| BackgroundAtherosclerosis is the result of a prolonged and excessive inflammatory process in the vascular wall (40–50% of patients with AS do not have established AS risk factors). Conventional cardiovascular risk factors such as high blood pressure and high cholesterol do not account fully for variation in AS disease. A considerable amount of evidence has shown that chronic mild stress (CMS) may play an important role in the etiology and progression of certain cardiovascular diseases such as atherosclerosis, and accumulating evidence also indicated CMS may induce a chronic immune inflammatory process culminating in atherosclerosis. These inflammatory events, caused by stress, may account for the approximately 40% of atherosclerotic patients with no other known risk factors. However, the underlying mechanisms are still largely unknown. Since the challenge of quantifying and defining the impact of CMS on human AS poses difficulties, attempts have been made to develop animal models to overcome these difficulties. In the present study, we examine the effect of CMS on the development of atherosclerosis in apoE-/- mice, since this strain is very sensitive to the unpredictable CMS protocol and develops atheroma similar in type and distribution to that found in human even when fed on normal diet.The innate immune response is the first line of defense in which highly conserved pathogen motifs, entitled pathogen-associated molecular patterns (PAMPs), are recognized. The receptors capable of recognizing these PAMPs are toll-like receptors (TLRs). As so far, 10 TLRs have been indentified in humans. Different members of TLRs are actived by different PAMPs. Ligand binding to TLR4 results in the recruitment of the adaptor molecule MyD88 to the Toll/IL-1 receptor domain of the receptor. Intracellular propagation of the signal leads to NF-κB activation and subsequent induction of proinflammatory cytokines, chemotatic factors and adhesion molecules. Moreover, a considerable of studies suggest that TLR4 may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis, and there is also accumulating evidence also indicates TLR4 may involve in immune response induced by chronic stress. The aim of the present study was to examine the effect of CMS on the development of atherosclerosis in adolescent apoE-/- mice, and investigate whether the TLR4 signaling pathway participates in the development of atherosclerosis induced by CMS.Aims1. To study the effects CMS on the development of atherosclerosis in apoE-/- mice.2. To study the effects CMS on the expressions of TLRs signaling pathway in apoE-/- mice3. To construct an expression vector of Ad-TLR4siRNA and study its'effects on the development of atherosclerosis and the expressions of TLR4/NF-κB-MCP-1,ICAM-1,IL-1βand TNF-αpathway in apoE-/- mice induced by CMS.Methods1. Construction of CMS atherosclerosis apoE-/- mice model and exploration of the effects of CMS on the expressions of TLRs signaling pathway in apoE-/- mice1.1 Chronic mild stress ProtocolOne hundred twenty male apoE-/- mice (from Peking University, China), 4 weeks old, weighing approximately 16 g upon arrival at the animal facilities, served as subjects in the present study. They were kept in the experimental room a week before the onset of the experiment in order to familiarize them with the testing environment. The stress scheme was slightly modified from that previously used for mice by Ipek Yalcin et al., and consisted of the following: two periods of continuous overnight illumination; two periods (7 and 17 h) of 45 degrees cage tile, one 17-h period in a soiled cage (100 ml water in sawdust bedding), two periods (9 and 15 h) of intermittent sound (a tone of 80 dB) and two periods of exposure to rat odour (removal of the cage containing the experimental mice into the procedure room and placing the experimental mice into cages in which rats had been held). The stressed mice received this stress protocol for 0 (n=20), 4 (n=20), and 12 weeks (n=20). The control mice were housed under identical conditions in a separate room, and had no contact with the stressed animals. All mice were fed a high-fat, high-cholesterol (atherogenic) diet containing 5% (wt/wt) fat and 1.0% cholesterol from 5 weeks of age through the duration of the experiment.1.2 Assessment of the serum corticosterone concentration and sucrose consumptionAt the start of the experiment, mice were first trained to consume a 1% sucrose solution. Sucrose consumption and body weight were monitored throughout the experiment. After a one-week period of adaptation, sucrose solution intake baseline tests were performed for all subjects. This was done in order to familiarize mice with the sucrose preference procedure. These tests involved an 8h period of food and water deprivation, followed by the offering of a sucrose solution for 1 h. Intake was determined by weighing the bottles containing sucrose solution at the beginning and at the end of each test. After this phase (7 days), mice were divided into two groups of normal control (n=60) and CMS (n=60).Serum corticosterone concentration was determined by solid-phase 125I radioimmunoassay using a commercially available reagent kit. Sucrose consumption and assessment of the serum corticosterone concentration were monitored throughout the experiment.1.3 Assessment of atherosclerosis in aortic trees and aortic sinusAfter anesthesia with pentobarbital sodium, the mice were perfusion-fixed with 4% paraformaldehyde, and their aortic trees were removed including the brachiocephalic region and the carotid, and femoral branches. Whole aortas were cleaned of adventitia and opened longitudinally from the aortic arch to the iliac bifurcation, mounted en face, and stained for lipids with Soudan IV. Serial sections 10μm thick were collected on slides for immunohistochemistry and staining with Oil red O. Cross sections of the aortic sinus and aortic valve were stained with Oil red O and counterstained with Gill III hematoxylin, and lesion areas were quantified with IMAGEPRO PLUS .1.4 Assessment of the expressions of TLR4/NF-κB-MCP-1,ICAM-1,IL-1βand TNF-αpathway in apoE-/- miceTLRs signaling pathway real-time PCR microarrays were used to determine the expressions of TLRs signaling pathway in aortas of apoE-/- mice, and Western blotting method was used to detect the expression of TLR4 and NF-κB in aortas of apoE-/- mice. Moreover, frozen sections of apoE-/- mice aortic root were immunostained with Rabbit anti-mouse TLR4 antibody according to the instructions, and the serum concentrations of MCP-1, sICAM-1, IL-1βand TNF-αwere measured utilizing a high-sensitivity enzyme-linked immunosorbent assay according to the manufacturer's instructions.2. Effects of Ad-TLR4siRNA on the development of atherosclerosis and the expressions of TLR4/NF-κB-MCP-1,ICAM-1,IL-1βand TNF-αpathway in apoE-/- mice induced by CMS2.1 The construction of an expression vector of Ad-TLR4 siRNA2.1.1 Construction and identification of siRNA vector for TLR4According to the design principles, two pairs of siRNA sequences were design. The positive recombinants were sequenced and called pRNAT-TLR4-1and pRNAT- TLR4-2. The empty vector of pRNAT-H1.1/Adeno was used as negative control vector.2.1.2 Selection of efficient siRNA vector for TLR4Total RNA or proteins from cells were extracted. Real-time PCR, western blotting showed the expression of TLR4 and selected the efficient siRNA.2.1.3 Proliferation and titration of TLR4 RNAi adenovirus293A cells were infected with pSuppressorAd-TLR4, pSuppressorAd-Neg. The culture supernatant and cells were collected respectively.The titer of concentrated virus was detected by hole-by-dilution titer assay2.2 The effects of Ad-TLR4 siRNA on the development of atherosclerosis in apoE-/- mice induced by CMS One hundred and twenty male apoE-/- mice (from Peking University, China), 4 weeks old, weighing approximately 16 g upon arrival