| BackgroundAtherosclerosis has been generally considered to be a continuous progress and irreversible for a long time. Little direct evidence was available until Amstrong demonstrated that the advanced AS lesions in the rhesus monkey could be markedly regressed by low-fat non-cholesterol diet in 1970. In recent years, evidence from experimental or clinical research has indicated that the progression of atherosclerosis can be delayed, prevented or even reversed. However, there are still many problems unsolved about regression of atherosclerosis. On one hand, the molecular biological mechanism by which regression occurs remains poorly understood; on the other hand, though several kinds of drugs has been indicated effective, unfortunately, however, an ideal drug for regressing atherosclerotic plaques is still lacking. Although statins have been demonstrated in animal studies to significantly decrease lipid contents and inflammatory cells in plaques by LDL-lowering and inflammation-inhibiting effects, the mechanisms are still not clear. Moreover, liver dysfunction as a side effect of statins caused some patients to withdraw from statin treatment. Consequently, drugs of herbal origin with low side effects are of high interest as alternatives and the traditional Chinese herbal medicine may provide a new therapy for regressing atherosclerotic plaques. Traditional Chinese medicine has been proved benefit in many experimental and clinical studies, but the mechanisms and relative effects compared to lipid-lowering drugs are still unknown. We proved previously that quyuxiaoban capsule could induce regression of atherosclerotic plaques, but the mechanisms are lacking.ObjectiveTo compare the relative effects of quyuxiaoban capsule and simvastatin on regressing atherosclerotic plaques, as well as to investigate the possible molecular mechanisms.Methods 1. Animal model: Aortic wall injuries were introduced using an intravascular balloon in 67 rabbits before they were fed on an atherogenic diet containing 1% cholesterol for 12 weeks. After 12 weeks of induction, euthanasia was performed using an overdose of intravenous pentobarbital in 8 rabbits from AS model group and 2 from normal group.2. Intervention methods: At the end of week 12, 45 rabbits were randomly divided into three groups and given drugs for another 12 weeks. The first group of rabbits (Group C, n=15) were fed with a normal diet plus 20ml distilled water and served as regression controls. The second group of rabbits (Group D, n=15) were given the same diet as in the first group plus quyuxiaoban capsule (at a dose of 1g·kg-1 once a day). The third group of rabbits (Group E, n=15) were fed the same diet as the other two groups but supplemented with simvastatin (5mg·kg-1 once a day).3. Body weight: Before the experiment, at the end of week 12 and week 24, body weight of all rabbits was measured, respectively.4. Lipid measurement: At the baseline, week 12 and week 24, blood samples were collected from all rabbits, respectively. Serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured.5. Aortic ultrasonography measurements: At the baseline, week 12 and week 24, the abdominal aorta was scanned using a high frequency duplex ultrasonographic system. The intima-media thickness (IMT), the end-diastolic luminal diameters (Dd), the end-systolic luminal diameter (Ds), the mean velocity (Vm), peak velocity (Vp), velocity integral (VTI) and the average image intensity (AII) of the abdominal aorta were measured.6. Intravascular ultrasound (IVUS) studies: IVUS studies were performed at the baseline, week 12 and week 24. The external elastic membrane area (EEMA), lumen area (LA), plaque area (PA) and plaque burden (PB) were measured.7. Histopathological analysis: The abdominal aorta was processed and examined by hematoxylin and eosin, Masson, Movat or oil red O staining.8. Immunohistochemical analysis: Immunohistochemical staining was performed and the expressions of RAM11,α-actin and PCNA were detected.9. Electron microscope: Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of abdominal aorta.10. RT-PCR: The mRNA expressions of bFGF and TGF-β1 in the abdominal aorta tissue were analyzed using RT-PCR technique.11. Western blot: The protein expressions of bFGF and TGF-β1 in the abdominal aorta tissue were analyzed using western blot technique.12. Statistical analysis: Continuous data