To Explore The Mechanism Of Guanxinkang Anti-atherosclerosis Based On ERK5-regulated Macrophage Efferocytosis

Objective:Through animal experiments,the expression of signaling molecules related to macrophage efferocytosis in ERK5-MKO/LDLR^-/-atherosclerosis mice improved by Guanxinkang,a traditional Chinese medicine compound,was observed.In particular,the possible mechanism of Guanxinkang anti-atherosclerosis was discussed based on ERK5-regulated macrophage efferocytosis.Methods:（1）Evaluation of ERK5-MKO/LDLR^-/-atherosclerosis mouse modelThe ERK5-MKO/LDLR^-/-atherosclerosis mouse model was established by feeding the high-fat diet for 24 weeks.The mouse model was evaluated by body weight,blood lipids,and aortic pathology.（2）Pathological analysisTo observe the change of the vulnerability of aortic root plaque after interventions,the following tests were used:H&E staining for the size of plaques,Oil red O staining for plaque lipid content,Masson staining for collagen content,and Immunohistochemical for the contents of macrophages and smooth muscle cells of plaque,Immunofluorescence was used to detect apoptotic cells（ACs）of plaque.（3）Real-Time Quantitative PCR（RT-q PCR）and Western blotThe expression of macrophage efferocytosis related signaling molecules in mouse aorta,including ERK5,S1P1,Annexin A1,Axl,Pro S,PPAR-δ,Tim4,C1qa,was detected by Western blot and PCR.Results:（1）Blood lipid level in miceCompared with Lys MCre⁺LDLR^-/-model group,the levels of TG,TC and HDL in ERK5-MKO/LDLR^-/-model group mice were significantly increased（p<0.05）,and the level of LDL was on the rise,which was not statistically increased（p>0.05）;After intervention with Guanxinkang,the levels of TC and LDL in Lys MCre⁺LDLR^-/-mice and ERK5-MKO/LDLR^-/-mice were significantly decreased（p<0.05）.（2）Pathological analysisCompared with Lys MCre⁺LDLR^-/-model group,PA/LA and NCA/PA of mouse aortic root plaques of ERK5-MKO/LDLR^-/-model group were significantly increased（p<0.05）,the contents of lipid and macrophages were increased（p<0.05）,the contents of collagen and smooth muscle cells were decreased（p<0.05）,and the contents of ACs were increased.After the intervention of Guanxinkang and simvastatin,PA/LA and NCA/PA of mouse aortic root plaques of Lys MCre⁺LDLR^-/-mice and ERK5-MKO/LDLR^-/-mice narrowed,the contents of lipid and macrophages decreased（p<0.05）,the contents of collagen and smooth muscle cells increased（p<0.05）,and the contents of ACs decreased.（3）RT-q PCR and Western blotCompared with Lys MCre⁺LDLR^-/-model group,the m RNA and protein contents of the signaling molecules of Axl,Pro S,PPAR-δ,Tim4,and C1qa related to macrophage efferocytosis in ERK5-MKO/LDLR^-/-model group statistically decreased（p<0.05）.After the intervention of Guanxinkang and simvastatin,the expression of Axl,Tim4,PPAR-δ,and C1qa were increased in both Lys MCre⁺LDLR^-/-mice and ERK5-MKO/LDLR^-/-mice（p<0.05）,while the contents of m RNA and protein of Pro S changed nothing.There were significant differences in the m RNA and protein of Axl,Tim4,PPAR-δand C1qa between ERK5-MKO/LDLR^-/-Guanxinkang group and Lys MCre⁺LDLR^-/-Guanxinkang group（P<0.05）.Conclusion:Knockout of the ERK5 gene aggravates the progression of AS,which may regulate macrophage efferocytosis in the aorta.Chinese herbal compound Guanxinkang and simvastatin can up-regulate macrophage efferocytosis by ERK5signaling pathway through the expression of Axl,Tim4,PPAR-δ,C1qa.In addition to ERK5,there may be other molecular mechanisms affecting the phagocytosis of Guanxinkang.