at the animal facilities, served as subjects in the present study. After 1% sucrose solution intake baseline tests (7 days), mice were divided into groups of CMS control, and CMS + empty vector and CMS +Ad-TLR4 siRNA (tail vein injection, 10μl/mouse; n=40 per group). All mice were fed a high-fat (5%, wt/w), high-cholesterol (1%, wt/wt) diet and received CMS protocol for 0 (n=20), 4 (n=20), and 12 weeks (n=20). Serial sections of the aortic sinus 10μm thick were collected on slides for immunohistochemistry and staining with Oil red O. Cross sections of the aortic sinus and aortic valve were stained with Oil red O and counterstained with Gill III hematoxylin, and lesion areas were quantified with IMAGEPRO PLUS .2.3 Effects of Ad-TLR4 siRNA on the expressions of TLR4/NF-κB-MCP-1,ICAM-1,IL-1βand TNF-αpathway in apoE-/- mice induced by CMSThe expressions of TLR4 and NF-κB mRNA in aortas of apoE-/- mice were determined with real-time PCR. Western blotting method was used to detect the expression of TLR4 and NF-κB in aortas of apoE-/- mice. The serum concentrations of MCP-1, sICAM-1, IL-1βand TNF-αwere measured utilizing a high-sensitivity enzyme-linked immunosorbent assay according to the manufacturer's instructions.Results1. Serum corticosterone concentrationAfter 4 weeks of CMS, Corticosterone concentration was significantly higher in the CMS group than that in the control group. The degree of increase in corticosterone concentration was related to the duration of stress such that there was an 11-fold increase in those mice exposed to 12 weeks of stress compared with controls (457.3±56.5ng/ml vs 40.2±7.2ng/ml, P < 0.001).2. Sucrose preference testsCMS induced a decrease in sucrose intake, relative to control conditions, which is indicative of operationally defined anhedonia. There was no baseline difference in sucrose intake between the two groups (P>0.05). The control group did not show a significant difference in the consumption of the sucrose solution over a period 12 weeks. However, the CMS group gradually reduced the consumption of the sucrose solution from the second week to the twelfth week. Two-way ANOVA was performed on sucrose intake. As expected, there was a significant stress×week interaction on sucrose intake [F (12,456) = 141.42, P < 0.001]. An ANOVA performed on sucrose intake yielded a main effect of group [F (1,456) = 236.47, P < 0.01].3. CMS effects on aortic atherosclerosis in apoE-/- miceResults showed that exposure to CMS resulted in a significant increase in atherosclerotic lesions area in entire aortic trees of apoE-/- mice. SoudanⅣstaining was absent in the normal vessels obtained from control and CMS apoE-/- mice at 0 week. The measurement of total aortic atherosclerotic lesion area showed that there was a significant difference in lesion area (% aorta covered by plaque) of mice exposured to CMS for 4, 12 weeks versus corresponding control mice respectively (10.37%±2.32%vs 2.61%±0.57%, P < 0.01; 24.58%±3.81%vs 6.36%±1.47%, P < 0.01).4. CMS effects on aortic sinus atherosclerotic lesion in apoE-/- miceResults showed that lesions were observed throughout the aorta in both two groups after 4, 12 weeks fed on the atherogenic diet, and lesions were more extensive in stressed mice than that in respective control ones. After 12 weeks fed on the atherogenic diet, the mean lesion area was 0.24±0.03 mm2 in the stressed group, meanwhile the atherosclerotic lesion was only 0.05±0.01 mm2 (P<0.01) in the control group. The atherosclerosis lesions both in aortic tress and sinuses of CMS mice were significantly increased linearly in response to duration of CMS exposure.5. Real-time PCR Array analysis of CMS induced gene expression of TLRs signaling pathwayAmong 87 genes analyzed, 15 genes were upregulated in stressed mice, especially TLR4, TNF-α, IL-6 and IL-1β, and 28 genes were downregulated compared with non-stressed mice. The increased expression of TLR4 mRNA was specific, while the