are presented as mean±SD. Values among the groups were compared with One-Way ANOVA. P<0.05 was considered statistically significant.Results1. General state of the experimental animals: 14 rabbits in group C, 15 rabbits in group D and 13 rabbits in group E completed the study.2. Body weight: At week 12, body weight in group A increased from (1.70±0.18)kg at baseline to (2.69±0.22)kg and body weight in group B increased from (1.71±0.17)kg at baseline to (3.14±0.29)kg. At the end of week 24, body weight in group E (4.02±0.15) was significantly higher than that in group A and group D (both P<0.01).3. Lipid measurement: Serum TC and LDL-C levels increased significantly in rabbits following a cholesterol rich diet (all P<0.001), among which TC increased from (2.63±0.33)mmol/L to (40.18±11.01)mmol/L, LDL-C from (1.77±0.29)mmol/L to (33.29±9.87)mmol/L. At week 24, simvastatin decreased TC by 46.48% and LDL-C by 49.61% (P<0.05 and 0.01, respectively) and quyuxiaoban capsule decreased LDL-C by 25.05%. The levels of TC and LDL-C in group E were significantly lower than that in group D (all P<0.05). In contrast, no significant differences were found between group C, group D and group E in terms of TG levels.4. Ultrasound Measurements: The maximum IMT of the abdominal aorta in group D and group E were significantly lower than that in group B or group C (all P<0.01), and the value of the maximum IMT in group E was lower than that in group D (P<0.05). No significant differences were found between the groups in terms of blood flow rate (all P>0.05).In contrast, corrected average ultrasonic intensity (AIIc%) value was 63.24±7.01 in group B and 69.71±6.71 in group C, and increased to 76.71±7.07 in group D and 83.38±8.18 in group E, respectively. The AIIc% value of the abdominal aorta in group D and group E were significantly higher than that in group B or group C (P<0.05～0.01), and the AIIc% value in group E was significantly higher than that in group D (P<0.05).5. IVUS Measurements: Quantitative IVUS analysis indicated that the PA values and PB% values in group B and group C were significantly lower than that in group D or group E, and PA and PB% in group E were significantly lower than that in group D (P<0.05～0.01). EEMA values in group D and group E were significantly lower than that in group B (both P<0.01), but no significant differences were found between group C, group D and group E. The LA values in the groups were not significantly different (P>0.05).6. Pathologic staining: Pathologic staining demonstrated that the intima was thin and complete in group A. A great quantity of widespread or scattered fatty plaque were seen in group B, in which intima thickened and foam cell accumulated obviously, with little collagen fiber hyperplasia. In contrast, more collagen fiber hyperplasia was seen in group C and in the other two groups, plaque thickness diminished and both quantity and volume of foam cell decreased and in which fat vacuolus reduced. Furthermore, prominent collagen fiber proliferation was present.Pathologic measurements revealed that the fibrous cap thickness and the ratio of fibrous cap thickness/IMT in group D and group E were significantly higher than the corresponding values in group B or group C (P<0.05～0.01), whereas IMT values in group D and group E were significantly lower than that in group B or group C. Comparison between group D and group E identified that the fibrous cap thickness and the ratio of fibrous cap thickness/IMT in group E was higher than that in group D (both P<0.01).7. Immunohistochemical staining: Immunohistochemical staining demonstrated that compared with group B, the expressions of macrophages in plaques were significantly higher in group C, group D and group E and the expressions of macrophages in group E was significant lower than that in group D.Theα-actin expressions in group B and group C were not significantly different. Compared with group B or group C, the a-actin expressions in group D and group E were significantly higher, whereas the PCNA expressions were significantly lower.8. Electron microscope examination: In group B and group C, the endothelial cells were damaged, muscle fiber spread chaoticly, and SMC showed a secretion type with few content of contractile element. But in group D and group E, the endothelial cells were relatively smooth and muscle fiber spread in uniformity, fatty