| Part1:Experimental StudyObjectiveTo observe the macrophage apoptosis and related signaling pathway in AS, and to observe the effect of baicalin on the macrophage apoptosis and related signaling pathway in AS, in order to provide theoretical basis for the use of heat-clearing and detoxifying method in AS related diseases.MethodsAfter7days adaptive feed,48New Zealand white rabbits were randomly divided into six groups:normal control group, high-cholesterol control group, high-cholesterol+endotoxin (LPS) model group, low-dose baicalin group, high-dose baicalin group and simvastatin group. Normal control group were fed with normal forage, other groups were fed with high-cholesterol forage, both for16weeks. High-cholesterol+LPS model, low-dose baicalin group, high-dose baicalin group and simvastatin group were injected LPS intramuscularly near the right ear artery, carotid artery and shallow.artery at week4,8and12. Baicalin low-dose and high-dose group were fed with50mg/kg/d and100mg/kg/d baicalin respectively after the first injection of LPS (5ml distilled water to dissolve). Simvastatin group were fed with1mg/kg/d of simvastatin lavage (5ml of distilled water to dissolve) after the first injection of LPS, Normal control group, high-cholesterol control group and high-cholesterol+LPS model group were fed with5ml of distilled water once per day, for12weeks. All the animals were euthanasia at week16and we detected the serum levels of lipid (TC, TG, LDL-C, HDL-C)(biochemical method), detected the serum level of LPS (limulus reagent), detected serum level of CRP, IL-1(ELISA). We also observed aorta organization form (HE staining), detected the protein expression of CD14, CHOP and JNK in aortic plaques (immunohistochemical method), and detected the mRNA expression of p38MAPK, JNK, SR-A, Bcl-2, Bax and Caspase3in aorta (fluorescence quantitative RT-PCR).ResultsThe serum levels of lipid in all high-cholesterol fed rabbits increased obviously, especially the TC and LCL-C levels, baicalin and simvastatin can significantly reduce the serum levels of TC and LCL-C. Repeated muscle injection of LPS can rise rabbit serum level of LPS, baicalin may have the effect on reduce the serum level of LPS. High cholesterol diet can rise serum level of inflammatory factors, CRP and IL-1, repeatedly LPS intramuscular injection can rise CRP levels, and baicalin and simvastatin can reduce serum levels of CRP and IL-1. HE staining show that aorta tissue structure was normal in normal control group, many plaques were found in aortic intima in high-cholesterol diet groups, and the aortic intima is local thickening and is not smooth, and lots of endothelial cells were falls off and discontinuous, and there were lots of foam cells deposited in plaques, ulcers on plaque surface, and the nucleus sizes of foam cells were not the same. In addition to above changes, there were more foam cells were deposited in intima plaques in cholesterol+LPS model group, and nucleus fragmentation of foam cells were observed. There were more relatively intact, continuous endothelial cells on the surface of plaques in baicalin high and low dose group and the size of foam cell nucleus were the same. There were less foam cells were observed in plaques in high-dose baicalin group and simvastatin group. Immunohistochemical tests showed that CD14, CHOP and JNK protein mainly expressed in macrophages within the aortic plaques,’and little in smooth muscle cells, high-cholesterol diet can enhance the expression of CD14, CHOP and JNK, and repeatedly LPS intramuscular injection can enhance the expression of CD14, high dose of baicalin can inhibit the expression of CHOP. Fluorescence quantitative RT-PCR test showed that the expression of aorta Caspase3, p38MAPK, JNK, SR-A and Bax mRNA were enhanced and the expression of aorta Bcl-2mRNA were inhibited in all the cholesterol-feed groups compared with normal control group. Repeated intramuscular injection of LPS can further enhance p38MAPK and JNK mRNA expression, baicalin can inhibit the expression of Caspase3, p38MAPK, JNK, SR-A and Bax mRNA and enhance the expression of Bcl-2mRNA, simvastatin can inhibit the expression of Caspase3, p38MAPK, JNK and Bax mRNA.Conelusion High cholesterol diet can lead to rabbit hyperlipidemia, promote AS