Study Effect Of Angong Niu Huang Pill On Early And Mid-term Atherosclerosis Model Of ApoE^-/- Mice By Immune Inflammatory Pathway

Objective:Cardiovascular disease is the number one killer of human health,and atherosclerosis?AS?is the pathological basis of cardiovascular disease,alleviating AS is critical to control the development of cardiovascular disease.Angong Niuhuang Pill?ANP?is famous Chinese patent medicine used in East Asia for centuries.It could clear heat,remove toxicity,relive convulsion and induce resuscitation.It's used to treat stroke,febrile seizures,meningitis and cerebral hemorrhage.However,there are few studies about AS of causing these critical complications.In this study,Apolipoprotein E deficient(ApoE^-/-)mice in a high-fat diet were used to replicate the AS model,to observe the effect of ANP on early and mid-term AS and elucidate its possible mechanisms.Methods and Results:Firstly,the experiment studied effect of ANP on ApoE^-/-mice early AS model.12SPF C57BL/6J male mice were randomly divided into control group,C57+ANP group?0.50 g/kg,6 mice for each group?;39 SPF ApoE^-/-male mice were randomly divided into ApoE^-/-negative control group(ApoE^-/-+normal diet,n=6),model group(ApoE^-/-+high-fat diet,n=6),simvastatin group?3.5 mg/kg,n=6?,low,medium and high-dose ANP groups?0.25,0.50,1.0 g/kg,7 mice for each group?.Model replication method:High-fat diet was fed continuously for 8 weeks.Method of administration:control group,ApoE^-/-negative control group and model group were administered with normal saline,0.1 m L/10g,once every other day for 8 weeks,administration of ANP was carried out simultaneously with the modeling.After 8 weeks,blood glucose,lipid detection and pathological examination were used to confirm the establishment of AS model;organ coefficient was used to evaluate the effect of ANP on liver and kidney;Treg cells and Th17 cells were analyzed by flow cytometry.The expressions of inflammatory factors and chemokines were measured by using RT-q PCR.protein expression was measured by using immunohistochemistry.The results showed that serum CHOL,TG,HDL and LDL was significant difference between the ApoE^-/-negative control group and the control group?p<0.05or 0.01?.There is a small amount of lipid plaque deposits in the aortic root and aorta.There was no significant difference between the C57+ANP group and the control group?p>0.05?,no obvious lipid plaque deposition in the aortic root and aorta.Compared with the control group,indicators of the model group had a significant difference?p<0.05 or 0.01?,obvious lipid plaque deposition in the aortic root and aorta.Compared with the ApoE^-/-negative control group,serum lipids of the model group were increased?p<0.05 or 0.01?.There was no significant difference in liver coefficient and kidney coefficient between the ANP groups and the model group.Compared with the model group,ANP had no significant difference on blood lipids and glucose.ANP could increase the proportion of Treg cells and decrease the proportion of Th17 cells?p<0.05or 0.01?.ANP down-regulated the expression of IL-6 and up-regulated the expression of TGF-?1 in the spleen?p<0.01 or 0.05?.ANP down-regulated the expression of IL-17A,IL-6,MCP-1,MCP-2,MCP-3,and CCR2,CXCR3 and up-regulated the expression of IL-10,TGF-?1 in the aorta,and ANP decreased protein expression of ICAM-1 and VCAM-1 in the aortic root?p<0.05 or 0.01?.The simvastatin decreased blood lipids,the proportion of Th17 cells,the levels of inflammatory factors and the protein expression of ICAM-1 and VCAM-1,increased the proportion of Treg cells in the aorta and aortic root.?p<0.05 or 0.01?.The above experiments preliminarily confirmed that ANP have effect of anti-atherosclerosis on ApoE^-/-mice early AS model,next experiment further studied the mechanisms of anti-atherosclerosis of ANP on ApoE^-/-mice AS model.12 SPF C57BL/6J male mice were control group;60 SPF ApoE^-/-male mice were randomly divided into model group,simvastatin group?3.0 mg/kg?,low,medium and high-dose ANP groups?0.25,0.50,1.0 g/kg?,12 mice for each group.Model replication method:High-fat diet was fed continuously for 10 weeks.Method of administration:the control group and the model group were administered with saline,0.1m L/10g,once every other day for 10 weeks,administration of ANP was carried out simultaneously with the modeling.After 10 weeks of administration,serum biochemical indicators were detected,Th17/Treg ratio was analyzed by flow cytometry in the spleen,RT-q PCR and Western blotting were used to detect the expression of inflammatory genes and proteins in the spleen and aorta,and tissue immunofluorescence was used to detect inflammatory cell infiltration in the aortic root.The results showed that ANP significantly increased thymus and spleen index,and decreased the ratio of LDL-C/HDL-C in the high-dose ANP group?p<0.05?.the ratio of Treg/CD4⁺T was increased,and the ratio of Th17/CD4⁺T and Th17/Treg was decreased in the ANP groups and the simvastatin group?p<0.05 or 0.01?.The expression of ROR?t was down-regulated,the expression of FOXP3 was up-regulated in the spleen,and the expression of IFN-?,TNF-a,IL-1?,CCL2,CCR2,CCL5,CCR5,MMP-2,MMP-9 was down-regulated?p<0.05 or 0.01?.The levels of serum IL-10were elevated,and the levels of IL-6 were reduced?p<0.05 or 0.01?.Masson staining showed that ANP could alleviate pathological changes,and immunofluorescence staining showed that ANP could alleviate inflammatory cell infiltration in the aortic root.The macrophages,dendritic cells and vascular smooth muscle cells was significantly reduced?p<0.01 or 0.001?.Conclusion:1.ApoE^-/-mice could spontaneously produce AS,but the process is quite long,and high-fat diet could accelerate the AS process in ApoE^-/-mice.The ApoE^-/-mice in a 8or 10 weeks high-fat diet could establish the early or mid-term AS model.2.ANP could recover Th17/Treg balance by regulating the expression of RORgt and Foxp3,and play anti-chronic inflammatory effect by inhibiting the expression of IL-6,IL-1?,TNF-a,IFN-g,CCL2,CCL5,CCR2,CCR5,ICAM-1,VCAM-1 and promoting expression of IL-10 and TGF-?1;maintain plaque stability by down-regulating the expression of MMP-2,MMP-9 and alleviating inflammatory cell infiltration.3.ANP regulate the Th17/Treg balance,inhibit chronic inflammation and maintain plaque stability to exert multi-channel anti-atherosclerosis in ApoE^-/-mice early and mid-term AS model.In addition,low-dose ANP focuses on anti-inflammatory effects,and high-dose ANP focuses on immunomodulatory effects.