vesicles reduced significantly and contractile element increased in SMC.9. RT-PCR: The mRNA expressions of bFGF in the atherosclerostic lesions of the abdominal aorta in group D and group E were significantly lower than those in group B and group C (P<0.05～0.01) and the value in group D was significantly lower than that in group E (P<0.05).The mRNA expressions of TGF-β1 in the atherosclerostic lesions of the abdominal aorta in group D and group E were significantly lower than those in group B and group C ( all P<0.05) but no significant difference was found between group D and group E.10. Western blotting analysis: The protein expressions of bFGF and TGF-β1 in the atherosclerostic lesions of the abdominal aorta in group D and group E were significantly lower than those in group B and group C (P<0.05～0.01) and the value in group D was significantly lower than that in group E (P<0.05).Conclusions: Quyuxiaoban capsule and simvastatin could induce regression of atherosclerosis in a rabbit model. The mechanisms of atherosclerotic regression were lipid-lowering effect, the decrease of macrophage, the reduced mRNA and protein expressions of several growth factors and the inhibition of the VSMC proliferation. BackgroundRecent studies have demonstrated that plaque rupture in the coronary artery is the major cause of acute cardiovascular events. Rupture of these atherosclerotic plaques induces partial or complete occlusion of lumens which results in acute myocardial ischemia or sudden death. Therefore, early detection and active treatment of vulnerable plaques have important clinical significance for the prevention of acute cardiovascular events.Unfortunately, however, an ideal drug for stabilizing vulnerable plaques is still lacking. Although statins have been demonstrated in animal studies to significantly decrease lipid contents and inflammatory cells and thicken the fibrous caps of plaques by LDL lowering and inflammation inhibiting effects, in the Prove-It Trial, 22.4% of patients experienced a coronary event despite an intensive statin therapy for 2 years. Moreover, liver dysfunction as a side effect of statins caused some patients to withdraw from statin treatment. Consequently, drugs of herbal origin with low side effects are of high interest as alternatives and the traditional Chinese herbal medicine may provide a new therapy for stabilizing plaques.The present study was designed to test the hypothesis that the Chinese medicine quyuxiaoban capsule has anti-atherosclerotic progression and plaque-stabilizing properties due to its anti-inflammation and lipid-lowering effects similar to simvastatin in a rabbit model of vulnerable atherosclerotic plaques.Objective(1) To establish an animal model of vulnerable atherosclerotic plaques that are mimic to human pathological changes and convenient for intervention; (2) To observe the imaging feature of vulnerable plaques using intravascular unltrasound technique and to approach the value of this technique in evaluating the development of atherosclerotic plaques; (3) To compare the effects of quyuxiaoban capsule and simvastatin on stabilizing vulnerable atherosclerotic plaques; (4) To investigate the possible molecular mechanisms of quyuxiaoban capsule and simvastatin on stabilizingvulnerable atherosclerotic plaques.Methods1. Animal model: Aortic wall injuries were introduced using an intravascular balloon in 48 rabbits before they were fed on an atherogenic diet containing 1% cholesterol for 12 weeks.2. Intervention methods: At the end of week 12, 47 rabbits were randomly divided into three groups and given drugs for another 12 weeks. The first group of rabbits (Group F, n=15) were fed with a normal diet plus 20ml distilled water and served as regression controls. The second group of rabbits (Group G, n=16) were given the same diet as in the first group plus quyuxiaoban capsule (at a dose of 1g·kg-1 once a day). The third group of rabbits (Group H, n=16) were fed the same diet as the other two groups but supplemented with simvastatin (5mg·kg-1 once a day).3. Gene transfection: At the end of week 24, laparotomy was conducted in all rabbits after having been anesthetized with an intravenous injection of sodium pentobarbital (30mg/kg) and an adenoviral vector containing recombinant p53 was transfected into these rabbits. These rabbits were maintained on a normal diet for additional 