| Objective: Vascular endothelial injury was induced with a variety of pathogenic factors in pathological events of atherosclerosis. Injured endothelial cells released chemotactic adhesive molecules which promoted mononuclear cells migrating to subendothelial layer, then transforming to macrophages, and foam cells and atheromatous plaques after phagocytizing lipids; polarized M1 macrophages released inflammatory factors, which injured endothelial cells, deteriorated arterial wall lesion and instability of plaque, leading to a vicious circle. Therefore, the interaction of endothelial cells with monocytes/macrophages existed significant effects on the formation, and instability of atheromatous plaque. In the light of the TCM collateral disease theory, the study aimed to observe the interventional effects of Tongxinluo on apoptosis of VECs induced by injured endothelial cells, monocyte adhesion and M1 macrophages and to provided experimental supports for the clinical prevention and treatment of atherosclerosis.Methods: The study includes two sections: 1 Study on the adhesion between injured endothelial cells and monocytes and the effects of Tongxinluo and ginsenoside Rb1In this section endothelial cells were injured with ox-LDL and treated with TXL and Rb1. Viability, mitochondrial membrane potential, intracellular calcium ion changes and LOX-1/NF-?B signal pathway related protein expression were detected to observe the protecive effect of TXL and Rb1. And the adhesion between endothelial cells and monocytes were observed the level of chemokines and adhesion molecules in endothelial cells cultivation supernatant were detected and relative receptors' expression of chemotaxis and adhesion molecules in monocytes were analysed to observe the effect of TXL and Rb1 on the adhesion of endothelial cells to monocytes. 2 Study on apoptosis of endothelial cells induced by M1 macrophages and the intervention effect of TXL and Rb1In this section THP-1 were intervented by PMA and IL-4 or LPS, and treated by TXL and Rb1.The level of M2 and M1 macrophages marker molecule in cell culture supernatant, the expression of Notch signaling pathway related protein, the apoptosis process of endothelial cells and the expression of apoptosis related protein were detected to observe the intervention effect of TXL and Rb1 to the apoptosis of endothelial cells induced by M1 macrophages.Results: 1 Study on the adhesion between damaged endothelial cells and monocytes and the intervention effect of TXL and Rb1 1.1 The intervention effect on endothelial cells damaged by ox-LDL of TXL and Rb1 1.1.1 TXL and Rb1 increased the viability of endothelial cellsCompared with Control group, ox-LDL significantly reduced the viability of endothelial cells,TXL and Rb1 improved the viability of endothelial cells injured by ox-LDL. 1.1.2 TXL and Rb1 inhibited the decrease of mitochondrial transmembrane potential induced by ox-LDLThe change of mitochondrial membrane potential(MTP) of endothelial cells were observed by dynamic imaging system. Mitochondria were labeled by green fluorescence and higher position of mitochondrial membrane potential were marked by red fluoresence. The red fluoresence persisted in control group and weaked rapidly in model group, the decrease of red fluoresence occurred later and lesser in TXL and Rb1 group. These results showed that TXL and Rb1 could reduce and delay the decrease of MTP induced by ox-LDL. 1.1.3 TXL and Rb1 inhibited the influx of calcium induced by ox-LDLThe influx of calcium ions were observed by dynamic imaging system, the calcium ions were labelled by green fluorescence. In control group, the green fluorescence located on the periphery of endothelial cells constantly and distributed lessly in the interior of endothelial cells. In model group, the distribution of green fluorescence was broad and strong.In TXL group and Rb1 group, the distribution of green fluorescence in the interior was less than that in model group, and similar to the distribution in control group. These results showed that TXL and Rb1 could inhibit the influx of calcium induced by ox-LDL. 1.1.4 TXL and Rb1 inhibited the expression of LOX-1 and activation of NF-?B signaling pathwayWestern blot results showed that, compared with control group, the protein expression of LOX-1, p38 MAPK, p I?B?, NF-?Bp65, pNF-?Bp65 increased and the expression of I?B? decreased.Compared with model group, the protein expression of LOX-1, p38 MAPK, pI?B?, NF-?Bp65, pNF-?Bp65 decreased and the expression of I?B? increased.These results showed that TXL and Rb1 could inhibit the expression of LOX-1 and activation of NF-?B signaling pathway induced by ox-LDL.The nuclear translocation of NF-?Bp65 was observed by immunofluorescence method, the results showed that TXL and Rb1 could inhibit the activation of NF-?B signaling pathway induced by ox-LDL. 1.2 The intervention effect on adhesion between endothelial cells and monocytes of TXL and Rb1 1.2.1 TXL and Rb1 inhibited the adhesion between endothelial cells and monocytesThe adhesion between endothelial cells and monocytes was observed by co-culture and live cell staining methods, the monocytes were stained by Hoechst-33343 blue nuclear dye. The results showed that, compared with control group, the number of monocytes adhered to endothelial cells increased significantly in model group. The number in TXL and Rb1 groups was reduced significantly. These results showed that TXL and Rb1 could inhibit the adhesion of endothelial cells and monocytes. 