| Objective: Coronary Heart Disease (CHD) is a common and frequent-happen illness in the clinical and it threatens the health of the people gravely. Therefore, the prevention and cure of CHD becomes one of the dominant topic for the domestic and foreign medical experts.The pathologic basis of CHD is atherosclerosis. At present, medicine studies on the pathogenesis of CHD relates to lipid infiltration , endothelial injury, thrombosis, especially to the oxidative stress and inflammation theory. In recent years, research shows that CHD is closely related to oxidative stress and inflammatory reaction, which take interaction and mutual influence, leads to the occurrence and development of CHD. Toxic heat doctrine is an important recognition of traditional Chinese medicine on the pathogenesis of CHD. It holds that toxic heat stasis in the venation, stagnating heart meridian and Qi-blood circulation, collateral vessels resistance leads to chest stuffiness and pains. Lots of researches confirmed that toxic heat related to the oxidative stress and inflammatory reaction. In the course of AS, variety of cytokine and lipid peroxide are categorized as"health poisoning evil"in traditional Chinese medicine.Endothelial dysfunction is now widely recognized as a key step of atherosclerosis. It not only leads to AS, but also plays an important role during the process of AS and its complications(e.g. Vein hyperkinesia, Thrombosis). The key point of anti-AS is to protect EC against invasion of all the risk factors and ox-LDL as a major carrier of oxygen free radicals,is the main mechanism for endothelial injury.From the results above,we established Reducing toxin and pushing blood Fang(RPF)according to the chest stuff-iness and heat-toxin doctrine,made its containing-serum act on the model of vascular endothelial cell injury induc- ed by ox-LDL ,detected the variation of indexes such as SOD,MDA,NO,ET,TNT-α, ICAM-1 in the culture medium,and studied the protective effects on VEC by the method of Reducing toxin and pushing blood in the view of anti-oxidative stress and anti-inflammation,which provides a theoretical and experimental basis for clinical trials.Methods:1 Observe the situation of the cell proliferation by MTT assay:Taked the cells in logarithmic growth phase, planted them into 96-well culture plate based on density of 2×10~5 cell/ml after digestion, waited until the attachment of cultured cells integrated at 80%, after synchronized culture for 24 hours, randomly divided them into 4 groups: (1) The contrast group (CG) :20% blank serum + 5% serum medium of newborn calf (2)The model group(MG) : 20% blank serum + 100mg/L ox-LDL + 5% serum medium of newborn calf (3)The low-dose group of the RPF containing-serum(LDG):10% RPF containing-serum + 10% blank serum + 100mg/L ox-LDL + 5% serum medium of newborn calf (4)The high-dose group of the RPF containing-serum(HDG):20% RPF containing-serum+ 100mg/L ox-LDL + 5% serum medium of newborn calf.After cultivated for 24 hours, observed the situation of the cell proliferation by MTT assay.2 Measure the content of SOD, MDA, NO, ET-1, TNF-αin supernatants: Taked the cells in logarithmic growth phase, planted them into 24-well culture plate based on density of 2×10~5 cell/ml after digestion. The remaining steps were the same as above. Measured the content of SOD, MDA, NO, ET-1, TNF-αin the supernatants after cultivated in different groups for 24 hours.3 Measure the expression of ICAM-1 by the method of immunohistochemistry: Taked the cells in logarithmic growth phase, digested and planted them into 6-well culture plate which contained pretreated slides in advance, based on density of2×10~4 cell/ml. Synchronizated 24 hours after cultured 24 hours, divided them into groups as above . Unfolded the glass coverslips after 24 hours, fixed the cells in cold acetone after washed two times by PBS, taked them out and dried them, then sticked to the glass slide with the neutral resin, measured the expression of ICAM-1 by the method of immunohistochemistry.Result:1 Observe the situation of the cell proliferation by MTT assay:Compared with MG(0.62±0.058), LDG(0.78±0.017),HDG(0.79±0.024) were significantly increased (P<0.01).2 SOD of supernatant :Compared with MG(58.26±1.34), LDG(63.57±1.63),HDG(67.63±2.95) were significantly increased (P<0.01).MDA of supernatant : Compared with MG (10.02±1.66),LDG(6.87±1.26),HDG(6.83±0.46) were significantly decreased (P<0.01).3 NO of supernatant: Compared with MG (2.34±0.18), LDG(1.86±0.19) were significantly decreased (P < 0.01). HDG(2.07±0.21),too (P<0.05).ET-1 of supernatant: Compared with MG(173.84±11.29), LDG(57.20±16.12),HDG(42.97±5.58) were signifycantly d-ecreased (P<0.01).4 TNF-a of supernatant: Compared with MG(0.68±0.09), LDG(0.58±0.03) were significantly decreased (P<0.05);HDG(0.43±0.04),too(P<0.01).5 The expression of intercellular adhesion molecule-1 (ICAM-1) of HUVEC was examined with immunohistoche-mistry: Compared with MG(0.507±0.064), LDG(0.446±0.020),HDG(0.400±0.043) were significantly decreased (P<0.01).Conclusion: 1 Through the experiment we can prove that the drugs choosed under the principle of reducing toxin and pushing blood has the effect of against the injure of ox-LDL to the vascular endothelial cell. The mechanism probably related to the anti-lipid peroxidation, depress or decrease the releasing of inflammatory factors. It provides a theoretical and experimental basis for prevention and cure of CHD in clinical trials.2 The experiment establishes theoretical and experimental basis for the further confirmation that toxic heat and congestion were the major pathogenesis of the CHD.3 CHD was a result of complex actions. The experiment only study the mechanism of prevention and cure of CHD by reducing toxin and pushing blood in the point of anti-inflammentory, anti-oxygen and protect function of EC. We need further investigation and consummation on if there are any other mechanisms . |