2 weeks.4. Pharmacological triggering: At the end of week 26, plaque rupture was induced by pharmacological triggering using Chinese Russell's Viper Venom and histamine. Rabbits were euthanized 24-48 hours after the above procedure.5. Body weight: Before the experiment, at the end of week 12 and week 26, body weight of all rabbits was measured, respectively.6. Lipid measurement: At the baseline, week 12 and week 26, blood samples were collected from all rabbits, respectively. Serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured.7. Biochemical studies: At the baseline, week 12 and week 26, blood samples were collected from all rabbits, respectively. Plasma fibrinogen was measured. Serum high sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1) and oxidized low-density lipoprotein (ox-LDL) were assayed using highly sensitive enzyme-linked immunosorbent assay (ELISA) kits. 8. Aortic ultrasonography measurements: At the baseline, week 12 and week 24, the abdominal aorta was scanned using a high frequency duplex ultrasonographic system. The intima-media thickness (IMT), the end-diastolic luminal diameters (Dd), the end-systolic luminal diameter (Ds), the mean velocity (Vm), peak velocity (Vp), velocity integral (VTI) and the average image intensity (AII) of the abdominal aorta were measured.9. Intravascular ultrasound (IVUS) Studies: IVUS studies were performed at the baseline, week 12 and week 26. The external elastic membrane area (EEMA), lumen area (LA), plaque area (PA) and plaque burden (PB) were measured.10. Histopathological analysis: The abdominal aorta was processed and examined by hematoxylin and eosin, Masson, Movat or oil red O staining.11 . Immunohistochemical staining was performed and the expressions of RAM11,α-actin, p53, ICAM-1, VCAM-1, MMP-9, TIMP-1 and MCP-1 were detected.12. Vulnerability index: Vulnerability index was calculated by dividing the area of (macrophages + extracellular lipids) by that of (smooth muscle cells + collagen fibers).13. Electron microscope: Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of the abdominal aorta.14. Real-time PCR: The mRNA expressions of p53, ICAM-1, VCAM-1, MMP-9, TIMP-1 and MCP-1 in the abdominal aorta tissue were analyzed using real-time PCR technique.15. Western blot: The protein expressions of p53, ICAM-1, VCAM-1, MMP-9, TIMP-1 and MCP-1 in the abdominal aorta tissue were analyzed using western blot technique.16. Statistical analysis: Data are expressed as mean±SD for continuous variables and by frequency count and percentage for qualitative variables. Incidence rate of plaque rupture was compared with Pearson Chi-Square test and other indexes were compared with One-Way ANOVA comparison test. P<0.05 was considered statistically significant.Results1. General state of the experimental animals: 15 rabbits in group F, 14 rabbits in group G and 14 rabbits in group H completed the study.2. Body weight: At baseline and week 12, body weights in the three groups were essentially equal. At the end of week 26, body weight in group G was significantly lower than that in group F and group H. Body weight in group F was not statistically different than that in group H.3. Incidence of Plaque Rupture: 12 rabbits in group F (12/15=80.00%), 6 in group G (6/14=42.86%) and 5 in group H (5/14=35.71%) had ruptured atherosclerotic plaques in the abdominal aorta. The incidence of rupture in group G and group H was significantly lower than that in group F (both P<0.05), while the plaque rupture rates in group G and group H were not statistically different.4. Lipid measurement: Compared with group F, simvastatin decreased TC by 38.84% and LDL-C by 58.37% (both P<0.01). The levels of LDL-C in group G were significantly lower than that in group F (P<0.01). Comparison between group G and group H showed that the levels of TC and LDL-C in group H were significantly lower than those in group G (P<0.05 or 0.01, respectively). In contrast, no significant differences were found between group C, group D and group E in terms of TG and HDL-C levels.5. Inflammatory markers: At week 26, the serum hsCRP levels in group G and group H were significantly lower than that in group F (both P<0.01), and there was no significant difference between the two drug groups.The sICAM-1 levels in group G were significantly lower than those in group F (P<0.01) and group H (P<0.05 for both), while no significant