| BackgroundAtherosclerosis (Atherosclerosis, AS) serious harm to human health, it raised the incidence of cardiovascular and cerebrovascular diseases increased year by year, and the incidence was getting younger and younger. The study, found that the instable atherosclerotic plaque which has a big lipid, a thin fibrous cap, fewer smooth muscle cells and collagen content, more macrophages, higher levels of inflammatory cells and inflammatory mediators, are closely related to acute cardiovascular eventsRecent studies have demonstrated that plaque angiogenesis have became a new measurement of plaque stability. In atherosclerotic plaque, the blood vessels from adventitia and outer middle-membrane of the normal blood vessels get richer, extend into the intima of atherosclerosis, thus form plaque neovascularization. These new blood vessels can promote accumulation of inflammatory cells, and can induce plaque hemorrhage and plaque rupture. Reduce plaque neovascularization has become the treatment of atherosclerotic plaque concern a therapeutic target.Traditional Chinese Medicine has made considerable progress in Prevention and treatment of atherosclerosis. In recent years, the study of traditional Chinese medicine on the atherosclerotic plaque angiogenesis also shows the effective inhibition. Previous work demonstrated that traditional Chinese medicine Shumai capsule can promote angiogenesis in ischemic myocardium. Our team will further observe the effect of Shumai Capsules on collagen, lipids, smooth muscle cells, macrophages and angiogenesis marker CD34in atherosclerotic plaque, to investigate the impact on the internal components of plaque and plaque neovascularization.AimTo observe the effect of Shumai Capsules on collagen, lipids, smooth muscle cells, macrophages and CD34in atherosclerotic plaque, to investigate the impact on the internal components of plaque and plaque neovascularization.MethodsMail50apolipoprotein E-knockout (apoE-/-) mice at8weeks of age were fed a high fat high cholesterol diet including15%fat and0.25%cholesterol. Mail10C57BL/6J mice at8weeks of age were fed a normal chow diet. At20weeks of age, apoE-/-mice were randomly divided into five groups, the ApoE-KO group(Model group), the apoE-/-+SMCH group (SMCH group), the ApoE-/-+SMCL group (SMCL group), the apoE-/-+total panax notoginsenoside group (PNS group) and the apoE-/-+Simvastatin group (Simvastatin group)(n=10for each group). C57BL/6J mice were the normal control group (Control group). Mice in SMCH and SMCL group were given SMC at3500mg/kg,700mg/kg, respectively. Mice in PNS group were given PNS at60mg/kg. And mice in Simvastatin group were given Simvastatin at3mg/kg. All drugs were given orally, once a day for12weeks. Mice in model and control groups were given0.2ml sterile distilled water, once a day for12weeks. After administered for12weeks, all mice were sacrificedDetection contents:(1)Serum levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and trigliyceride (TG) was detected by oxidation enzyme method.(2)Histopathological analysis:Sections were stained with hematoxylin and eosin, oil red0staining and Sirius red staining.(3) Immunohistochemical analysis:Immunohistochemical staining were performed and the expressions of MOMA-2, a-SMA,CD34were detected.Results1. Comparison of serum lipid concentrationThe serum TC, TG and LDL-C concentration was significantly higher in the Model group than that in Control group (P＜0.001). Lipid levels in SMCH, SMCL, Simvastatin and PNS group were significantly lower compared with that in Model group (P＜0.05). The levels of TG and LDL-C in SMCH group decreased the most significant, while Simvastatin group significantly reduce the level of TC than other groups. There was a significant difference between SMCH group and SMCL group (P＜0.05).2. Histological and Morphology analysisHematoxylin and eosin staining:A normal aorta wall was observed in Control group mice. Atherosclerotic lesions were observed in all apoE-/-mice and the lumens were narrowed in a different degree. The ratio of plaque area to lumen area was54.64±9.31%(Model group),43.21±6.69%(SMCL group),24.16±7.88%(SMCH group),25.85±6.66%(Simvastatin group) and31.90±9.79%(PNS group). The ratio of plaque area to lumen area reduced to varying significant degrees in each treatment group compare with that in Model group (P＜0.01or P＜0.001). SMCH, Simvastatin and PNS group differ statistically significant compared to SMCL group(P<0.05or P＜0.01).Oil red0staining:There was no lipid deposition in normal aortic wall. There were a lot of red dye plaque lipid deposition in model group. The lipid content within plaque was25.85±3.17%(model group),20.95±3.24%(SMCL group),12.33±3..13%(SMCH group),13.80±3.92%(Simvastatin group) and12.67±5.52%(PNS group). Compared with model group, lipid infiltration in plaques were lower in each treatment group(P＜0.05or P＜0.001). There was a significant difference between SMCH group and SMCL group(P＜0.01). There was no significant difference between SMCH, Simvastatin and PNS