| Background:Cardiovascular disease is the major cause leading to mortality and morbidity throughout the world and its basic pathogenesis is atherosclerosis. Lipid-lowering therapy is a kind of conventional treatment. However, as the commonly used lipid-lowering drugs are synthesized by chemical methods and have side-effects and adverse reactions, the drugs having better curative effects and being safer are urgently needed in clinic. Lactobacillus has been used in fermenting foods since the third century, AD. The long term used history proves that Lactobacillus is a kind of safe probiotics and owns variety effects. In1960’s and1970’s, people began to paid attention to its cholesterol-lowering effect, which is also rapidly becoming the hot field of probiotics research area. Yet there has been controversy on the cholesterol-lowering effect of Lactobacillus. In addition, the research about the effect of Lactobacillus on cardiovascular disease is limited and fewer studies have investigated the role of Lactobacillus directly in atherogenesis.Objective (1) To investigate the effect of Lactobacilus (L.) acidophilus ATCC4356on the development of atherosclerosis (As) and serum cholesterol in ApoE-/-mice. The underlying mechanisms were further studied through detecting the modulation of oxidative and inflammatory process, and the regulation of intestinal microflora.Methods (1) Grouping and feeding of mice:8week-old male mice were randomly assigned to4groups:(a)WT group: Wild Type C57BL/6J mice were given normal saline (NS) as normal control group (WT);(b) Vehicle group:ApoE-/-mice were treated with NS as model control group;(c) La. L group:ApoE-/-mice treated with low dose (5×107CFU) of L. acidophilus ATCC4356daily;(d) La. H group:ApoE-/-mice treated with high dose (5×10CFU) of L. acidophilus ATCC4356daily. C57BL/6J mice were fed with normal chow, while all the ApoE-/-mice were fed with high fat and high cholesterol diet (including15%fat and0.25%cholesterol). The volume of NS and L. acidophilus ATCC4356was0.5mL/day per mouse. The experimental time was12weeks.(3) During the course of experiment, mice body weights were monitored once a week. After12weeks of intervention the mice were sacrificed, the needed materials were drawn from responding organ and frozen in liquid nitrogen or-80℃.(4) Detecting the effects of L. acidophilus ATCC4356on mice serum cholesterol:Serum lipid levels, including total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), were tested by automatic biochemical analyzer.(5) Testing the development of As in mice aorta: Aortic en face analysis and cryosections of aortic root were stained with Oil Red O to examine the aortic atherosclerotic lesions; The morphologic changes in the atherosclerotic lesions were identified by hematoxylin-eosin (H&E) staining.(6) Examining the oxidative status: Eenzyme-linked immunosorbent assay (ELISA) kits was used to test the serum levels of oxidized low density lipoprotein (ox-LDL); Malondialdehyde (MDA) levels, superoxide dismutase (SOD) activities and glutathione (GSH) concentrations were detected by biochemical analyzed kits to show the oxidative status.(7) Detecting the changes of inflammatory factors:The protein levels of tumor necrosis factor alpha (TNF-α) and interleukin10(IL-10) in mice serum were assessed by ELISA kits; the mRNA levels of TNF-a and IL-10in aorta were analyzed with quantitative real-time PCR.(8) Testing the activation of nuclear factor kappa-B P65(NF-κB p65) signaling pathway:The expression levels of nuclear factor-kappa-B inhibitor alpha (IκB-α), cytoplasmic and nuclear NF-κB p65in aorta were determined by western blot.(9) Analyzing the changes of intestinal microflora:The contents of colon were homogenized and diluted with NS, then certain diluted contents were plated on responding selective media to distinguish bacterial and count the number of colony forming units (CFU). The effect of L. acidophilus ATCC4356on proliferation of Caco-2cells was detected by MTT method.Results (1) The atherosclerotic animal models were successfully established.(2) The changes of mice body weights:Body increments in high fat and high cholesterol fed ApoE-/-mice were higher than those in normal chow fed wide type C57BL/6J mice, while L. acidophilus ATCC4356could not affect the body weights.(3) The effect of L. acidophilus ATCC4356on mice serum cholesterol:The serum levels of TC, LDL-C and HDL-C in ApoE-/-mice were significantly higher that those in WT group mice, but there were no differences among ApoE-/-mcie treated with or without L. acidophilus ATCC4356.(4) Atherosclerotic lesion area:At the end of experiment, the Vehicle group mice could develop apparent atherosclerotic lesions in aorta. However, ApoE-/-mice treated with L. acidophilus ATCC4356showed decreased atherosclerotic lesion size in both en face aorta and aorta root. H&E staining also showed attenuated morphologic severity in La.H group.(5) The results of oxidative status examination:Compared to WT mice, ApoE-/-mice in Vehicle group showed higher levels of serum ox-LDL and MDA, but lower levels of SOD. Administration of L. acidophilus ATCC4356could decrease the levels of ox-LDL and MDA while increase the activities of SOD in a dose-dependent manner. However, the levels of GSH showed no changes.(6) The results of inflammatory molecules detection:Serum levels of TNF-α were increased while IL-10levels were reduced in Vehicle group compared with WT group. The aortic mRNA expression of TNF-α and IL-10also showed the same trend. Treatment with L. acidophilus ATCC4356could reverse these trends.(7) The activation of NF-κB p65signal pathway was suppressed:The results of Western blot demonstrated that compared to ApoE-/-mice in Vehicle group, the degradation of aortic IκB-α was suppressed, expression of cytoplasmic NF-κB was upregulated while nuclear NF-κB p65was downregulated in ApoE-/-mice treated with L. acidophilus ATCC4356, suggesting the inhibition of NF-κB p65nuclear translocation.(8) The changes of intestinal microflora:The numbers of both intestinal Lactobacillus and Bifidobacterium were lowered in high fat and high cholesterol fed Vehicle group mice than those in WT mice. Administration of L. acidophilus ATCC4356to ApoE-/-mice showed significant increases in the numbers of intestinal Lactobacillus, Bifidobacterium and Enterococcus spp. colonies as compared to ApoE-/-mice treated with vehicle only.1×108CFU/mL L. acidophilus ATCC4356could augment the proliferation of Caco-2cells.Conclusions Administration of L.acidophilus ATCC4356in ApoE-/-mice can attenuate the development of atherosclerotic lesions through the mechanisms of reducing oxidative stress, anti-inflammatory response and regulating intestinal microflora, rather than lowering cholesterol levels. The effects on preventing oxidative stress and inflammation are possably associated with inhibiting the activation of NF-κB singal pathway. L.acidophilus ATCC4356can promote the proliferation of Caco-2cells to strengthen the intestinal barrier function, which is the possible underlying mechanism of regulating gut flora. Figures:33, Table:1, References:115... |
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