Explolation Of The Effects And Mechanisms Of Musca Domestica Anti-atherosclerotic

Introduction:Atherosclerosis (AS) is one of the most direct cause of cardiovascular and cerebrovascular disease, its pathogenesis are mainly involved in lipid metabolism disorders and lipid infiltration, oxidative damage, inflammation injury and other theories, but the exact etiology and pathogenesis has not yet been fully elucidated. In further studies, the occurrence and development of AS is the dynamic response of the body such as vascular endothelial injury produced a range of chronic inflammation. The pathological changes including the classic inflammatory response of tissue degeneration, necrosis, mononuclear macrophages/inflammatory cells.Therefore, anti-inflammatory and immunomodulatory have became a new way in treatment of AS. Vascular endothelial cell is an important component of the immune system. There was a direct impact on the growth, proliferation and migration of immune response in the microenvironment of the distribution and orientation. This suggests that inflammatory and immune throughout the the whole process when AS occurrence and development. Therefore, inflammation and immune regulation has become the new direction in treatment of AS.The Musca domestica known as the maggots, also called the grain insects, is one of the traditional Chinese medicinal materials, which have the functions of clearing heat and phlegm, strengthening the spleen and stomach and so on. And also be used for the treatment of impetigo, boil and bacterial infections in clinic. The secretions of Musca domestica could inhibit human mononuclear cells expression of inflammatory factor released and induced differentiation of monocytes into macrophages in vitro and slso could promote the healing of bedsores and skin transplant patients through enhance the body’s immune system. This suggests that Musca domestica may be rich in regulating inflammatory immune active substances. We obtained the protein extracts of Musca domestica through tissue homogenate and high-speed centrifugal. The protein extracts (PE) could reduce the levels of blood lipids and inflammatory factors both in rat and mouse, and suppress the occurrence and development of AS. When macrophage was induced by bacterial lipopolysaccharide (LPS), the relase of inflammatory factor has been significant inhibited by PE.However, the composition of the PE and the specific mechanism of anti-AS are unclear. So this paper focused on these two issues further depth. Designed to provide a theoretical basis for new drugs from traditional Chinese medicine Musca domestica in order to develop an effective treatment of AS.Methods:1. Musca domestica anti-AS screening in vitro and efficacy of the active site verification in vivo(1) Preparation and separation of PEWe got Musca domestica third instar larvaes by captivity and enriched the active ingredient through hot and cold stimulation. After cleaning, disinfection, high-speed tissue homogenates, filtration, sonication, low-temperature high-speed centrifugation, vacuum freeze-drying and Soxhlet defattedget we got PE. The PE was separated by dextran filtration gel chromatography further, and merged into different fractions according to molecular size. (2) Screening of the Musca domestica anti-AS active site in vitroThe inflammatory and immune regulation activity was screened in vitro through LPS-induced macrophage platform. The expression and releasing of inflammatory cytokines and macrophage activation were detected. The most effective part (sample X) was found by this way.(3) Verify the efficacy of anti-AS active site of Musca domestica in vivoMouse atherosclerosis model by established by LPS intramuscular injection and high cholesterol diet.The effectiveness of the anti-atherosclerosis in animals was verified. TC, TG and LDL and HDL, TNFa and IL-1α levels were test. The animal’s liver, heart and thoracic aorta were taken, and the pathological changes were observed by HE staining. After determined the model was constructed successfully, in vivo validation experiments were carried out. The detection of animal serum lipids and inflammatory factors were carried out. The animal’s liver, heart and thoracic aorta were taken, and the pathological changes were observed by HE staining in order observe the pathology situation of cardiac and thoracic aortic.(4) Clear the molecular weight and composition of effective partThe molecular weight and composition of the effective part was test by Tris-tricine-SDS-PAGE.2. Musca domestica inflammation regulation mechanismThe LPS-induced macrophage was experimental subjects. The nuclear transfer of p65was test by immunofluorescence techniques combined with fluorescence microscopy and laser scanning confocal microscope. The expression of p65protein was test by western blotting and RT-PCR to clear the influence of Musca domestica on NF-κB signaling pathway.3. Immune regulation mechanism of Musca domestica (1) Effect of Musca domestica on the interaction of macrophages and endothelial cellsThe human macrophages THP-1were induced by phorbol and co-cultured with LPS for different times (4h and24h). Then mixed culture and Transwell chamber separated culture with endothelial cells ECV304. Cell cycle and apoptosis were test by flow cytometry and cell migration wsa test by cell wound healing assay and Transwell assay chemotactic. The impact of Musca domestica on the growth, proliferation and migration of endothelial cell which co-cultured with macrophages was detected. The macrophage invasion was test by immunofluorescence in the cardiac and thoracic aortic plaque in the mouse atherosclerosis model.