group(P＞0.05).Sirius red staining:Under polarized light microscopy AS plaque shows red or brown and green patches of collagen type Ⅰ and Ⅲ. Compared with the Model group, the treatment group had increased the collagen content of the plaque (P＜0.05). There was no significant difference in collagen content between SMCH group and Simvastatin group (P＞0.05). There was a significant difference between SMCH group and SMCL group(P＜0.05).3. Immunohistochemical analysis Immunohistochemical staining showed that the expression of both local MOMA-2(macrophages) and a-SMA (smooth muscle cells) within the plaque. Compared with the Model group, the SMCH, Simvastatin and PNS groups were significantly reduced macrophage expression(P＜0.01). But there was no difference between SMCL and model group in reducing macrophage expression(P＞0.05). Meanwhile, the expressions of smooth muscle cells in SMCH, Simvastatin and PNS group treatment groups were significantly higher than that in Model group(P＜0.05). But there was no difference between SMCL and model group in increasing smooth muscle cells expression(P＞0.05). Comparison between the two groups, There was no significant difference between SMCH, Simvastatin and PNS group(P＞0.05).CD34-positive staining neovascularization in each treatment group significantly decreased compared with that in model group(P＜0.05). There was no difference between SMCH, Simvastatin and PNS group(P＞0.05). CD34-positive staining areas were lower in SMCH, Simvastatin and PNS groups than those in SMHL group(P＜0.05).Conclusion1. Shumai capsule could decrease blood lipid level in apoE-/-mice. It had an obvious effect in decreasing TG and LDL-C levels.2. Shumai capsule could effectively inhibit the formation of atherosclerotic plaques. SMC could reduce atherosclerotic plaque area, affect the internal components of the plaque, reduce the amount of plaque macrophages and lipids, increase expression of smooth muscle cells and collagen in plaque. Thereby increased the stability of the plaque.3. Shumai capsule could reduce neovascularization within atherosclerotic plaques. And the affection was dose-dependent. BackgroundThe development of atherosclerosis (Atherosclerosis, AS) disease on human health have had a serious harm, is an important clinical cardiovascular events pathological basis. In recent years, numerous studies have demonstrated that angiogenesis and atherosclerotic plaque progression are closely related.The mechanism is very complex of angiogenesis. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is the most critical for angiogenesis promoting factors currently known. It can specifically acts on endothelial cells and induce endothelial cell proliferation, migration, increased vascular permeability, plays an important role in physiological and pathological angiogenesis process. VEGF play a role via its receptor. Currently there are three VEGF receptors found: VEGFR-1, VEGFR2and VEGFR3. And increased vascular permeability neovascular endothelial cell proliferation and VEGF induced expression of adhesion molecules, mainly through VEGFR2to achieve this important medium.Hypoxia is the basic cause of plaque angiogenesis. In Hypoxic conditions, hypoxia-inducible factor-1α (HIF-la), as a important transcription factor, plays a major role in the regulation VEGF. It can increase the expression VEGF, the downstream of pro-angiogenic substances. Reactive oxygen species (Reactive oxygen species, ROS) play an important role in the angiogenic process. The excess of reactive oxygen species involved senescence and apoptosis of endothelial cells and stem cells, resulting in the generation of new blood vessels immature.Nicotinamide adenine dinucleotide phosphate oxidase (NADPH Oxidase) is the main body of the enzyme to produce ROS in vascular endothelial cells. NADPH oxidase-derived ROS plays an important role in the angiogenic endothelial cells NOX4mainly expressed in vascular endothelial cells. The study found that ROS sources from NOX4activation plays an important role in the expression of VEGF.Traditional Chinese Medicine has made considerable progress in Prevention and treatment of atherosclerosis. Previous work had demonstrated that traditional Chinese medicine Shumai capsule can effectively reduce AS plaque angiogenesis. Our research will further investigate the mechanism of Shumai capsule on atherosclerotic plaque. Real-time quantitative RT-PCR technique to detect the plaque VEGF, VEGFR2, HIF-la and NOX4mRNA expression. Western blot technique to detect VEGF, VEGFR2,HIF-la and NOX4protein expression,AimTo detect VEGF, VEGFR2, HIF-la and NOX4mRNA expression by Real-time quantitative RT-PCR technique. To detect VEGF, VEGFR2, HIF-la and NOX4protein expression by Western blot technique. To investigate the mechanism of Shumai capsule on atherosclerotic plaque angiogenesis.MethodsMail50apolipoprotein E-knockout (apoE-/-) mice at8weeks of age were fed a high fat high cholesterol diet including15%fat and0.25%cholesterol. Mail10C57BL/6J mice at8weeks of age were fed a