difference was found between group F and group H. The sVCAM-1 levels in group G were significantly lower than those in group F (P<0.01), while compared with group F or group G, the sVCAM-1 levels in group H were not significantly different.Serum ox-LDL and plasma fibrinogen levels in group G and group H were significantly lower (P<0.01 and 0.05, respectively) than those in group F, and the levels in group G was significantly lower than that in group H (P<0.05 and 0.01, respectively).6. Ultrasound measurements: The maximum IMT of the abdominal aorta in group G and group H were significantly lower than that in group F (all P<0.01), and the value of the maximum IMT in group H was lower than that in group G (P<0.05). No significant differences were found between the groups in terms of blood flow rate (all P>0.05).In contrast, corrected average ultrasonic intensity (AIIc%) value was 75.58±7.56 in group F and increased to 81.43±8.30 in group G and 82.09±6.55 in group H, respectively. The AIIc% value of the abdominal aorta in group G and group H were significantly higher than that in group F (P<0.05～0.01), but the value in group G and group H was not significantly different (P<0.05).7. IVUS measurements: Quantitative IVUS analysis indicated that the EEMA values in group G and group H were lower than group F but only the difference between group F and group H was significant (P<0.01).The PA values in group G and group H were significantly lower than that in group F (both P<0.01), and PA in group H was significantly lower than in group G (P<0.05).The PB% values in group G and group H were comparable and both were significantly lower compared with that in group F (both P<0.01).8. Pathologic staining: Most rabbits in group F developed plaque rupture and intravascular thrombosis with a dense infiltration of inflammatory cells. In contrast, the atherosclerotic lesions in group G and group H were similar in that plaque thickness was diminished, foam cells decreased and collagen proliferation present.Pathologic measurements revealed that the fibrous cap thickness and the ratio of fibrous cap thickness/IMT in group G (P<0.05) and group H (P<0.01) were significantly higher than the corresponding values in group F, whereas IMT values in group G and group H were significantly lower (P<0.05 and 0.01, respectively) than group F. Comparison between the two drug groups identified that the fibrous cap thickness in group H was higher than that in group G (P<0.01).9. Immunohistochemical staining: Immunohistochemical staining demonstrated that the expressions of macrophages, SMC, ICAM-1, VCAM-1, MMP-9, TEVIP-1 and MCP-1 in plaques were higher in group F than those in the two drug groups.10 . Vulnerability index: Both drug groups showed significantly lower macrophage expressions and lipids positive staining area, and significantly higher SMC expressions and collagen positive staining area than the control group, thus giving rise to a lower vulnerability index than group F (both P<0.01). No significant difference was found in vulnerability index between group G and group H.11. Electron microscope examination: In group F, the damage of smooth muscle cells were lessened, muscle fiber spread chaoticly. But in group G and group H, the endothelial cells were relatively smooth, muscle fiber spread in uniformity, fatty vesicles reduced significantly and heterochromatins assemble in edges.12. Real-time PCR: The mRNA expressions of ICAM-1, VCAM-1, MMP-9, TIMP-1 and MCP-1 in the atherosclerostic lesions of the abdominal aorta in group G and group H were significantly lower than those in group F (P<0.01 or 0.05). Comparison of the two drug groups revealed that group G had lower VCAM-1 and higher MMP-9 and MCP-1 mRNA expressions than group H (P<0.01 and 0.05, respectively) while the mRNA expression levels of other inflammatory factors were not significantly different between the two groups.13. Western blot: The protein expressions of ICAM-1, VCAM-1, MMP-9, TIMP-1 and MCP-1 in the atherosclerostic lesions of the abdominal aorta in group G and group H were significantly lower (P<0.01 or 0.05) than those in group F. Comparison between the two drug groups revealed that group G had lower VCAM-1 and higher MMP-9 protein expressions than group H (P<0.01 and 0.05, respectively) while the other protein expressions were not significantly different between the two