| Atherosclerosis (AS) is a common type of cardiovascular disease, refers to the artery and its branches in the artery wall intima and intimal lipid calm under (mainly cholesterol and cholesterol esters), accompanied by the middle smooth muscle cell migration to the intima proliferation, intimal thickening, the formation of yellow or greyish yellow patch like a porridge-like substance. Atherosclerosis is the No. 1 cause of death in developed countries, is the most important human health hazards of disease, disability and death of human primary reasons. In China, with the increasing standard of living and population aging, hyperlipidemia, and consequent cardiovascular disease showed a yearly increase in morbidity and mortality and morbidity "younger" trend, this country and the people suffered a tremendous loss of life and property, to health care resources has brought about tremendous burden. Although the current medical standards and technology to be developed, but it has yet to find effective drugs to control these diseases, only as a secondary means of prevention. Since prevention is a long-term chronic process, so the drug should be used without side effects. Currently, a number of widely used clinical anti-atherosclerosis drugs such as simvastatin, probucol in the prevention and treatment of diseases such as occurred during the toxic side effects can not be ignored, and its long-term clinical efficacy in patients has gradually proved to be not significant. Therefore, the search for natural, non-toxic and effective drug to inhibit atherosclerosis in the genesis and development of clinical pharmacy has been a hot spot.Housefly maggot(HM) with a wide range of uses, it can reduce cholesterol level, improve the immune capacity, control diabetes, reduce blood pressure, etc. According to preliminary experiments in our laboratory data show that Housefly maggo(tHM) to reduce the SD rats with blood, the prevention of atherosclerosis hardening effect. From the last century to the present, the country has more universities; research institutes conducted Housefly maggot(HM)breeding research and development. Research can be divided into two types: one is the development of antimicrobial peptide, chitin, etc., for human health food, pharmaceutical products; and those that deal with livestock manure using Housefly maggot(HM)to get high-quality organic fertilizer and protein feed. But the study of anti-bot role of atherosclerosis and abroad is extremely rare. Therefore, in order to further explore the Housefly maggot(HM)on the role of anti-atherosclerosis, this study internationally recognized AS model——ApoE knockout (ApoE-/-) mice were studied to explore the Housefly maggot(HM)against atherosclerosis mechanisms of atherosclerosis, including lipid-lowering mechanisms, antioxidant mechanism, anticoagulation mechanism. Also, macrophage in atherosclerosis of the physiological and pathological processes play an important role in plaque formation is the dominant factor. Housefly maggot(HM) meal can affect the migration of macrophages, thereby delaying the formation of plaque, you need to verify in the experiment.Research Methods:Housefly maggo(tHM) and Their Homogenate. Sterile second- and third-instar larvae of L. sericata were produced, maggots were collected. and dry maggots were blent into Housefly maggot(HM) homogenate for 3 h with water to produce 50% serofluid as previously describedAnimals and Treatment. Male ApoE-/- mice were were shifted to a Western diet consisting of 0.5% cholic acid, 1.0% cholesterol, and 18% fat for 8 weeks. Experimental group received the 50% Housefly maggot(HM) homogenate, without heating at a dose of 8g/kg/day. The control group was received isovolumic normal saline (NS). Both groups were given administration by gavage. The experiment lasted 8 weeks. Retro-orbital bleeds were performed after 8, 10, 12,14,and 16 weeks on diet and mice were euthanized at 16 weeks. All animal experiments were performed in accordance with institutional guidelines. Atherosclerosis Quantification. We used En face and Cross-section to estimate the extent of atherosclerosis for each animal.Elastic fiber in arterial