The Effect And Mechanism Of Persimmon Leaves Total Flavonoids On Atherosclerotic Lesions Of ApoE^-/- Mice

Objective:The flavones is the effective parts extracted from Persimmon Leaves. Modern research shows Persimmon leaves total flavonoids(PLTF)has shown promise in softening blood vessels, enhancing the body immunity, hemostasis, sterilization and so on, however, the effect of PLTF on atherosclerotic plaque formation have not been reported. So in our study, in order to investigate PLTF for treatment of atherosclerosis and its possible mechanism , AS model of apoE-/- mice was established to observe the effect of PLTF on the lipid levels, the concentration of MDA and the activity of SOD. Besides that, HE staining and oil red O staining was used to observe the histomorphological change and measure the area of atherosclerotic plaques, immunohistochemistry analysis was performed to detect the expressions of P-Selectin, ICAM-1, VCAM-1 in thoracoabdominal aortic atherosclerotic lesions andα-actin, MMP-9 in brachicephalic arteries atherosclerotic lesions.Methods:Part I: The Effect and Mechanism of Persimmon Leaves Total flavonoids on Thoracoabdominal Aortic Atherosclerotic Lesions of apoE-/- Mice8-week old male ApoE-/- mice were divided randomly into model group (normal saline,30mg/kg/day) and PLTF treated group (PLTF,30 mg/kg/day), all the mice were fed with high fat diet. Eight 8-week old male C57BL/6 mice were set as the control group, given normal rodent diet everyday. At the 16th week with continuous intragastric administration, the serum and aortas were taken for the determination of a series of indicators. Plasma lipids were determined by biochemical methods, HE staining and oil red O staining of the proximal end was used to observe the histomorphological change and measure the area of atherosclerotic plaques, Immunohistochemistry analysis was performed to detect the expressions and distribution of P-Selectin, ICAM-1 and VCAM-1. All the images were measured by"HMIAS-2000"software.Part II: The Effect and Mechanism of Persimmon Leaves Total flavonoids on Stability of Brachicephalic Arteries Atherosclerotic Plaque of apoE-/- Mice8-week old male apoE-/- mice were divided randomly into three groups: model group (normal saline, 30 mg/kg/day) , PLTF treated group (PLTF, 30 mg/kg/day), simvastatin treated group (simvastatin, 30 mg/kg/day), all the mice were fed with high fat diet. Eight 8-week old male C57BL/6 mice were set as the control group, given normal rodent diet everyday. At the 12th week with continuous intragastric administration, the serum and brachicephalic arteries were taken for the determination of the plasma lipids levels and histomorphological changes almost same as the first part. Elastic fiber dyeing shows the number of the fibrous caps. Immunohistochemistry analysis was performed to detect the expressions ofα-actin and MMP-9. All the images were measured by"HMIAS-2000"software, too.Results:Part I: The Effect and Mechanism of Persimmon Leaves Total flavonoids on Thoracoabdominal Aortic Atherosclerotic Lesions of apoE-/- Mice1. Effect of PLTF on the plasma lipids levels: Serum TC, TG and VLDL levels in model group and PLTF treated group were significantly higher than that in control group (P<0.05), while these indicators didn't differ significantly between model group and PLTF treated group (P>0.05).2. Effect of PLTF on MDA and SOD: The concentration of MDA in PLTF treated group and control group were significantly lower than that in model group (P<0.05). The activity of SOD in PLTF treated group and model group didn't differ significantly, however, both lower than that in control group(P<0.05).3. Effect of PLTF on histomorphological change of lesions in thoracoabdominal aorta: The aorta endomembrane of control group was of smooth and thinness without plaque; In model group, some plaques were bigger and broke into the lumen, aortic cavity was narrower. In PLTF treated group, the thoracoabdominal aorta had a more limited plaque lesion and a slighter narrowing of aortic cavity. The ratio of plaque area to aortic cavity area was significantly smaller in PLTF treated group than that in model group(P<0.05).4. Effect of PLTF on adhesion molecules: A wide range of expressions of P-Selectin, ICAM-1 and VCAM-1 were found in model group compared with that in PLTF treated group, showing significantly difference between this two groups(P<0.05).Part II: The Effect and Mechanism of Persimmon Leaves Total flavonoids on Stability of Brachicephalic Arteries Atherosclerotic Plaque of apoE-/- Mice1. Effect of PLTF on the plasma lipids levels: Serum TC, LDL levels were significantly increased in the model group relative to that in control group(P<0.05), while HDL level was significantly decreased(P<0.05). However, all these plasma lipid levels in PLTF treated group and simvastatin treated group didn't differ significantly compared with that in model group.2. Effect of PLTF on MDA and SOD: The concentration of MDA in model group were significantly higher than that in other groups(P<0.05). The activity of SOD in model group, PLTF treated group and simvastatin treated group were significantly decreased relative to that in control group(P<0.05).3. Effect of PLTF on histomorphological change of lesions in brachiocephalic artery: Advanced AS lesions were found in brachiocephalic arteries of all the groups except the control group, however, In model group, some plaques were bigger and the ratio of plaque area to aortic cavity area was significantly higher than that in other groups(P<0.05).4. Effect of PLTF on stability of brachicephalic arteries atherosclerotic plaque: The numbers of the buried fibrous caps in model group was more than that in PLTF treated group and simvastatin treated group(P<0.05), as well as the expression of the MMP-9. Besides that, an increased expression of MMP-9 was found in the unstable plaque groups versus stable ones (P<0.05).Conclusions:1. The PLTF can inhibit to some extent the progress of atherosclerotic plaque in apoE-/- mice, which might not be significantly related to its effect of decreasing mice blood lipid.2. PLTF can inhibit the progress of atherosclerotic plaque in apoE-/- mice through the effect of anti-oxidative, but not through the enhancement of SOD activity to participate in the protective mechanisms.3. The weakened expression of P-Selectin, ICAM-1, VCAM-1 in the PLTF group indicated that PLTF can inhibit the progress of atherosclerotic plaques by reducing the expression of adhesion molecules. Besides that PLTF played an important role in the instabilizing plaques through the effect of inhibiting the expression of MMP-9.