Effect Of Jiedu Huoxue Formula On Plaque Stability Of ApoE^—/— Mice By Regulating CD40L/NF-κB Signaling Pathway

Research purpose:A large number of experimental and clinical studies have proved that the method of detoxification and activating blood circulation is effective in the treatment of atherosclerosis（AS）vulnerable plaques and can inhibit the inflammatory response.The preliminary experimental study of our research team showed that Jie du Huo xue Formula can interfere with inflammatory response by inhibiting the TLR-4/NF-κB signaling pathway,and thus inhibit the intimal hyperproliferation and restenosis after percutaneous coronary intervention（PCI）.On this basis,we believe that the use of Jiedu Huoxue Formula to intervene vulnerable plaques and Acute coronary syndrome（ACS）which is the basis of pathology is the key to the pathogenesis.This project intends to start with the inhibition of inflammatory response in vulnerable plaques and take CD40L/NF-κB signaling pathway as the entry point to observe the effect of Jiedu Huoxue Formula on inflammatory response in vulnerable plaques of ApoE^—/—mice,and confirm the effectiveness of Jiedu Huoxue Formula on inflammatory response by inhibiting CD40L/NF-κB signaling pathway.To provide a new way of thinking and treatment for the pathogenesis of ACS.Methods:Forty 8-week-old male ApoE^—/—mice were selected,and 6 of them were fed with normal diet only as the control group.After modeling,they were given0.2 m L/d of normal saline.During the feeding process,4 mice died due to severe biting,and the remaining 30 mice were only fed with high-fat diet for 5 weeks,and then randomly divided into 5 groups:A.Model group was given 0.2 m L/d normal saline without drug administration after modeling;B.Positive control group was given atorvastatin 3.0mg/kg.d;C.TCM low-dose group,5.35g/kg.d;D.Medium-dose group,10.7g/kg.d;E.In the high-dose group,21.4g/kg.d.Chinese herbal decoction was given by gavage.In addition,8 C57BL/6J mice（same genetic background）of the same sex and week of age were selected and fed with ordinary diet and set as blank group.After modeling,0.2 m L/d of normal saline was given by gavage.Mice in the above 7 groups were given free water（acidified water:p H2.8～3.0）for5 consecutive weeks.During the experiment,they were weighed once every two weeks,and the dosage was adjusted according to the body weight of the mice during the administration.After administration,blood samples were collected by venous plexus method,and serum lipidemia,inflammatory markers IL-1βand ICAM-1 were measured.The heart and aorta tissues were dissected and separated under microscope.After the aorta tissues were stained with oil red,the ratio of the plaque area to the full-length aorta area was calculated using the Image J image analysis system.The expression levels of CD40L and NF-κB protein in aortic root were detected by immunohistochemistry.Result:1.Changes in body weight of mice in each group:mice in the C57BL/6J group and the control group were in good mental state and their fur was bright and lush;In contrast,mice in the model group were mentally poor,less active,and obese.The active degree of each Chinese medicine group and positive control group was general,and the fur was slightly sparse.Compared with the initial body weight at the end of the experiment,the body weight of model group increased significantly（P<0.05）.2.Changes of blood lipid in each group:the blood lipid level in the control group was significantly higher than that in the C57BL/6J group（P<0.05）,but lower than that in the model group（P<0.05）.Compared with model group,TC value in low,medium and high dose TCM groups decreased significantly（P<0.05）,but no significant difference was found in low dose TCM group（P>0.05）.TG value in high-dose group decreased significantly（P<0.05）,but there was no significant difference in the decrease of TG value between low-dose and medium-dose groups（P>0.05）.HDL value in TCM groups was significantly increased（P<0.05）;LDL values in TCM groups were significantly decreased（P<0.05）.3.The expression of serum inflammatory cytokines in each group:（1）Compared with the control group,the expression of ICAM-1 in C57BL/6J group was significantly decreased（P<0.05）,but the expression of ICAM-1 in model group was significantly increased（P<0.05）.Compared with model group,the level of ICAM-1in medium-dose and high-dose groups was significantly decreased（P<0.05）,but there was no significant difference in the level of ICAM-1 in low-dose group（P>0.05）.（2）Compared with the control group,there was no significant difference in the level of IL-1βin C57BL/6J group（P>0.05）,but the level of IL-1βin model group was significantly increased（P<0.05）.Compared with model group,the level of IL-1βin TCM groups was significantly decreased（P<0.05）.4.Oil red O staining data showed that compared with the control group,the plaque area proportion of C57BL/6J group decreased（P<0.05）,while the plaque area proportion of model group increased（P<0.05）.Compared with model group,the proportion of plaque area in TCM groups decreased（P<0.05）.5.Immunohistochemical detection of CD40L and NF-κB protein expression in aortic root:（1）Comparison of CD40L protein expression area:Compared with the control group,there was no significant difference in the expression of C57BL/6J histone（P>0.05）,but the expression of histone in model was significantly increased（P<0.05）.Compared with model group,low dose histone expression had no significant difference（P<0.05）,while medium and high dose histone expression decreased significantly（P<0.05）.（2）Comparison of the positive expression area of NF-κB protein:compared with the control group,there was no significant difference in the expression of C57BL/6J histone（P>0.05）,but the expression of histone in the model was significantly increased（P<0.05）.Compared with model group,histone expression of low,medium and high doses was significantly decreased（P<0.05）.Conclusion:1.Jiedu Huoxue Formula can effectively reduce the levels of TG,TC and LDL in ApoE^—/—atherosclerotic mice,improve the level of HDL,and reduce the body weight of mice,and the effect is most obvious in the group of high dose of traditional Chinese medicine.2.Jiedu Huoxue Formula can inhibit the expression of inflammatory factors ICAM-1 and IL-1βin serum of ApoE^-/-atherosclerotic mice,reduce the inflammatory response in plaques and slow down the process of AS plaque formation.3.Jiedu Huoxue Formula can effectively inhibit the formation of plaque in ApoE^—/—atherosclerotic mice and reduce the plaque area.4.Jiedu Huoxue Formula can reduce the expression of CD40L and NF-κB protein in ApoE^—/—atherosclerotic plaques of mice,block the activation and transmission of CD40L/NF-κB signaling pathway,reduce the release of downstream inflammatory factors,and have a stabilizing effect on AS-vulnerable plaques.