| ObjectiveCoronary heart disease (CHD) is one kind of chronic diseases with the highest human fatality rate, which is a common type caused by atherosclerosis. With the rapid development of social economic and the changing of lifestyles, the morbidity of CHD increased continuously with the average onset age decreased gradually. Control the incidence of cardiovascular events and mitigating the harm has become a major public health issues. Lipid metabolism disorders is the basis of CHD and is closely related to the onset risk. Although methods of surgeries and medication can, to some extent, reduce the incidence of coronary events, residual cardiovascular risk still persists and side effects cannot be ignored. CHD associated with elevated blood lipids is attributable to "CHD phlegm retention with blood stagnation syndrome", which is a common syndrome of coronary heart disease. Although a lot of Chinese patent medicine aimed at CHD are being used currently, there are less medicine focused on "CHD phlegm retention with blood stagnation syndrome" with proofed mechanism evidence. Huoxue Huatan prescription is an experiential effective recipe based on years of clinical practice, with exact effect for the treatment. In this study, via the establishment of hyperlipidemic rats myocardial ischemia-reperfusion injury model, role of heart protection and inflammatory mechanism of Huoxue Huatan formula was observed in order to provide data support for the control of "CHD phlegm retention with blood stagnation syndrome"treatments.Method1.Theoretical researchBased on the historic evolution and modern researches of relative theories, "CHD phlegm retention with blood stagnation syndrome" is confirmed to be related to certain pathogenesis such as invasion of pathogenic cold, improper diet, moodiness, tiredess, weakness with the age, etc. Clinical syndromes include chocking feeling or pain in the chest, shortness of breath and palpitation, obesity, hyperlipidemia, physical fatigue, profuse phlegm with sweat taste in the mouth, tiredness, irritability and insomnia, slimy and greasy fur or with ecchymosis, choppy or slippery or wiry pulse. Based on the insufficiency of medicine focused on "CHD phlegm retention with blood stagnation syndrome", Huoxue Huatan formula was designed to activate blood circulation to remove blood stasis and resolving phlegm to relieving chest stuffiness, thus is suitable for "CHD phlegm retention with blood stagnation syndrome".2. Experimental research(1)210 male wistar rats(120-140g) were randomly divided into 7 groups:general diet model group, hyperlipid diet-sham group, hyperlipid diet model group, rosuvastatin group, high dose group, middle dose group and low dose group. After 7 weeks of hyperlipid diet feeding, blood was collected from eye socket and the blood lipid was tested. After 7 days of medicine given by intragastric administration(general diet model group, hyperlipid diet-sham group were give 0.25% CMC; rosuvastatin group was given rosuvastatin; Huoxue huatan prescription groups were give individual dose of huoxue huatan suspension), MIRI moedel was established. EF values were recorded before, immediately after and 24h after the reperfusion. AT the same time spot, myocardial infarct size was calculated via Evans-TTC blue staining. Hs-CRP、IL-1β、IL-10、IL-18、TNF-αI、CAM-1、VCAM-1 were tested via ELISA, the best dose was determined then.(2) 80 male wistar rats was used to establish the same hyperlipidemia-MIRI model.2h after the reperfusion, heart was cut for HE dyeing. Protein expression level of TLR4、NF- κBP56、IL-1β、IL-18、NLRP3 were detected via WB, mRNA expression level of TLR4、 NF-κBP56、NLRP3、microRNA-223 were detected via qt-PCR.Result1.Pharmacodynamic effects of Huoxue Huatan formula(1)myocardial infarct size:percentage of infarct size of hyperlipid diet model group is higher than other groups, with highly significant difference with rosuvastatin group and high dose group (P<0.01), significant difference with low dose group and general diet model group (P<0.05) and no significant difference with middle dose group (P>0.05). Percentage of infarct size of high dose group is lower than low dose group (P>0.05) and middle dose group (P<0.05)(2)EF value:There is no differences among groups before surgery. EF value of hyperlipid diet model is lower than other groups immediately after reperfusion, with significant difference with lower dose group (P<0.05), highly significant difference with hyperlipid diet sham group (P<0.01) and no significant difference with other groups (P>0.05). EF value of hyperlipid diet model is lower than other groups 24h after reperfusion, with significant difference with middle dose group (P<0.05) and highly significant with other groups (P<0.01).High dose group was better than middle and low dose group (P>0.05)(3) hs-CRP:hs-CRP value of hyperlipid diet group is higher than other groups, with highly significant difference with high and low dose group (P<0.01). High dose group is better than rosuvastatin、middle and low dose group (P>0.05)2. Effects of Huoxue Huatan formula on inflammatory factors and cells.