1.2.2 TXL and Rb1 reduced the level of chemokines and adhesion molecules in the culture supernatants of impaired endothelial cellsELISA results showed that,compared with control group, the level of MCP-1, sICAM-1 and sVCAM-1 increased in cell culture supernatants of model group. Compared with model group, the level decreased in TXL and Rb1 group. These results showed that TXL and Rb1 can inhibit the expression of chemokines and adhesion molecules induced by ox-LDL. 1.2.3 TXL and Rb1 inhibited the expression of the receptors of chemokines and adhesion molecules in monocytes cultured by endothelial cell conditioned mediumqRT-PCR results showed that,compared with control group, the expression of CCR2, Mac-1 and VLA4 mRNA increased in THP-1 cells in model group. Compared with model group, the expression of CCR2, Mac-1 and VLA4 mRNA decreased in TXL and Rb1 group.Western blot results showed that, compared with control group,the protein expression of CCR2, Mac-1 and VLA4 increased in THP-1 cells in model group. Compared with model group, the protein expression of CCR2, Mac-1 and VLA4 decreased in TXL and Rb1 group. These results showed that TXL and Rb1 could inhibit the expression of CCR2, Mac-1, VLA4 in THP-1 cultured by endothelial cell conditioned medium. 2 Study on apoptosis of endothelial cells induced by M1 macrophages and the intervention effect of TXL and Rb1 2.1 The intervention effect on the polarization of macrophage to M1 type of TXL and Rb1 2.1.1 TXL and Rb1 inhibited the expression of M1 macrophages marker moleculesELISA results showed that there was no statistical difference on the level of IL-10,IL-6 and TNF-? between control group and PMA group, indicating that experimental concentration of PMA had no effect on the polarization phenotype of macrophage. Compared with control group, the level of IL-10 increased in M2 group and there was no statistical difference on the level of IL-6 and TNF-?; Compared with control group and M2 group, the level of IL-10 decreased and IL-6, TNF-? increased in M1 group; theses results showed the establishment of M2 and M1 macrophage model. Compared with M1 group, the level of IL-10 increased and IL-6,TNF-? decreased in TXL and Rb1 group, indicating that TXL and Rb1 can inhibit the polarization of macrophage to M1 type induced by LPS. 2.1.2 TXL and Rb1 inhibited the activation of Notch signaling pathwayWestern blot results showed that there was no statistical difference on the expression of Notch-1, active Notch-1, DLL4 and Hes-1 among control group, PMA group and M2 group, indicating that experimental concentration of PMA has no effect on Notch signaling pathway. Compared with control and M2 group, the expession of Notch-1 has no statistical difference and the expression of active Notch-1, Dll4 and HES-1 increased significantly in M1 group. Compared with the M1 group, the expression of active Notch-1, Dll4 and HES-1 decreased significantly in TXL and Rb1 group. These results suggest that TXL and Rb1 could inhibit the activation of Notch signinaling pathway induced by LPS. 2.2 The intervention effect on the apoptosis of endothelial cells induced by M1 macrophage conditioned medium of TXL and Rb1 2.2.1 TXL and Rb1 inhibited the apoptosis of endothelial cells induced by M1 macrophage conditioned mediumThe apoptosis process was observed by co-culture method and dynamic imaging system. The results showed that in control group, the nucleus with regular shape, clear edge and the fluorescence intensity distributed homogeneously, meanwhile, cell proliferation was observed. There was no significant difference among PMA group, M2 group and control group, nuclei pyknosis and fragmentation was observed at the later stage. In M1 group,the nuclei become hyperchromatic, pyknoticcataclastic and marginalized early. Compared with M1 group, the nuclear showed neat shape and clear edge, nuclear hyperchromatic, pyknotic, cataclastic and marginalized occured lately and cells proliferation was also observed in TXL and Rb1 group.Flow cytometry results showed that there was no statistical difference among control group, PMA group and M2 group. Compared with control group, the apoptosis rate increased significantly in M1 group.Compared with M1 group, the apoptosis rate decreased significantly in TXL and Rb1 group. 2.2.2 TXL and Rb1 inhibited the expression of apoptosis related protein induced by M1 macrophage conditioned mediumWestern blot results showed that there was no statistical difference among control group, PMA group and M2 group.Compared with control group, the expression of caspase3, bax increased and bcl-2 decreased in M1 group. Compared with M1 group, the expression of caspase3, bax decreased and bcl-2 increased in TXL and Rb1 group.Conclusions:1 TXL and Rb1 can inhibited the expression of LOX-1 and the activtion of NF-?B signaling pathway, and partially prevent the decline of mitochondrial membrane potential and the influx of calcium ions, and improve the function of endothelial cells impaierd by ox-LDL, and reduce the expression of chemokines and adhesion molecules. TXL and Rb1 could futher reduced the adhesion between injuried endothelial cells and monocytes,and reduced the receptors of chemokines and adhesion molecules in monocytes cultured by endothelial cell conditioned medium.2 TXL and Rb1 can inhibited the activation of Notch signaling pathway in macrophages induced by LPS, and partially prevent the polarization of macrophages to M1 type, and futher reduced the expression of inflammatory factors, and then inhibited the apoptosis of endothelial cells induced by M1 macrophages conditioned medium.3 By directly protecting injured endothelial cells and inhibiting the polarization of macrophages to M1 type, TXL and Rb1 indirectly inhibited the chemotaxis adhesion of monocyte to damaged vascular endothelia and the apoptosis of endothelial cells, thus to played a role in anti-atherosclerosis by inhibiting the plaque formation and stabilizing plaque, and further to provide experimental evidence for TXL in the prevention and treatment of atherosclerosis4 Ginsenoside Rb1, as one composition of TXL,has similar role with TXL in this experiment,suggesting that ginsenoside Rb1 may be one of the substance basis of the function of TXL.The specific mechanism of TXL and ginsenoside Rb1 and if ginsenoside Rb1 and TXL has other similar roles need further study. |
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