aorta was used to quantified atherosclerosis of aortic.Plasma Lipid. Mice fasted over-night by retro-orbital blood drawing and then sera were separated by centrifugation for 10 min , 3000rpm.Sera from each group at the designated time points were harvested and Total cholesterol (TC) and triglyceride (TG) concentrations in plasma were determined enzymatically. High-density lipoprotein-cholesterol (HDL) and Low-density lipoprotein-cholesterol (LDL) were measured by homogeneous assay. Detection of Anti-Oxidized-LDL (Ox-LDL) Antibodies by ELISA. Mice plasma was measured as described by the manufacturers and every sample tested in duplicate. Anti-oxLDL levels were calculated as standard deviation units (SDΜ) according to the formula.Malondialdehyde Measurement. Plasma lipid peroxide as measured malondialdehyde (MDA) was determined spectra photo metrically using Bioxytech LPO-586 kit (R&D System).Immunohistochemical Analysis. The frozen sectioned were harvested just similar to the cross-section method, except for the thickness were 5μm. Endogenous peroxides was blocked by 3% hydrogen peroxide before the primary antibody (Mac3 for macrophages,1:200 over night (4°C), goat anti-mouse, BD) was applied. Digital images were taken for quantitative morphometric analysis. Mac-3+ areas were determined using Adobe Photoshop V6.0 by tracing.Results:Atherosclerosis, determined by the lesion size at the aortic sinus, was significantly decrease in Housefly maggot (HM) treated mice (mean areas, 216272.27±28649μm2 versus 131998.17±24165μm2; P<0.01). The aortic atherosclerotic lesion area was distinct in both experimental groups (21.34% and 13.58% of total aortic area in the control and Housefly maggot (HM) treated group, respectively; P<0.01).Lipoprotein. There were significantly differences on lipoprotein in the two group mice. 3.18±0.21 vs. 1.71±0.17 for HDL; 8.32±0.69 vs. 15.07±0.74 for LDL (mmol/L; n=12;P<0.01).) Meanwhile, our data shows that Housefly maggot (HM) has an anti-oxidant effect and reduce anti–oxidatively modified low density lipoproteins (Ox-LDL) antibody levels on lipoprotein or on plasma (P<0.005). Taken together, these data provide strong evidence that the anti-atherogenic and anti-oxidant effect of Housefly maggot (HM) in ApoE-/- mice.Immunohistochemical Analysis. The local accumulation of Ox-LDL attracts monocytes to influx into the vessel wall, differentiate into macrophages and develop into foam cells, which is the major component of atherosclerotic plaque. To confirm the reducing Ox-LDL by MH, we use monoclonal antibody for MAC-3, the marker of macrophage, to detect the macrophage quantity and infiltration in atherosclerotic plaque. We also observed that the MAC-3 stated on the surface of atherosclerotic plaques in control group mice. However, in MH-gavaged mice, we could noted that the MAC-3 disperse in the atherosclerotic plaques. It indicated that MH is related to the infiltration of macrophages.Conclusion:I. Taken together, these data provide strong evidence that the anti-atherogenic and anti-oxidant effect of Housefly maggot (HM) in ApoE-/- mice,including reduced atherosclerotic plaque areas of the aorta and abdominal aorta and the heart tricuspid valve in ApoE-/ - mice. At the same time, hyperplasia of elastic fiber in the aorta also be relieved.II. Housefly maggot (HM) may be infected regulation of blood lipids, on the one hand, by increased levels of bile flow and total bile acid (TBA) synthesis for the body to accelerate cholesterol excretion. On the other hand, through competitive inhibition of cholesterol synthesis rate-limiting enzyme in the system——3-hydroxy-3-methyl glutaryl coenzyme A reduetase(HMG-CoA), inhibition of cholesterol biosynthesis.III. Housefly maggot (HM) played as a strong antioxidant in the ApoE-/- mice, the one hand, by raising the HDL level of the body, protecting LDL from oxidation as the Ox-LDL. On the another hand,, reducing oxidative stress in the body, thereby reducing the formation of Ox-LDL, and ultimately reduce arterial plaque. Housefly maggot(HM) could inhibit macrophage migration to the vessel wall and thus affect the formation of foam plaque.
|
PDF Full Text Request