| 1BackgroundThe heart is on the top of the five internal organs, belongs to fire, moves in the summer, is yang within yang. Although the pathological mechanism and clinical manifestations of heart diseases are different, but the main pathological basis is heart-Yang deficiency, just seize the main pathological basis will be able to grasp the main contradiction of heart disease.Danggui Buxue Decoction which is initiated by Li Dongyuan is consist of astragalus and angelica with5:1proportion. The prescription is used to the treatment of internal Laojuan injuries, Qi and blood deficiency, Yang floating on the outside. Astragalus is used for tonifying Qi, Qi belongs to Yang which is strong in warming heart Qi, so with large dose for Jun. The heart relies on blood nourishing, and the blood is the carrier of Qi to run through the whole body,in other words,it is mutual for rooting of yin-yang, yang can’t change without yin, so it needs small dose angelica for the minister. The two herbs are taken together is a curative effect for the treatment of cardiovascular diseases.2ObjectiveBy means of antiing AS and inhibiting the apoptosis of myocardial cells from Danggui Buxue Decoction and its effective components (include astragaloside, ferulic acid, total saponins of Astragalus, volatile oil of the angelica)to investigate the effect of Danggui Buxue Decoction and its effective components. Finding out the phagocytosis of RAW264.7cells from the source of mice under the action of OX-LDL. Discussing the excretion of inflammatory factors like TNF-α and IL-1β, the protein expressions of TNF-a, ICAM-1, P38-MAPK, NF-kBP65; Studying the effects on cell survival rate of myocardial cells under oxygen stress state, the influence of mitochondrial apoptosis pathway related regulatory proteins with the expressions of Hsp-70, Bcl-2, Bax, Caspase-3. Get the main effective components of Danggui Buxue Decoction on antiing AS and inhibiting myocardial cell apoptosis to provide experimental basis for extracting effective ingredients of treatment in cardiovascular diseases, and also Danggui Buxue Decoction.3Methods3.1Effect of Danggui Buxue Decoction and its active ingredients on the effect of OX-LDL on RAW264.7cellsThe astragalus and angelica famous-region drug were according to the mass ratio of1:1,2:1,3:1,5:1to be boiled with soft fire and concentrated into1.04g/mL decoction containing crude drug.The blank serum group was gavaged with saline. Every group was with a dose of20mL/kg orally, daily two times, lasting for3days. In the morning of the fourth day, every group was given a dose of30mL/kg once, then took the orbital venous plexus blood after1h. After standing2-4hours, all the samples were taken to centrifuge with10000rpm for10mins, collected the supernatant. The standard of astragaloside IV and ferulic acid was purchased from Guangzhou food and Drug Inspection Institute. The total saponins of Astragalus was purchased from Shanghai Aladdin chemical company. The volatile oil was extracted by water. It was used Angelica500g and distilled water4000mL to boil6-8h. The extracted material was purified by centrifuge with10000rpm,10mins. Finally it was obtained volatile oil360mg, just sealed and preserved in4℃.The RAW264.7cells were cultured in DMEM high glucose medium with10%fetal bovine serum (FBS).The liquid was changed every other day.The cells were passaged every2days. The cells were cultivated with8000/mL density in96holes plate, synchronized24h in serum-free medium after overnight incubation. MTT method was used to detect the survival rate of RAW264.7cells with Danggui buxue decoction containing drug serum and its effective components. The foam cells were induced by50mg/LOX-LDL with RAW264.7cells48h.It was used oil red0staining method to detect the phagocytosis of OX-LDL on RAW264.7cells with Danggui buxue decoction containing drug serum and its effective components. Using OX-LDL effected on RAW264.7cells24h, ELISA method to detect the excretion of TNF-α and IL-1β by the effective components of Danggui buxue decoction. Using OX-LDL effected on RAW264.7cells24h, Western-blotting method to detect the expression of P38-MAPK, NF-kBP65, TNF-a, ICAM-1with Danggui buxue decoction containing drug serum and its effective components.3.2Effect of Danggui Buxue Decoction and active components on myocardial cells’apoptosis induced by H202The1-3d SD rats were disinfected with75%ethanol solution, opened chest to get the heart, then taken into the clean platform. The hearts were cut into lmm3with sterile sharp ophthalmic scissors. The cell suspension was collected by adding compound enzyme digestion (0.125mg/L trypsin and0. lmg/L collagenase type I) to digest again and again. The digestive enzyme was terminated by the medium containing serum. Then the liquid was