process, and repeated intramuscular injection of LPS may increase the AS plaques formation and instability, however, baicalin and simvastatin may reduce the AS plaque formation and instability. High-cholesterol diet can increase macrophage apoptosis in aortic plaques, baicalin and simvastatin can reduce macrophage apoptosis in aortic plaques. High-cholesterol diet may induce macrophage apoptosis by activating endoplasmic reticulum stress and unfolded protein reaction, thus activating JNK—Bcl-2/Bax—Caspase-3pathways and p38-CHOP—Bcl-2/Bax—Caspase-3pathway and enhance the SR-A express. Baicalin may reduce macrophage apoptosis by inhibiting endoplasmic reticulum stress, thus inhibiting JNK—Bcl-2/Bax—Caspase-3pathways and p38-CHOP—Bcl-2/Bax—Caspase-3pathway and inhibiting the SR-A express. Simvastatin may reduced plaque macrophage apoptosis by inhibiting JNK—Bax—Caspase-3pathways and p38—Bax—Caspase-3pathway.Part2: Glinical StudyObjectiveTo investigate the clinical curative effect and safety of heat-clearing-detoxifying method, on the basis of conventional western medicine therapy and activating-blood method, in patients with acute coronary syndrome (ACS).Methods94cases of hospitalized patients with ACS and incorporated with the standard of toxic heat and blood stasis syndrome differentiation, were randomly divided into three groups using randomized controlled methods: western medicine group, huoxue group and qingrejiedu huoxue group. Western medicine group were treated with routine western medicine, huoxue group were treated with conventional western medicine combined Guanxin II decoction, qingrejiedu huoxue group were treated with conventional western medicine combined qingre-huoxue decoction, and the course is1week. TCM syndrome score, TCM syndrome curative effect, serum level of lipoprotein associated phospholipase A2(LP-PLA2) and safety index. were observed before and after treatment.ResultsBefore treatment, obvious heat symptom were observed in each groups ACS patients, and the score of irritated heat, bitter mouth, yellow short urine, stem stool and yellow tongue coating symptoms were high before treatment. After treatment, the symptoms of chest pain and chest tightness in huoxue group and qingrejiedu huoxue group were improved compared with western medicine group (P<0.01), furthermore, the effect in qingrejiedu huoxue group was better than huoxue group (P<0.05). The symptoms of palpitations, shortness of breath, fatigue, chills were improved in huoxue group and qingrejiedu huoxue group compared with western medicine group (P<0.01), but there is not difference between huoxue group and qingrejiedu huoxue group. The symptoms of irritated heat, bitter mouth, yellow short urine, stem stool and yellow tongue coating symptoms were improved in qingrejiedu huoxue group compared with western medicine group and huoxue group (P<0.01and P<0.05). Total TCM syndrome score in qingrejiedu huoxue group was lower than in western medicine group and huoxue group (P<0.01), and TCM syndrome curative effect in qingrejiedu huoxue group was better than in western medicine group (P<0.05). Serum levels of LP-PLA2in qingrejiedu huoxue group were lower than in western medicine group and huoxue group (P<0.01and P<0.05). There were not obvious adverse reactions were observed in three groups.ConclusionToxic heat symptoms are obvious in patients with ACS. On the basis of western medicine and activating blood method, combine use of heat-clearing-detoxifying method can further improve TCM symptoms, especially the symptoms of irritated heat, bitter mouth, yellow short urine, stem stool and yellow tongue coating symptoms, in patients with ACS. What is most importtent, combine use of heat-clearing-detoxifying method will not the symptoms of shortness of breath, fatigue and chills. On the basis of western medicine and activating blood method, combine use of heat-clearing-detoxifying method can further reduce the serum level of LP-PLA2, which indicate that heat-clearing-detoxifying method may reduced plaque macrophages apoptosis and Improving the prognosis of ACS. In a word, on the basis of western medicine and activating blood method, combine use of heat-clearing-detoxifying method is effective and safe. |
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