(1) Interleukin:IL-10 level of hyperlipid diet group is lower than other groups, with highly significant difference with rosovastatin and high dose group (P<0.01). IL-1β、IL-18 level of hyperlipid diet group is higher than other groups, with highly significant difference or significant difference with rosovastatin and high dose group. There is no significant difference between rosovastatin and high dose group (P>0.05). High dose group is the best group while middle dose group is the worst.(2) TNF-a:TNF-a level of hyperlipid diet group is higher than other groups, with highly significant difference with general model、hyperlipid diet sham、rosovastatin and high dose group (P<0.01). high dose group is worse than rosovastatin (P<0.05) but better than middle and lower dose group (P<0.05)(3)CAM:Both ICAM-1、VCAM-1 level of hyperlipid diet group is higher than other groups, which has highly significant difference in ICAM-1 level between hyperlipid diet group with general model、hyperlipid diet sham and rosovastatin (P<0.01), highly significant difference in VC AM-1 level between hyperlipid diet group with general diet model-. hyperlipid diet sham、rosovastatin and high dose group (P<0.01) and significant difference in VCAM-1 level between hyperlipid diet group with low dose model. There is no significant difference between osovastatin and high dose group (P>0.05). High dose group is better than middle and low dose group.(4) HE dyeing:High dose group and rosuvastatin group are both better than hyperlipid diet group in myocardial fibers arrangement, myocyte necrosis, karyolysis, changing of cell configuration and leukocyte infiltration.3. Effects of Huoxue Huatan formula on miR-223/ppr inflammatory pathways(1)TLR4/NF-κB pathway: Protein and mRNA expression level of NF-κBp56、TLR4 of Huoxue Huatan formula group is highly significantly lower than hyperlipid diet group (P<0.01), NF-κBp56 protein expression level of Huoxue Huatan formula group is lower than rosuvastatin group while TLR4 protein expression level is higher (P>0.05)(2) NLRP3 pathway:NLRP3 protein and mRNA expression level of hyperlipid diet group is higher than other groups, with significant difference with rosuvastatin group and Huoxue Huatan formula group (P<0.05).There is no significant difference between rosuvastatin group and Huoxue Huatan formula group (P>0.05). IL-1、IL-18 protein expression level of hyperlipid diet group is higher than other groups, with significant difference or highly significant difference. IL-1β protein expression level of Huoxue Huatan formula group is higher than rosuvastatin group (P<0.05) while IL-18 shows the opposite (P>0.05)(3) miR-223 expression level:miR-223 expression level of hyperlipid diet group is higher than other groups, with highly significant difference with Huoxue Huatan formula-, general diet model and hyperlipid diet sham group (P<0.01), also with significant difference with rosuvastatin group (P<0.05). Expression level of Huoxue Huatan formula group is lower than rosuvastatin group (P>0.05)Conclusion1. Huoxue Huatan formula which was designed according to the theory of remove blood stasis and resolving phlegm and combination of symptoms and syndromes could activate blood circulation to remove blood stasis and resolving phlegm to relieving chest stuffiness, thus is suitable for "CHD phlegm retention with blood stagnation syndrome".2. hyperlipidemia disease could reduce cardiac function, increase the percentage of infarct size, myocardial cell degeneration, aggravate inflammatory cells infiltration, increase levels of inflammatory cytokines, increase the gene level and related PPRs, thus exacerbate inflammation injury of MIRI, objectively indicates the essence of "CHD phlegm retention with blood stagnation syndrome".3. Huoxue Huatan formula could effectively reduce the percentage of infarct size, improve cardiac function of hyperlipidemia-MIRI rat model, decrease level of inflammatory parameter, alleviate myocardial cell degeneration, thus exert obvious protective effect on myocardial ischemia.4. Huoxue Huatan formula could play its role effectively in inflammatory cells infiltration, improve IL level, TNF, CAM and other inflammatory factor levels. Then could play a role in the inflammation of the protection by improving the level of gene expression level and the inflammasome and other PPR pathway.5. Chinese compound formula based on the combination of symptoms and syndromes theory could play its role through multiple pathways via multiple targets, which fully reflects the characteristics and advantages of Chinese medicine. |
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