expression of the other 8 TLRs was either downregulated or unchanged in the CMS model. This result indicates that a differential physiological regulation of the several members of the TLRs family occurs. There was a significant difference in the expression of IL-1βof CMS mice versus corresponding control mice (P=0.008).6. Effect of CMS on TLR4 expressions in aorta of apoE-/- miceWestern blotting showed that there was no difference in the expression of TLR4 between CMS and control group mice (0.21±0.03 vs 0.22±0.05, P >0.05) at the stage of 0 week. However,after exposure to CMS for 4, 12 weeks, TLR4 levels in CMS were markedly increased, and there was a significantly difference in CMS mice, compared with control mice respectively (0.25±0.08 vs 0.74±0.06, P <0.05; 0.47±0.06 vs 1.13±0.11, P<0.05). Immunohistochemistry staining indicated that TLR4 immunoreactivity was markedly observed in the aortic root atherosclerotic lesions, and TLR4 expression in CMS for 4, 12 weeks was much stronger than that of control mice at the same stage (P < 0.01).7. Effect of CMS on NF-κB expressions in aorta of apoE-/- miceWestern blotting showed that the expression of NF-κB both in CMS and control mice was low at the stage of 0 week, and there was no difference in CMS and control mice. However,after exposure to CMS for 4, 12 weeks, NF-κB levels in CMS were significantly increased, and there was a markedly difference in CMS mice, compared with control mice respectively (0.18±0.04 vs 0.45±0.06, P<0.05; 0.42±0.07 vs 0.95±0.13, P <0.05).8. ELISA analysis of cytokinesELISA showed that there was no baseline difference in the expressions of MCP-1, sICAM-1, IL-1βand TNF-a leves both in CMS and control mice at the stage of 0 week respectively (P >0.05). However,after exposure to CMS for 4weeks, there were significant increases in the expressions of MCP-1, sICAM-1, IL-1βand TNF-a leves in CMS compared with control mice respectively (137.38±28.37pg/ml vs 348.36±42.06pg/ml ,P<0.05; 82.53±16.37ng/ml vs 176.62±24.36ng/ml, P<0.05; 2.65±0.36ng/ml vs 9.77±1.16ng/ml, P<0.05; 3.16±0.46ng/ml vs 7.51±1.06ng/ml, P <0.05). Exposure to CMS 12 weeks later, there were more significant increases in the expressions of MCP-1, sICAM-1, IL-1βand TNF-a leves in CMS than that in control mice respectively (274.92±50.18pg/ml vs 827.33±96.62pg/ml,P<0.05;265.31±36.42 ng/ml vs 586.24±86.53ng/ml, P<0.05;4.28±0.83ng/ml vs 21.36±3.17ng/ml, P<0.01; 5.14±0.93ng/ml vs 17.84±2.28ng/ml, P <0.05).9. The effection of TLR4 siRNA on the expression of TLR4 and titration detection of TLR4 RNAi adenovirusReal-time PCR and Western blotting showed that the expression of TLR4 mRNA and protein levels were significantly downregulated in 293A-pTLR4-2 cells when compared with those of other groups. We construct an expression vector of TLR4 and screen the positive clone successfully. pSuppressorAd-TLR4 and pSuppressorAd-Neg were amplified in scale. The hole-by-dilution titer assay showed that viral titer was 3.4×1011 TU/ml.10. The effects of Ad-TLR4siRNA treatment on the development of AS in apoE-/- mice induced by CMSResults displayed that there was no AS lesion in apoE-/- mice at the experiment stage of 0 week. However, after 4, 12 weeks fed on the atherogenic diet, AS lesions were observed throughout the aorta in all groups, and lesions of TLR4siRNA group were significantly decreased than that of other two groups at the same experiment stage respectively. There was a significantly difference in AS lesions between TLR4siRNA group and respective CMS control group after 4, 12 weeks fed on the atherogenic diet (1.53±0.22% vs 3.14±0.37%,P<0.05;5.77±0.73% vs 26.82±2.14%,P<0.01). There was no difference in AS lesions between CMS+ empty vector group and respective CMS control group (P >0.05).11. The effects of Ad-TLR4 siRNA treatment on the expressions of TLR4 mRNA and protein in aortas of CMS apoE-/- miceReal-time PCR and Western blotting showed there was less expression of TLR4 mRNA and