centrifuged with1000rpm for10min to get cellular precipitation which was suspended with complete medium. The myocardial cells were separated and purified by90min differential adhension, and then cultured by the complete medium, in37℃,5%CO2conditions. The liquid was changed after48h, and the cells were used to the experiment after72h. The cardiomyocyte apoptosis was induced by150μmol/L H2O220h. MTT method was used to detect the survival rate of myocardial cells induced apoptosis by H202with Dangguibuxue Decoction containing drug serum and its effective components. Immunocytochemistry and Western-blotting methods were used to observe the effects of Danggui Buxue Decoction containing drug serum and its effective components on the expressions of cardiomyocytes apoptosis associated protein of Hsp-70, Caspase-3, Bcl-2, Bax.4Results4.1Effect of Danggui Buxue Decoction and its effective components on RAW264.7cells with OX-LDL4.1.1Control to the normal group, the blank serum group had no effect on cell proliferation(P>0.05), the serum containing Danggui Buxue Decoction with different proportions had effect on promoting apoptosis of RAW264.7cells(P<0.05or P<0.01), with a ratio of5:1on RAW264.7cell apoptosis is the most significant.The astragaloside IV concentration of100and200mg/L had no effect on promoting proliferation of RAW264.7cells(P>0.05),the concentration of300mg/L and above promoted apoptosis in RAW264.7cells significantly(P<0.05or P<0.01).The concentration of100and200mg/L of ferulic acid and the total saponins of Astragalus had an effect on promoting proliferation of RAW264.7cells(P<0.01), the concentration of300mg/L had no effect on promoting proliferation of RAW264.7cells(P>0.05), there was an effect on promoting apoptosis significantly(P<0.01).The volatile oil of the angelica on concentration of40and50mg/L had no effect on promoting proliferation of RAW264.7cells(P>0.05), the concentration of60mg/L and above promoted apoptosis in RAW264.7cells significantly(P<0.05or P<0.01).4.1.2It was observed that the cells were cultured with OX-LDL after48h could induce to foam cells even with50mg/L concentration, so the concentration was used to subsequent trials. From the Oil red0staining results, it was found that the different proportions of Danggui Buxue Decoction drug-containing serum groups were all enhanced the phagocytosis, and a significant degradation effect on the foam cells with the5:1group.The experiment was also observed that in different drugs of Astragaloside IV, ferulic acid, total saponins of Astragalus and essential oil of Angelica sinensis groups were enhanced the phagocytosis of OX-LDL.4.1.3In the ELISA test, it was found that the expression of IL-1β between model group and control group, Astragaloside IV group, ferulic acid group, Astragaloside IV with ferulic acid group, the total saponins of Astragalus and essential oil of Angelica sinensis groups were not difference(P>0.05), the model group was lower than the total saponins of Astragalus with essential oil of Angelica sinensis group. In the expression of TNF-a, it was no difference between the model group and the control group, Astragaloside IV group, ferulic acid group, Astragaloside IV with ferulic acid group (P>0.05). The model group was lower than the total saponins of Astragalus, essential oil of Angelica sinensis group, the total saponins of Astragalus with essential oil of Angelica sinensis group.4.1.4From the Western-blotting test, it was found that the control group and different proportions of Danggui Buxue Decoction were expressed higher than the model group in P38-MAPK, NF-kBP65, TNF-a and ICAM-1. Control to the model group, the expression of P38-MAPK, NF-kBP65, TNF-a and ICAM-1in ferulic acid and total saponins of Astragalus groups were lower, the expression of ICAM-1in Astragaloside IV, ferulic acid with Astragaloside IV and essentialoil of Angelica sinensis groups were lower, but all these groups were lower than normal group.4.2Effect of Danggui Buxue Decoction and its ingredients on the myocardial apoptosis induced by H2024.2.1It was observed that the cells were spherical and had good refraction in cell culture after just digested under the inverted microscope. The cells were rod-shaped adherent on the culture flask after24h and some were beating spontaneously. There were a lot of cells extended pseudopods to link together and beat rhythmically after cultured48h. When the cells were cultured72h, they were beat rhythmically and synchronously, and were gather into clusters which could be used to experiments.4.2.2It was found that the100and200μmol/L densities of H202could induce myocardial apoptosis significantly(P<0.01), the concentration of300μmol/L and above it were leaded to myocardial necrosis significantly(P<0.01) after the cells were