Effects Of XinMaiTong On Expressing Of IL-6,AngⅡ And Its Receptor In Cardiocytes Harmed By Overoxidation

Objective:Harmed cardiopathy is one of the most serious illnesses of humans. OFR(oxgen free radical) isproduced when myocardial ischemia or ischemia -reperfusion occurs, the injury of OFR is the principle mechanism of disease caused by myocardial ischemia or ischemia-reperfusion. A compound recipe XinMaiTong composed of traditional Chinese medicine has an effect on supplying Qi and nourishing blood, enhancing blood circulation and removing stasis. It is proved that XinMaiTong has an effect against injury of myocardial ischemia clinically and experimentally. In this study, a model of analogous injury by OFR produced in the case of myocardial ischemia induced by using of H₂O₂ was made. An effect of XinMaiTong containing serum on heart injury by myocardial ischemia and related possible mechanism have been studied at cellular and molecular level by detection of expressing of IL-6, Angâ…¡and AT₁ in cardiocytes under the injury caused by H₂O₂.Methods:1. Choose a best time of H₂O₂ working and researching the XinMaiTong-medicated mouse serum's effect agaist injury.Pour the traditional Chinese medicine compound recipe XinMaiTong solution into the mouse stomachus, withdrawal the blood from abdominal aorta of mouse, make herb-medicated mouse serum.Prepare cardiocytes of neonate rats by enzyme digestion, depurating cardiocytes by adherence of unequal velocity. Put cardiocytes into the 96 wells culture plates on 1.5×10⁴/ml, cardiocytes were randomly divided into 3 groups: control group, model group, XinMaiTong group. 6 wells were allocated to each group. On the third day, control group and model group were cultured by DMEM containing 15% normal mouse serum for 24h; XinMaiTong group was cultured by DMEM containing 15% herb-medicated mouse serum for 24h. ARer 24h, put H₂O₂ into each well (the final concentration of H₂O₂ was 400umol/L). After 1h, 3h, 6h the working time of H₂O₂, detect the figure of MTT.2. Staining by ICC(immunocytochemistry) and detecting and comparing the expression of IL-6, Angâ…¡, AT₁ by image processing and analysis systemCulture cardiocytes on the way experiment 1, put cadiocytes cardiocytes into 6 wells culture plates (coverslips have been put into the well previeously) to make cardiocytes adhere the coverslip. On the third day, cardiocytes were divided to 4 groups randomly, control group; model group; XinMaiTong's preadministration group; XinMaiTong's treating group. 3 wells were allocated to each group. Control group: culture with DMEM containing 15% normal mouse serum were put into wells for 24h, then thay were changed to be DMEM without serum, which took 6hs. Model group: culture with DMEM containing 15% normal mouse serum were put into wells for 24h, then thay were changed to be DMEM without serum and put H₂O₂ into wells (the final concentration of H₂O₂ was 400umol/L) which took 6h. XinMaiTong's preadministration group: culture with DMEM containing 15% herb-medicated mouse serum were put into wells for 24h, then thay were changed to be DMEM without serum, put H₂O₂ into wells (the final concentration of H₂O₂ was 400umol/L) which take 6h. XinMaiTong's treating group: culture with DMEM without serum and H₂O₂ were put into wells(the final concentration of H₂O₂ is 400umol/L) which take 6h. Then, change culture with DMEM containing 15% herb-medicated mouse serum and culture cells for 24h. At the same time, all 4 groups were taken out from culturing box and stained by ICC ways. Block the coverslips and take pictures, 20 pictures were taken for each sample and detect its greylevel, extract 10 pictures sandomly to compute their mean which was taken as the greylevel of one sample. Compare different expressing of IL-6, Angâ…¡, AT₁ in different groups through comparing their greylevel.Results:1. Choose a best time of H₂O₂ working and researching the XinMaiTong-medicated mouse serum's effect agaist injury of H₂O₂.â‘ . Injury effect of H₂O₂: when the time of H₂O₂ working was 1h, the changs of cadiocytes was not obvious, their beating rate descend. When the time extend, the pseudopodium shrink, beating rate descend and even beating disappear, connection between cells break, the refractive index decrease, all these occur obviously when the H₂O₂ working time extend. These hint the injury effect of H₂O₂ increase when it work longer.