groups.Conclusions: (1) The method of balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet, gene transfection and drug triggering is a simple, efficient and time-saveing way to establish rabbit vulnerable plaque model which is mimic to human disease and useful for interventions; (2) Intravascular unltrasound technique can quantificationally evaluate vulnerable plaques and is an realiable technique in monitoring the atherosclerotic plaque progression and evaluating therapeutic effect; (3) The effects of quyuxiaoban capsule and simvastatin on stabilizing vulnerable atherosclerotic plaques are similar and the net effects of the two drugs are similar; (4) Different mechanisms of quyuxiaoban capsule and simvastatin contribute to the plaque stabilization produced by the two drugs. Quyuxiaoban capsule has an efficient effect on decreasing serum lipids and inflammatory factors, reducing plasma fibrinogen, lowering lipid peroxidation, decreasing lipid content and inhibiting the expressions of inflammatory factors in plaques. Simvastatin enhances the stability of vulnerable plaques via its effects on lipid lowering and anti-inflammation. BackgroundAtherosclerosis has a complex, multigenic basis. Recent investigations have increasingly suggested inflammatory and immune mechanisms, activated by noninfectious and possibly even infectious agents, might be important in the development and/or destabilization of atherosclerotic plaque. Inflammatory cells, such as T lymphocyte and macrophage play an important role in the progression of atherosclerosis. Several kinds of cells, including vascular endothelial cell, smooth muscle cell and fibroblast and mast cell in adventitia can produce proinflammatory cytokine as immunological cells. However, the molecular mechanisms of the correlation between inflammatory factor and immunological reaction are still not clear, therefore, it is important to investigate the inflammatory and immunologic mechanism in atherosclerosis and take effective interventions to prevent its pathogenesis.Particular interests have focused on the interaction of toll-like receptors (TLRs) signaling with established pathologic determinants of plaque development and destabilization. TLRs are a family of pattern-recognition receptors and play an important role in the transduction of cellular signaling. Currently, more than 10 members of the TLR family have been identified in mammals. Ligation of these receptors initiates the activation of nuclear factor-KB (NF-kB), resulting in the expression of a wide array of inflammatory genes. TLR-2 and TLR-4 play an important role in the innate immune and inflammatory responses and several reports have documented the expression of TLR2 and TLR4 in atherosclerotic lesions. More and more evidence suggest that TLRs, especially TLR4, are bridges between immunological reaction, chronic inflammation and metabolic disorder of lipids and have considerable importance in atherosclerosis. However, a comprehensive understanding of the correlation between TLRs and atherosclerosis in molecular detail has proven elusive. Intervention studies with vaccinations and antimicrobial strategies have yielded mixed results in humans. Hence finding drugs of herbal origin with no side-effects is of high priority. It is important to study deeply the effects of traditional drugs on blocking the pathway of TLRs in atherosclerosis which may be useful in exploring specific and effective drugs.At present, the investigations on TLR signal in atherosclerosis are still in initial phase and the exact mechanisms of TLR/MyD88/NF-κB signal in the development of atherosclerosis are still not clear. The present study was conducted to approach the role of TLR/MyD88/NF-κB signal in atherosclerosis and to evaluate the effects of quyuxiaoban capsule and simvastatin on progression and stability of atherosclerosis in a rabbit model of unstable atherosclerotic plaques verified previously in our laboratory as well as to investigate the possible molecular mechanisms.Objective(1) To prove the existence of TLR/MyD88/NF-κB pathway in atherosclerosis and to explore the effects of this pathway on the occurrence and development of atherosclerosis in molecular level; (2) To evaluate the effects of quyuxiaoban capsule and simvastatin on TLR/MyD88/NF-KB pathway and on the progression and stability of atherosclerosis in rabbit model of unstable