Mechanism Of The Inhibitory Effect Of Tongxinluo On Atherosclerosis

Atherosclerosis is one of the basis of the coronary artery disease(CAD), which is the leading cause of death in the world. Endothelial activation is considered a first step in the development of atherosclerotic lesions, followed by the invasion of proinflammatory cells and the proliferation of smooth muscle cells. Several risk factors such as hypercholesterolemia promote atherosclerosis.ROS are also involved in the pathogenesis of atherosclerosis. Oxidative stress is involved in the pathophysiological mechanisms of atherosclerosis. It has also been reported that low plasma antioxidant activity is associated with high concentration lipoproteins. Smooth muscle cell(SMC) proliferation and migration are known to play a critical role in the development of atherosclerosis. Oxidized low-density lipoprotein(ox LDL) is involved in the generation of atherosclerotic lesions. Recent studies have indicated that ox LDL is a well-established risk factor for atherosclerosis that induces VSMC proliferation and migration.The concept of mild chronic vascular inflammation as part of the pathophysiology of atherosclerosis has been well accepted. Indeed there are links between vascular inflammation, endothelial dysfunction and oxidative stress. Inflammation induced by Ox LDL plays an important role in the pathogenesis of atherosclerosis. Intravascular plaques, the pathologic lesions of atherosclerosis, are largely composed of cholesterol-laden luminal macrophage-rich infiltrates within a fibrous cap. Ox LDL due to its role in endothelial dysfunction and foam cell formation. Tissue-resident cells such as macrophages and mast cells release inflammatory mediators upon activation that in turn cause endothelial activation and monocyte adhesion.Collateral disease theoty of Traditional Chinese Medicine consider that AS is related to obstruction of collaterals and Qi stagnation. At present, the treatment for AS is given priority to use statins and proprietary Chinese medicine combination. The side effects of western drug can be significantly reduced and improve drug efficacy.Tongxinluo(TXL) is a traditional Chinese medicine which is extracted, concentrated, freeze-dried and standardized from a mixture of 12 medicinal constituents, is composed of ginseng, Hirudo, Buthus martensi, Eupolyphaga seu steleophaga, Periostracum cicadae, Scolopendra subspinipes, Radix paeoniae rubra, Lignum dalbergiae odoriferae, Semen ziziphi spinosae, Lignum santali albi, and Borneolum syntheticum. The previous studies have revealed that treatment with TXL can improve endothelial cell function, lower lipid, reduce inflammation and apoptosis, inhibit neointima hyperplasia and promote angiogenesis. However, the effects of TXL treatment on atherosclerosis and its action mechanisms are still unclear. Therefore, in this study, we used atherosclerosis model in apo E-/- mice and to human cardiac microvascular endothelial cells investigate whether and how TXL exerts a protective effect on atherosclerosis in vivo and in vitro. PartⅠ TXL inhibits plaques formation of atherosclerosis model in apo E-/-miceObjective: To observe the role of TXL in protecting against plaques formation of atherosclerosis model in apo E-/- mice.Methods: 1 Wild-type C57BL/6(WT) and apolipoprotein E gene knockout(Apo E-/-) mice were fed with a high-fat diet(HFD) or a HFD supplemented with TXL or Atorvastatin, for 12 weeks. 2 Micro-ultrasound imaging was performed using the Vevo770 high-resolution micro-ultrasound imaging system to measure vascular diameter. 3 The serum cholesterol and triglyceride levels were performed. 4 Plaque extensions on tissue sections of the aortic root were determined on pentachrome-stained sections, lipid core sizes were determined by measuring the area of Oil Red O staining. 5 Immunohistochemical staining was performed to examine CD31.Results:1 TXL inhibits plaques formation of atherosclerosis model in apo E-/- miceAtherosclerotic plaques were observed clearly in the aortic sections of apo E-/- mice fed HFD. TXL and AT treatment significantly decreased plaque area. B-mode ultrasonic examination got the same results. The effect of TXL and AT on plaque formation in the aorta was evaluated by analyzing lipid deposition on Oil Red O-stained tissue sections of the aortic root. The results showed that TXL and AT treatment significantly decreased plaque area. There was no significant difference between TXL and AT.2 TXL significantly reduced serum TG, TC and LDL levelsSerum lipid levels were detected by semi-automatic biochemical analyzer. Treatment with TXL and AT significantly reduced serum TG, TC and LDL levels, compared with model group. However, there were no statistical differences in the amounts of HDL. These results suggested that TXL and AT decrease the development of atherosclerotic plaque through reducing serum lipid levels.3 TXL inhibited atherosclerotic plaque formation