(2) Effect of the Musca domestica on growth, proliferation and migration of endothelial cell induced by TNFaEndothelial cell was co-cocultured with TNFa.The effect of Musca domestica on cell cycle and apoptosis were test by flow cytometry and cell migration was test by cell scratch assay.4. Date statistical analysisDate statistical analysis was carried out by the software SPSS13.0. Comparison between two sets of data, the independent samples t test was used. Comparison between multiple sets of data, the one-way anova test was used. When the p value bigger than0.05, the LSD method was used, and when the p value smaller than0.05the Dunnett T3method was used. P<0.05indicated statistical significance.Results:1. Musca domestica anti-AS screening in vitro and efficacy of the active site verification in vivoPE was isolated by dextran gel filtration chromatography and combined three main components based on the molecular weight range which were named as sample 1. sample2and sample3. Relatively samples1and2, sample3had more effectively reduce TNFα and MCP-1levels in the cell culture supernatant which induced by LPS in macrophage and also had the function of reducing the proportion of activated macrophages. Mouse atherosclerosis model could be successfully established by intramuscular injection of LPS combined with high cholesterol solution gavage treatment. In vivo validation experiment results showed that sample3can effectively reduce the TC, TG and LDL and TNFa and IL-1α, and increased HDL levels and improve the structure of myocardial cells and inhibited the atherosclerotic lesions of arteries. Further Tris-tricine-SDS-PAGE vertical electrophoresis results showed that the molecular weight range of sample3is mainly about concentrated in the4.1KD.2. Effect of Musca domestica on NF-κB signaling pathwaysSample3could effectively inhibit p65nuclear transfer. Western Blotting results showed that the sample3could reduce p65expression in the cytoplasm and the nucleus. RT-PCR results showed that p65anti-transcription level decreased significantly in sample3treatment group. So we inferred that the sample3may inhibit the inflammatory response by blocking NF-κB signaling pathway.3. Effect of Musca domestica on endothelial cell growth and proliferation co-cultured with LPS-induced macrophages.The flow cytometry results showed that the endothelial cells co-cultured with macrophages which induced by LPS4h and24h after treated with sample3for24h, the proportion of S phase cells reduced. And the S phase of endothelial cells which induced4h by LPS was decreased significantly (P<0.01). Compared to negative control group, sample3could reduce the proportion of endothelial cell apoptosis (P <0.01). And the proportion of apoptotic cells which treated for24h cells were increased. 4. Effect of Musca domestica on endothelial cell migration co-cultured with LPS-induced macrophages.The cell scratch experimental results showed that after separated co-cultured24h with macrophages which induced by LPS for4h, significant migration occurs. The sample3could significantly inhibit cell migration. And the rate of migration after separated co-cultured24h with macrophages which induced by LPS for24h induced compared to the LPS-induced macrophages4h group significantly reduced, and there were almost no migration in sample3treated group. Transwell results showed that the endothelial cells induced by LPS in macrophages for4h co-culture, and the sample3can significantly inhibit endothelial cell migration. The migration rate was reduced in group of LPS-induced co-cultured24h macrophages group.5. Effect of Musca domestica on endothelial cell growth, proliferation, and migration induced by TNFa.The cell scratches testing showed that the apparent of endothelial cells ECV304which induced by TNF a migration was changed. Compared with the normal control group and the negative control group cells appeard many pseudopods shape structure, and the mobility increased significantly. The migration in sample3processing group was restrained.Conclusions:The PE was treated by further glucan filter gel chromatography separation, three main samples were own. Anti-inflammatory activity screening was carried out by LPS-induced macrophages, and the sample3was found as the most effective ingredients. Mouse atherosclerosis model were constructed by LPS muscle injection and high cholesterol diet. After treatment, sample3could reduce the serum lipid and inflammation factors levels and improving heart and thoracic aorta pathology. Sample3had the function of inhibiting the expression and nuclear transfer of p65protein, further more influenced the NF-kappa B signal pathway. When the endothelial cell was co-cultured with macrophage which induced by4and24hours, there were different effects on late apoptosis and proliferation. In4h treatment group, the S-phase cells were increased, and the late apoptosis were more significant. When the endothelial cells were mixed co-cultured with macrophages, the influences were more significant. Animal experiments showed that the samples could effectively inhibit macrophage invasion to the myocardial tissue and arterial. Sample3had good inhibition of endothelial cell proliferation, apoptosis and migration which induced by TNFa. A further study found, and its mechanism may be reducing the downstream expression of inflammatory factors through inhibition of NF-κB signaling pathway and regulation of the mutual relationship between macrophages and endothelial cells and regulation of endothelial cell growth, protectionmembrane integrity.