Mechanism Study For Effects Of Ningxintong Granule On Atherosclerosis Vulnerable Plaques In Rabbit Model

Mechanism study for effects of Ningxintong granule on atherosclerosis vulnerable plaques in rabbit modelObjective:To investigate the effect of Ningxintong granule (NXTG, a self-made drug composed from Chinese natural medicine of Radix Astragali, Radix Puerariae, Rhizoma Chuanxiong, Radix Ilecis Pubescens and Herba Asari, each pack contains 1Og of compound) on stabilizing the vulnerable plaques and address the mechanisms of therapeutic effects of NXTG on stabilization of vulnerable plaques.Methods:42 of healthy male new Zealand white rabbits were randomized into the group A (n=6), and the group B (n=36), rabbits in group B were divided into four subgroups randomly: group B1 was treated with high dose of NXTG (n=9,2g-kg-1·-d-1), B2 was treated with normal dose of NXTG (n=9, 1g-kg-1·d-1), group B3 was administered with Xuezhikang (n=9, 60mg-kg-1·d-1) and group B4 was as baseline control group (n=9). Rabbits in group A were fed with standard chow. Rabbits in group B were fed with the high cholesterol diet for 2 weeks, then underwent the ballon-induced abdominal aortic wall injury. At 3rd week, first of drug intervention was added during the process. Rabbits in group B were still fed by the cholesterol-rich diet. At the end of the 8th week, recombinant adenovirus, which carrying the p53 gene was injected through a catheter into the aortic segments rich in plaques in group B. After two weeks, all of the remaining rabbits in group B underwent pharmacological triggering with injection of Chinese Russell's viper venom (CRVV) and histamine. At the beginning of the experiment and 12th week, blood samples were collected for measuring the lipid profile. The Fibrinogen level on grouping, pre-pharmacological triggering and post-pharmacological triggering, the concentrations of hs-CRP, IL6, MMP-1, TIMP-1, ICAM-1, VCAM-1 on grouping, pre-pharmacological triggering and post-pharmacological triggering were measured using the ELISA method.The abdominal aorta were processed and examined by HE staining. Using transmission microscope to observe the ultra structure of the AS segments. The levels of NF-κB, MCP-1, MMP-1, TIMP-1, ICAM-1 and VCAM-1 were determined by means of immunohistochemistry. The protein expressions of NF-κB, MCP-1, MMP-1 and TIMP-1 in the lesions were examined using Western Blot analysis The mRNA expressions of IκB, MCP-1, VCAM-1, MMP-1 and TIMP-1 in the abdominal arterial atherosclerosis lesions were measured using the real-time fluorescence quantitative PCR. SMC apoptosis was detected by TUNEL assay.Results:1. Establishment of vulnerable plaques animal model:Rabbits were treated with high cholesterol diet followed by balloon-induced abdominal aortic wall injury, then, tranfected the human wild-type p53 gene in the location of plaques to build the vulnerable plaques animal model.2. Compared with Group B4, the fibrous cap thickness(FCT) in medication groups (group B1, B2 and B3) was significantly thicker and the intima-media thickness(IMT) was significantly decreased (P<0.05). The ratio of FCT and IMT was significantly different between Group B4 and treatment groups (P<0.05). There were no significant difference in the parameter such as FCT, IMT, ratio of FCT and IMT between groupB1 and group B3. The ratio of FCT and IMT was significantly lower in group B2 than group B1 (P<0.05).3. Transmission microscopy showed that there was no significant difference in ultra structure of the AS segments among four model groups.4. Lipid profile:After 12 weeks of high lipid diet, the serum lipid levels (TCH, TG,LDL-C level and HDL-C)in group B1, B2, B3 and B4 significantly increased (P<0.05). Compared with group B4, the levels of TCH, TG and LDL-C in group B1 were decreased significantly(P<0.05), Compared with the group B4, the levels of TCH, LDL-C and HDL-C in group B3 were decreased significantly(P<0.05), There is no significant differences in TCH, TG, LDL-C and HDL-C between group B1 and B3.5. Fibrinogen level:Compared with the beginning of the experiment, the level of fibrinogen in group B1, B2, B3 and B4 was increased significantly before pharmacological triggering and after pharmacological triggering. There was no significant difference among these four groups.6. Compared with the beginning of the experiment, the concentrations of hs-CRP, IL-6, MMP-1, TIMP-1, ICAM-1 and VCAM-1 in group B1, B2, B3 B4 was increased significantly before pharmacological triggering and after pharmacological triggering (P<0.05). Compared with group B4,the concentrations of hs-CRP, IL-6, MMP-1, TIMP-1, ICAM-1 and VCAM-1 in group B1, B3 were decreased significantly after pharmacological triggering (P<0.05), but there were no significant difference between group B1 and B3. The concentrations of hs-CRP, IL-6 and TIMP-1 was significantly lower in group B1 than in group B2 (P<0.05).7. Immunohistochemistry analysis showed that expressions of NF-κB, MCP-1, MMP-1, TIMP-1, ICAM-1 and VCAM-1 were found in the atherosclerotic plaques. Compared with group B4, the expressions of NF-κB, MCP-1, MMP-1, ICAM-1 and VCAM-1 detected by immunohistochemistry in the group B1 and B3 were much lower (P<0.05), but there was no significant difference between group B1 and group B3. Compared with group B4, TIMP-1 positive cells was lower in group B1 (P<0.05). The expressions of NF-κB and ICAM-1 was significantly lower in group B1 than in group B2 (P＜0.05).8. Western Blot analysis showed, that the protein expressions of NF-κB, MCP-1, MMP-land TIMP-1 were detected in the atherosclerosis lesions. Compared with group B4, the protein expressions of NF-κB, MCP-1, MMP-land TIMP-1 in group B1, B2 and B3 were much lower in the atherosclerosis lesions (P<0.05), but there was no significant difference between group B1 and group B3. The expressions of NF-κB, MCP-1, MMP-land TIMP-1 in the group B2 were higher compared with group Bl (P<0.05).9. The expressions of IκB, MCP-1, MMP-land TIMP-1 mRNA in Group B4 were significantly higher than Group A (P<0.05).Compared with group B4, the mRNA expressions of IκB, MCP-1, MMP-land TIMP-1 in group B1 and B3 were significantly lower in the atherosclerosis lesions (P<0.05), but there was no significant difference between group B1 and group B3. The mRNA expression of VCAM-1 was significantly lower in group B3 than group B4(P<0.05), but there was no significant difference of VCAM-1 mRNA expression between group B1 and group B4. The mRNA expressions of MMP-1, and IκB in the group B2 were higher compared with group B1(P<0.05).10. TUNEL analysis showed the positive cells in four groups. Compared with group B4, positive cells in group B1, B2 and B3 were significantly different(P<0.05). The positive cells in group B1 was slightly lower than group B3, but there was no significant difference between the two groups.Conclusion:NXTG has effects of anti-inflammation, decreasing the level of lipid and leading to stabilize the vulnerable plaques.