â‘¡. The best time of H₂O₂ working: When the time of H₂O₂ working waslh, the different OD figure of each group was (0.234±0.0117, 0.215±0.0120, 0.22±0.0170). The difference between control group and other groups through comparing had not statistic meaning(P＞0.05), suggesting cadiocytes were harmed lightly by H₂O₂, normal cadiocytes could resist and repair the injury. When the time of H₂O₂ working was 3h(0.204±0.0341, 0.165±0.0120, 0.154±0.0170), The difference between control group and other groups through comparing had statistic meaning(P＜0.05), these hint that 3h working time of H₂O₂ could make a obvious injury result, but at the same time, the difference between model group and other groups using herbs through comparing had not statistic meaning, these hint that resisting and repairing injury effect of herbs to 3h working time of H₂O₂ was not different from them of cadiocytes themselves. When the time of H₂O₂ working was 6h, the different OD figure of each group was(0.238±0.025, 0.177±0.008, 0.210±0.032,). The difference between control group and other groups through comparing had statistic meaning(P＜0.05), the figure of control group was higher than it of others; The difference between model group and other groups through comparing had statistic meaning(P＜0.05), the figure of model group was-lower than others, these hint that the result of injury effect of H₂O₂ and resisting injury effect of herbs for 6hs all were obvious—6h were sure to be the best time of H₂O₂ working.2. Staining by ICC(immunocytochemistry) and detecting and comparing the expression of IL-6, Angâ…¡, AT₁ by image processing and analysis system.â‘ . Expressing of IL-6 in the different groups: The pictures of control group showed there were not brown part in pictures which hint that normal cardiocytes don't express IL-6. The statistic handling showed he difference between control group and other groups had statistic meaning (P＜0.05), control group's figure of greylevel (160.96±1.144)was obviously higher than others(102.21±3.509, 116.99±1.286, 111.62±1.086), suggesting IL-6 express strongly in harmed cardiocytes by OFR producing from H₂O₂ working. The difference between model group and other groups through comparing had statistic meaning (P＜0.05), the greylevel of model group(102.21±3.509)was lower than groups of using herbs, suggesting XinMaiTong had the effect against injury caused by OFR producing from H₂O₂ working, their mechnism could be reducing or inhibiting the expressing of IL-6 in the harmed cardiocytes. The difference between XinMaiTong's preadministration and XinMaiTong's treating group, had statistic meaning (P=0.005,＜0.05), greylevel of XinMaiTong's preadministration was higher than XinMaiTong's treating group(111.62±1.086), suggesting: about reducing or inhibiting the expressing of IL-6 in the harmed cardiocytes, the way preadministration was better than the way treating after injury occurs.â‘¡. Expressing of Angâ…¡in the different groups: The pictures of control group show there were brown parts in pictures, which hints that normal cardiocytes express Angâ…¡, too. The statistic handling show the difference between control group and other groups had statistic meaning (P＜0.05), control group's figure of greylevel (111.279±1.369) was obviously higher than others(90.55±1.526, 107.92±1.732,97.35±1.438)which hinted that Angâ…¡express strongly in ischeminc cardiocytes by OFR producing from H₂O₂ working. The difference between model group and other using herbs groups through comparing have statistic meaning (P＜0.05), the greylevel of model group(90.55±1.526)is lower than groups of using herbs, which hinted that through reducing or inhibiting the expressing of Angâ…¡in harmed cardiocytes, do the two herbs resist and repair injury caused by OFR producing from H₂O₂ working. The difference between XinMaiTong's preadministration and XinMaiTong's treating group, had statistic meaning (P=0.000,＜0.05), greylevel of XinMaiTong's preadministration(107.92±1.732) was higher than XinMaiTong's treating group(97.35±1.438), which hinted: about reducing or inhibiting expressing of Angâ…¡in the harmed cadiocytes, the way preadministration was better than the way treating after injury occurs.â‘¢. Expressing of ATlin the different groups: The pictures of control group showed there were brown parts in pictures which hints that normal cardiocytes express AT₁, too. The statistic handling showed the difference between control group and other groups had statistic meaning (P＜0.05), control group's figure of greylevel (106.09±4.051) was obviously higher than others (83.57±2.518, 94.93±0.369, 88.09±a:0.370) which hinted that AT₁ express strongly in harmed cadiocytes by OFR producing from H₂O₂ working. The difference between model group and other using herbs groups through comparing had statistic meaning(P＜0.05), the greylevel of model group(83.57±2.518)was lower than groups of using herbs, which hinted that through reducing or inhibiting the expressing of AT₁ in harmed cadiocytes, do the two herbs resist and repair injury caused by OFR producing from H₂O₂ working. The difference between XinMaiTong's preadministration and XinMaiTong's treating group had statistic meanings, greylevels of XinMaiTong's preadministration (94.93±0.369) was higher than XinMaiTong's treating group, which hints: about reducing or inhibiting expressing of AT₁in the harmed cardiocytes, the way preadministration was better than the way treating after injury occurs.Conclusion:The injury caused by OFR such as H₂O₂ working could evoke or promote expressing of IL-6, Angâ…¡, AT₁ in the cardiocytes. Chinese medicine herbs compound recipe XinMaiTong-medicated mouse serum have the effect against injury of cardiocytes caused by OFR such as H₂O₂ working, its mechnism could relate to reducing or inhibiting the expressing of IL-6, Angâ…¡, AT₁ in the cardiocytes.