protein levels in aortas of apoE-/- mice at the experiment stage of 0 week, and there was no difference in the expression of TLR4 among three groups (P >0.05). However,after exposure to CMS for 4 weeks, TLR4 mRNA and protein levels in TLR4 siRNA group were markedly decreased, and there was a significantly difference in TLR4 mRNA and protein levels compared with CMS group respectively (0.25±0.03 vs 0.61±0.05,P <0.05; 0.23±0.03 vs 0.58±0.07,P <0.05 ). After 12 weeks CMS, TLR4 mRNA and protein levels in TLR4 siRNA group were more markedly decreased than those in CMS group (0.24±0.02 vs 0.91±0.10, P <0.01; 0.22±0.03 vs 0.79±0.08, P <0.01). There was no difference in TLR4 levels between CMS + empty vector group and respective CMS control group (P >0.05).12. The effects of Ad-TLR4 siRNA treatment on the expressions of NF-κB mRNA and protein in aortas of CMS apoE-/- miceReal-time PCR and Western blotting suggested that there was less expression of NF-κB mRNA and protein levels in aortas of apoE-/- mice at the experiment stage of 0 week, and there was no difference in the expression of NF-κB among three groups (P >0.05). However,after exposure to CMS for 4 weeks, NF-κB mRNA and protein levels in TLR4 siRNA group were markedly decreased, and there was a significantly difference in NF-κB mRNA and protein levels compared with CMS group respectively (0.21±0.03 vs 0.45±0.06,P <0.05; 0.24±0.03 vs 0.53±0.05,P <0.05). After 12 weeks CMS, NF-κB mRNA and protein levels in TLR4 siRNA group were more markedly decreased than those in CMS group (0.24±0.04 vs 0.63±0.07, P <0.05; 0.27±0.03 vs 0.67±0.07, P <0.05). There was no difference in NF-κB levels between CMS+ empty vector group and respective CMS control group (P >0.05).13. The effects of Ad-TLR4 siRNA treatment on the expressions of MCP-1, sICAM-1, IL-1βand TNF-αin serum of CMS apoE-/- miceELISA suggested that the expressions of MCP-1, sICAM-1, IL-1βand TNF-αin serum of apoE-/- mice were fairly low at the experiment stage of 0 week, and there were no difference in the expressions of MCP-1, sICAM-1, IL-1βand TNF-αamong three groups respectively (P >0.05). However,after exposure to CMS for 4 weeks, MCP-1, sICAM-1, IL-1βand TNF-αlevels in TLR4 siRNA group were markedly decreased, and there was a significantly difference in MCP-1, sICAM-1, IL-1βand TNF-αlevels compared with CMS group respectively (94.27±12.26pg/ml vs 326.63±29.17pg/ml , P<0.05; 63.72±6.81ng/m vs 195.83±19.67ng/ml , P<0.05; 2.27ng/ml±0.28ng/ml vs 10.61 ng/ml±1.17ng/,P <0.05; 3.26ng/ml±0.31ng/ml vs 7.18 ng/ml±0.78ng/ml, P <0.05 ). After 12 weeks CMS, MCP-1, sICAM-1, IL-1βand TNF-αlevels levels in TLR4 siRNA group were more markedly decreased than those in CMS group (90.62±13.82pg/ml vs 754.07±61.26pg/ml, P <0.01; 58.16±6.03ng/ml vs 436.29±46.52ng/ml, P <0.01; 2.38ng/ml±0.32ng/ml vs 18.83ng/ml±1.64ng/ml, P <0.01; 3.02ng/ml±0.37ng/ml vs 15.87ng/ml±1.47ng/ml, P <0.01). There was no difference in MCP-1, sICAM-1, IL-1βand TNF-αlevels between CMS+ empty vector group and respective CMS control group (P >0.05).Conclusions1. We constructed a CMS atherosclerosis apoE-/- mice model successfully.2. CMS can change the expressions of TLRs signaling pathway differentially. TLR4 /NF-κB signaling pathway may involve in the acceleration of the development of AS in apoE-/- mice induced by CMS.3. We constructed a siRNA vector for TLR4 (pRNT-TLR4-2) successfully.4. Intervention with TLR4 siRNA can markedly inhibit the development of AS in apoE-/- mice induced by CMS.5. Intervention with TLR4 siRNA can markedly decrease the expressions of TLR4 and NF-κB in CMS apoE-/- mice. Intervention with TLR4-siRNA can markedly decrease the expressions of MCP-1,sICAM-1,IL-1βand TNF-αproduced by TLR4 /NF-κB signaling pathway.6. TLR4 /NF-κB-MCP-1, sICAM-1, IL-1βand TNF-αsignaling pathway may take an important role in the acceleration of the development of AS in apoE-/- mice induced by CMS.
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