| ObjectiveTo observe the effect of Hirudinaria manillensis on relative protein and gene expression in atherosclerotic plaque of ApoE-/- mice, as well as the lipid level in ApoE-/- mice. To investigate the mechanism of Hirudinaria manillensis on anti-atherosclerosis.Methodsmale ApoE-e-knockout(ApoE-/-) mice( 15-week-old) were adopted with high fat cholesterol diet for model. Male C57BL/6J mice(15-week-old) were fed a normal chow diet. At 20 weeks of age, ApoE-/- mices were assigned into six groups randomly, the model group, the BDH group, the BDH group, the Sim group, the HPH group, and the HPL group. C57BL/6J mice were the Con group.(n=8, for each group). mice in HPH, HPL group were given HP at 200mg/kg/d, 75mg/kg/d, respectively in orally, mice in BDH, BDL group were given BD at 400U/kg/d, 200U/kg/d in i.p way. Mice in Sim group were given Simvastatin at 3mg/kg/d orally. Mice in Con group were given 0.2mL sterile distilled water orally. After 10 weeks, the following items were measured:1、Triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C);2、Blood vessel at aortic root by HE staining.;3、 Collagen content of the plaques by Masson staining;4、 The expression of CD34, VEGF, VEGFR2 and HIF-1αat aortic root by immunohistochemical analysis;5、 The expression of VEGFmRNA, VEGFR2mRNA and HIF-1αmRNA in aortic tissue by RT-PCR analysis;ResultsTC,TG and LDL-C level in Mol group are significantly higher than in Con group(P<0.001); TC,TG and LDL-C level in BDH, BDL, HPH, HPL, Sim are significantly lower than Mol(P<0.001 or P<0.05); Sim, HPH, HPL reduce the level of HDL-C compared with Mol(P<0.001 or P<0.05); while BDH, BDL group significantly increase the level of HDL-C compared with Mol group(P<0.05)Compare to the Con group, Atherosclerotic plaques were observed clearly in the aortic sections of ApoE-/- mice .Compared to the Mol group, Sim, BHD,BDH, HPH and HPL group could significantly decreased plaque areas.The collage content with plague was 21.5 ± 2.44%(Mol),51.7 ±2.47%(BDH),45.9 ±4.12%(BDL),37.7± 3.33%(Sim),31.6 ±4.80%(HPH),26.0± 2.10%(HPL).Compare to the Mol group, all treatment group had increased the collagen content of the plaque(P<0.01).CD34-positive areas in each treatment group significantly decrease compare with Mol (P<0.01 ). the difference between the HPL and HPH also show significant.(P<0.05).VEGF-positive areas in each treatment group significantly decrease compare with Mol (P<0.01). VEGF-positive areas in all ApoE-/- group significantly increased compare with Con group(P<0.01 or P<0.05).there is a significant difference between BDH and BDL(P<0.01). Compared with Sim,both HPH(P<0.01) and HPL(P<0.05) incrased the areas.VEGFR2-positive areas in each treatment group significantly decrease compare with Mol (P<0.01) VEGFR2-positive areas in all ApoE-/- groug significantly increased compare with Con group(P<0.01).HIF-la-positive areas in each treatment group significantly decrease compare with Mol (P<0.01) HIF-1α-positive areas in all ApoE-/- groug significantly increased compare -with Con group(P<0.01).there is a significant difference between BDH and BDL(P<0.01). However, there is no difference between HPH and HPL(P>0.05).Comparison of expression of VEGF mRNA: The expression of VEGF mRNA in the Mol group was the higher than that in Con group (P<0.01). The treatment group Sim BDH, BDL, HPH, HPL was decreased compared with Mol.HPH and HPL are both increaed compared with Sim (P<0.01).Comparison of expression of VEGFR2 mRNA: The expression of VEGFR2 mRNA in the Mol group was higher than that in Con group (P<0.01).The treatment group Sim BDH, BDL, HPH, HPL was decreased compared with Mol(P<0.01 or P<0.01).Comparison of expression of HIF-lamRNA: The expression of HIF-lamRNA in the Mol group was higher than that in Con group (P<0.01).The treatment group Sim BDH, BDL, HPH, HPL was decreased compared withMol. HPH and HPL both increaed compared with Sim (P<0.01). HIF-1αmRNA is the highest in BDH(P<0.01).ConclusionsHirudinaria manillensis could decreased lipid level in ApoE-/- mice.Hirudinaria manillensis could inhibite atherosclerotic plaque, increase the collagen in plaque, thereby increased the stability of the plaque.Hirudinaria manillensis could reduce neovascularization in plagues,suppress the expression of VEGF,VEGFR2 , HIF-1αand their mRNA in ApoE-/-mice. |
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