cultured with different concentrations of H20220hours. Finally it was chosed150μmol/L for the latter tests after morphological observation and with the character of easyly to decompose when saw the light and heat.4.2.3In the test, different ratios of Danggui Buxue Decoction were improved the survival rate of myocardial cells (P<0.01) and significantly with the blank serum group(P<0.01). And the5:1group woked best.Different concentrations of Astragaloside IV, ferulic acid and total saponins of Astragalus had obvious protective effect in myocardial cells(P<0.05or P<0.01), and with the increase of drug concentration, the survival rate of myocardial cells increased as well. Different concentrations of essential oil of Angelica sinensis had no protective effect in myocardial cells and aggravated myocardial cells apoptosis. There were no difference in different concentrations.4.2.4The results from immunocytochemical method:compared with the normal control group, the expressions of myocardial cells in Hsp-70, Bcl-2were increased and Bax, Caspase-3proteins were significantly increased after150μmol/LH2O2treatment20h. Control to the model group, the Danggui Buxue Decoction(5:1) serum group could significantly promote the expression of Hsp-70, Bcl-2, decreased the expression of Caspase-3, Bax, and also with pretreatment of Astragaloside IV, ferulic acid and total saponins of Astragalus.4.2.5From the western-blotting results, it was showed that compared with the normal control group, the expression of myocardial cells in Caspase-3, Bax were increased significantly and Hsp-70is increased slightly, the expression of Bcl-2were decreased significantly. The Danggui Buxue Decoction drug-containing serum groups were increased the expression of Hsp-70, Bcl-2significantly and decreased the expression of Caspase-3, Bax compared with model group, and also with the drug pretreatment of Astragaloside IV, ferulic acid, Astragaloside IV and ferulic acid, total saponins of Astragalus, essential oil of Angelica sinensis (small dose),total saponins of Astragalus and essential oil of Angelica sinensis.5Conclusions5.1It was by reducing the number of macrophage cells and degradating the foam cells of Danggui Buxue Decoction serum for the treatment of atherosclerosis.5.2It was by promoting the apoptosis of macrophages of large dose of Astragaloside IV, ferulic acid, total saponins of Astragalus and essential oil of Angelica sinensis to play a role of resistance to the AS.5.3It was not by phagocyting OX-LDL of macrophagocytes from Danggui Buxue Decoction, Astragaloside IV, ferulic acid, total saponins of Astragalus and essential oil of Angelica sinensis to play a role of resistance to the AS.5.4It couldn’t think that Astragaloside IV, ferulic acid, total saponins of Astragalus and essential oil of Angelica sinensis play a role in resisting the AS by inhibiting the inflammatory factor expressions of TNF-α, IL-1β.5.5It couldn’t think that Danggui Buxue Decoction and its components play a role in resisting the AS by inhibiting the protein expressions of TNF-α、 ICAM-1、NF-Kbp65、P38-MAPK.5.6Danggui Buxue Decoction and Astragaloside IV, ferulic acid, total saponins of Astragalus have the effect of protecting myocardial cells by inhibiting the apoptosis of myocardial cells induced by H2O2.5.7It may be the mechanism of Danggui Buxue Decoction and Astragaloside IV, ferulic acid, total saponins of Astragalus for the apoptosis resistance by up regulating Hsp-70, Bcl-2expression and down regulating Caspase-3, Bax expression. So they play a role in the treatment of cardiovascular disease by preventing oxidative damage of myocardial cells.6innovation points6.1It is discussed Danggui Buxue Decoction’s rationality of compatibility of herbs via’Heart Yang deficiency’which is the main pathogenesis of heart.6.2The study is through the formation of atherosclerotic plaque-the impact of macrophage on the phagocytosis of OX-LDL to discuss the mechanism of action on antiing AS with Danggui Buxue Decoction and effective components.6.3At the same time to study the effective protection on myocardial cells with the action of Danggui Buxue Decoction against AS which has an effect on treatment and prevention of cardiovascular disease from multiple targets.7Prospects and shortageWith the development of modern biomedical technology and in-depth study on cardiovascular disease, it is expected to break this difficulty to improve people’s quality of life and reduce the economic burden of the people.The selection of experimental indexes in the experiment is limited, but it may play a role in treatment by other means with the Chinese medicine’s characteristic which has a multi-targets, multi-channels. It is need to study further in-depth. |
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