through protecting the endothelial cellsTo further investigate whether decreased serum lipid level by TXL and AT protected endothelial in apo E-/- mice fed HFD, the expression of CD31(endothelial-specific marker) was detected by immunohistochemisty. The results showed that TXL and AT treatment significantly increased CD31 expression in atherosclerotic plaque of apo E-/- mice. There was no significant difference between TXL and AT. These results prompt us to assume that TXL inhibited atherosclerotic plaque formation through protecting the endothelial cells.Summary:TXL inhibited atherosclerotic plaque formation through protecting the endothelial cells. Part II TXL exerted anti-oxidative effect in endothelial cellsObjective: To examine whether whether TXL exerts anti-oxidative effect in endothelial cells induced by C16.Methods: 1 Cultured human cardiac microvascular endothelial cells were preincubated with TXL and then treated with C16. 2 The level of ROS was detected by DHE. 3 The levels of SOD, NO and MDA was detected by diagnostic kits. 4 q RT-PCR was performed to examine p22 phox, p47 phox and HO-1 expression. 5 Western Blot was performed to examine p22 phox, p47 phox and HO-1 expression. 6 Immunohistochemical staining was performed to examine p22 phox, p47 phox and HO-1 expression.Results:1 TXL suppresses ROS production induced by C16 in endothelial cellsTo further explore whether the role of TXL in improving endothelial cell function was related to its anti-oxidative properties, anti-oxidative effect of TXL was analyzed using C16 as an oxidative stress inducer. The results showed that the ROS levels in the endothelial cells treated with C16 were significantly increased. The TXL treatment significantly reduced levels of ROS suggest that TXL had markedly antioxidative effect in endothelial cells.2 TXL suppresses MDA levels induced by C16 TXL and exerted anti-oxidative effect in endothelial cellsThe results showed that the MDA levels in the endothelial cells treated with 600μmol/L of C16 were significantly increased, while the NO and SOD level was significantly decreased when compared with the cells treated without C16. The TXL treatment significantly reduced levels of MDA. By contrast, the levels of NO and SOD were increased in dose-dependent manner. These results suggest that TXL had markedly antioxidative effect in endothelial cells.3 TXL inhibited expression of NADPH oxidase and HO-1 in endothelial cellsTo examine whether antioxidative effect of TXL is mediated by NADPH oxidase and HO-1, HCMECs were pre-incubated with the different doses of TXL for 24 h and then treated with C16. The results showed that C16-treated endothelial cells showed significantly higher p22 phox, p47 phox and HO-1 m RNA expression levels than the cells treated without C16. Treatment with different doses of TXL significantly decreased p22 phox, p47 phox and HO-1 m RNA expression in dose-dependent manner. Accordingly, the expression of p22 phox, p47 phox and HO-1 proteins was detected by Western blotting. The results showed that oxidative stress inducer C16 dose-dependently induced the expression of p22 phox, p47 phox and HO-1 proteins, while TXL treatment markedly decreased their expression in dose–dependent manner, especially when 600 μg/ml and 800 μg/ml TXL were used.4 TXL inhibited expression of NADPH oxidase and HO-1 in aop E-/- miceTo further investigate the effect of TXL on the expression of NADPH oxidase and HO-1 proteins in vivo, apo E-/- mice were used to examine p22 phox, p47 phox and HO-1 expression by immunohistochemical staining. Atherosclerotic mice showed significantly higher p22 phox, p47 phox and HO-1 expression levels than the control groups. Treatment with TXL(0.75 g/kg/day) markedly decreased p22 phox, p47 phox and HO-1 expression as evidenced by immunohistochemisty and RT-PCR, without significant difference between TXL and atorvastatin(AT). These results indicated that TXL exerts its anti-oxidative effect through regulating the expression of NADPH oxidase subunits.Summary:TXL inhibited oxidative stress by decreasing the expression of NADPH oxidase in endothelial cells, increasing the expression of SOD, suppressing the ROS production and so on. Part Ⅲ TXL suppresses inflammatory response.Objective: To explore the molecular mechanisms whereby TXL regulates inflammatory response.Methods: 1 Cultured human cardiac microvascular endothelial cells were preincubated with TXL and then treated with C16. 2 The level of TNF-α and IL-1β was detected by ELISA. 3 q RT-PCR was performed to examine NF-κB and e NOS expression. 