Bax and casepase3 influenced by Hcy were tested by western blot. The results showed that stimulation of NNMR by arecoline, carbachol and PPVP can increase the protein expression of Bcl-2 attenuated by Hcy and can inhibit the protein expression of Bax, and casepase3 stimulated by Hcy. Atropine can reverse the effect of arecoline. It indicates the stimulation of NNMR can affect the mitochondria and endoplasmic apoptosis passway, and then affect the endothelial cells apoptosis.