normal chow diet. At20weeks of age, apoE-/-mice were randomly divided into five groups, the ApoE-KO group(Model group), the apoE-/-+SMCH group (SMCH group), the ApoE-/-+SMCL group (SMCL group), the apoE-/-+total panax notoginsenoside group (PNS group) and the apoE-/-+Simvastatin group (Simvastatin group)(n=10for each group). C57BL/6J mice were the normal control group (Control group). Mice in SMCH and SMCL group were given SMC at3500mg/kg,700mg/kg, respectively. Mice in PNS group were given PNS at60mg/kg. And mice in Simvastatin group were given Simvastatin at3mg/kg. All drugs were given orally, once a day for12weeks. Mice in model and control groups were given0.2ml sterile distilled water, once a day for12weeks. After administered for12weeks, all mice were sacrificedDetection contents:(1) Immunohistochemical analysis:Immunohistochemical staining were performed and the expressions of VEGF were detected.(2) RT-PCR: The mRNA expressions of VEGF, VEGF-R2, HIF-1α and NOX4in the aortic tissue were analyzed by using RT-PCR technique.(3) Western blot:The protein expressions of VEGF, VEGF-R2, HIF-la and NOX4in the aortic tissue were analyzed with Western blot technique.Results1. Immunohistochemical analysisThere was a significant difference between Model group and Control group in reducing VEGF expression(P＜0.001). VEGF positive staining area in each treatment group significantly decreased compared with that in Model group(P＜0.01or P＜0.05). SMCH and Simvastatin group exerted the most effective action, and there was a significant difference compared with SMCL group(P＜0.01).2. RT-PCR analysis(1) Comparison of expression of VEGF mRNA:The expression of VEGF mRNA in the model group was higher than that in the normal group (P＜0.001). After treatment with SMC, Simvastatin and PNS, VEGF mRNA was decreased in a different degree compared with the model group(P＜0.01), and among those treatment groups, the Simvastatin group had the lowest VEGF mRNA expression (P＜0.001). There was a significant difference between SMCH group and SMCL group(P＜0.01). There was also a significant difference between Simvastatin and PNS group(P＜0.01).(2) Comparison of expression of VEGFR2mRNA:The expression of VEGFR2mRNA in the model group was higher than that in the normal group (P＜0.001). VEGFR2mRNA was decreased in a different degree in drugs treatment groups compared with the model group (P＜0.01). There was a significant difference between SMCH group and SMCL group (P＜0.01).(3) Comparison of expression of HIF-1α mRNA:The expression of NOX4mRNA in the model group was higher than that in the normal group (P＜0.001). HIF-1α mRNA was decreased in a different degree in drugs treatment groups compared with the model group (P＜0.01). There was no difference between all drugs treatment groups (P＞0.05).(4) Comparison of expression of NOX4mRNA:The expression of NOX4mRNA in the model group was higher than that in the normal group (P＜0.001), After treatment with SMC, Simvastatin and PNS, NOX4mRNA was decreased in a different degree compared with the model group(P＜0.01). Among those treatment groups, PNS group and SMCH group decreased the most significant, and there was a significant difference of those two groups compared with Simvastatin and SMCL group (P＜0.01).3. Western blot analysis(1) Comparison of expression of VEGF protein:The expression of VEGF protein in Model group was higher than that in Control group (P＜0.001). The expression of VEGF protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01). There was no significant difference between SMCH and Simvastatin group (P＞0.05). SMCH and Simvastatin group had significant differences compared with PNS group and SMCL group(P＜0.05).(2) Comparison of expression of VEGFR2protein:The expression of VEGFR2protein in Model group was higher than that in Control group (P＜0.001). The expression of VEGFR2protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01or P＜0.05). The expression of VEGFR2protein was lower in SMCH, Simvastatin and PNS groups than that in SMHL group (P＜0.01).(3) Comparison of expression of HIF-la protein:The expression of HIF-la protein in Model group was higher than that in Control group (P＜0.001). The expression of HIF-la protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01). Simvastatin group had the lowest HIF-1α protein expression(P＜0.01).(4) Comparison of expression of NOX4protein:The expression of NOX4protein in Model group was higher than that in Control group (P＜0.001). After treatment with SMC, Simvastatin and PNS, NOX4protein expression was decreased in a different degree compared with the Model group(P＜0.01). PNS group had the lowest NOX4protein expression(P＜0.01). SMCH group decreased NOX4protein expression in a different degree compared with Simvastatin and SMCL group (P＜0.01).ConclusionShumai capsule could inhibit plaque angiogenesis and promote plaque stabilization. The mechanisms were possibly associated with the suppressive effect of VEGF, VEGFR2, HIF-la and NOX4expression in aortas of apoE-/-mice. |
PDF Full Text Request