atherosclerotic plaques, with the anticipation to offer new targets for the treatment of atherosclerosis.Methods1. Animal model: Aortic wall injuries were introduced in 48 male New Zealand White rabbits using an intravascular balloon in these rabbits before they were fed on an atherogenic diet containing 1% cholesterol for 12 weeks.2. Intervention methods: At the end of week 12, 47 rabbits were randomly divided into three groups and given drugs for another 12 weeks. The first group of rabbits (Group F, n=15) were fed with a normal diet plus 20ml distilled water and served as regression controls. The second group of rabbits (Group G, n=16) were given the same diet as in the first group plus quyuxiaoban capsule (1g·kg-1 once a day). The third group of rabbits (Group H, n=16) were fed similarly as the other two groups but supplemented with simvastatin (5mg·kg-1 once a day).3. Gene transfection: At the end of week 24, laparotomy was conducted in all rabbits after having been anesthetized with an intravenous injection of sodium pentobarbital (30mg/kg) and an adenoviral vector containing recombinant p53 was transfected into these rabbits. These rabbits were maintained on a normal diet for additional 2 weeks.4. Pharmacological triggering: At the end of week 26, plaque rupture was induced by pharmacological triggering using Chinese Russell's Viper Venom and histamine. Rabbits were euthanized 24-48 hours after the above procedure.5. Immunohistochemical analysis: Immunohistochemical staining was performed and the expressions of TLR2, TLR4, NF-κB, MyD88, Fas, FasL and IL-6 were detected.6. Real-time PCR: The mRNA expressions of TLR2, TLR4, NF-κB, Fas, IL-6 and a housekeeping gene (GAPDH) in the abdominal aorta tissue were analyzed using real-time PCR technique.7. Western blot: The protein expressions of TLR2, TLR4, NF-κB, MyD88, Fas, FasL and IL-6 in the abdominal aorta tissue were analyzed using western blot technique.8. Apoptosis of smooth muscle cells: The cell cycle and apoptosis ratio of smooth muscle cells were measured using flow cytometer.9. Statistical analysis: Continuous data are presented as mean±SD. Values among the three groups was compared with One-Way ANOVA. P<0.05 was considered statistically significant.Results1. General state of the experimental animals: 15 rabbits in group F, 14 rabbits in group G and 14 rabbits in group H completed the study.2. Immunohistochemical analysis: Immunohistochemical staining demonstrated that the expressions of TLR2, TLR4, NF-κB, MyD88, Fas, FasL and IL-6 were all detected. The expressions of TLR2, TLR4 and NF-κB in plaques were significantly lower in group G and group H than those in group F (all P<0.01), with much lower expressions in group H compared with group G (all P<0.01). Both drug groups showed significantly lower MyD88, Fas, FasL and IL-6 expressions (P<0.01 and 0.05, respectively) and no significant difference was found between the two drug groups.3. Real-time RT-PCR: The mRNA expressions of TLR2, TLR4, NF-κB p65, Fas and IL-6 in the atherosclerostic lesions of the abdominal aorta in group G and group H were significantly lower than those in group F (P<0.01 or 0.05) with no significantly different between group G and group H.4. Western blot: The protein expressions of TLR2, TLR4, NF-κB p65, MyD88, Fas, FasL and IL-6 in the atherosclerostic lesions of the abdominal aorta in group G and group H were significantly lower than those in the control group (P<0.01 or 0.05), whereas comparison between group G and group H revealed that the protein expressions were not significantly different between the two groups.5. Apoptosis of smooth muscle cells: The apoptosis ratios of smooth muscle cells in group F, group G and group H were 15.60%±1.71%, 8.35%±1.29% and 6.31%±1.34%, respectively. Compared with group F, the cellular apoptosis in group G and group H was significantly lower (both P<0.01), with much lower values in group H than in group G. (P<0.01)Conclusions(1) TLR/MyD88/NF-κB signaling pathway existed in vulnerable atherosclerostic plaques in a rabbit model and showed obvious correlation with the development of vulnerable plaques; (2) Quyuxiaoban capsule and simvastatin can prevent the progression of vulnerable plaque and the blocking of signaling molecules in the TLR/MyD88/NF-κB signaling pathway was an important mechanism.
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