4 Western Blot was performed to examine NF-κB, e NOS, phos-p22 phox, phos-p47 phox and phos-NF-κB expression.Results:1 TXL inhibited inflammation in HCMECs induced by C16Except for oxidative stress, the inflammation is also involved in the atherogenesis. To detect the effect of TXL on inflammation, HCMECs were treated with C16 and the expression of IL-1β and TNFα was detected. The results showed that C16 treatment increased the level of proinflammatory cytokines IL-1β and TNFα in the cultured medium of HCMECs in a dose-dependent manner. Treatment with different doses of TXL dose-dependently decreased the release of IL-1β and TNFα to the medium, suggesting that TXL decreases the inflammation induced by C16 in HCMECs.2 TXL inhibited the expression of NF-κB and e NOS in apo E-/- miceTo gain mechanistic insight into how TXL modulates the expression of proinflammatory cytokines, NF-κB expression in C16-treated HCMECs was examined. We found that C16 increased, but TXL decreased NF-κB m RNA expression and protein expression in dose-dependent manner. Conversely, TXL increased the expression of e NOS in C16-treated HCMECs, suggesting that TXL can improve HCMECs function. Atherosclerotic mice also showed that treatment with TXL and AT could decrease NF-κB m RNA expression. These results make us believe that inhibitory effect of TXL on atherogenesis may be related to its anti-inflammation by decreasing the expression of NF-κB and proinflammatory cytokines in HCMECs.3 TXL decreased inflammation oxidative stress by inhibiting the activation of NF-κB and p47 induced by C16We and others have shown that NF-κB plays an important role in the regulation of the expression of multiple genes involved in the inflammatory responses. To gain mechanistic insight into how TXL modulates inflammation in endothelial cells, HCMECs were pre-incubated with TXL and then treated with C16. The results showed that the phosphorylation of p22 phox, p47 phox and NF-κB was significantly increased in HCMECs, following stimulation with C16. TXL pre-treatment decreased C16-stimulated p47 phox and NF-κB phosphorylation, but phosphorylation of p22 phox was little changed by the TXL treatment, suggesting that TXL treatment leads to the suppression of p47 phox and NF-κB activation by C16, subsequently reducing the inflammation and oxidative stress generation.Summary:TXL inhibits the expression and releases of inflammatory factors. The mechaism is related to p47 phox and NF-κB signaling pathway. Part IV The effect of mi R-155 on atherosclerosisObjective: To explore the effect of mi R-155 on atherosclerosis.Methods: 1 Real-time PCR analysis the expression of mi R-155 in the blood of cornary artery disease. 2 Plaque extensions on tissue sections of the aortic root were determined on pentachrome-stained sections, lipid core sizes were determined by measuring the area of Oil Red O staining. 3 Immunohistochemical staining was performed to examine Mac-2 and KLF5.Results:1 mi R-155 was associated with coronary artery diseaseQuantity real-time PCR results showed that circulating mi R-155 levels in patients with coronary artery disease decreased significantly, compared with the normal group.To verify the reduction of circulating mi R-155 in patients with CAD and confirm the factors influencing the level of circulating mi R-155, we prospectively measured circulating mi R-155 in a validation cohort. The results showed that high cholesterol and diabetes were associated with the reduction of circulating mi R-155 level.2 mi R-155 inhibited the formation of atherosclerotic plaquesAtherosclerotic plaques were observed clearly in the aortic sections of apo E-/- mice fed HFD. mi R-155-/-apo E-/- mice significantly decreased plaque area. The effect of mi R-155 on plaque formation in the aorta was evaluated by analyzing lipid deposition on Oil Red O-stained tissue sections of the aortic root. The results showed that mi R-155 deficiency significantly decreased plaque area.3 mi R-155 inhibited macrophage infiltration and KLF5 expressionMacrophage infiltration was observed in apo E-/- mice fed with HFD, which was strongly reduced in mi R-155-/-apo E-/- mice, and the expression of KLF5 was barely observed in mi R-155-/-apo E-/- mice. These results suggested that mi R-155 deficiency inhibited macrophage infiltration, meanwhile decreased the expression of KLF5, which is one of the mechanism of atherosclerosis.Summary:High cholesterol and diabetes were associated with the reduction of circulating mi R-155 level; mi R-155 deficiency inhibited macrophage infiltration, meanwhile decreased the expression of KLF5, which is one of the mechanism of atherosclerosis.Conclusion:1 TXL inhibited atherosclerotic plaque formation through regulating the function of endothelial cells.2 TXL inhibited oxidative stress by decreasing the expression of P22 phox, P47 phox in endothelial cells, increasing the expression of SOD, supressing the ROS production and so on.3 TXL inhibits the expression and releases of inflammatory factors. The mechaism is related to p47 phox and NF-κB signaling pathway.4 High cholesterol and diabetes were associated with the reduction of circulating mi R-155 level.5 mi R-155 deficiency inhibited macrophage infiltration, meanwhile decreased the expression of KLF5, which is one of the mechanism of atherosclerosis.