| In vivo,excess lipids are deposited in the arterial wall,forming atherosclerotic plaques.Changes in the polarization phenotype of macrophages and the accompanying inflammatory response play an important regulatory role in the formation of atherosclerotic plaques.Macrophage autophagy contributes to the restoration of cellular homeostasis and increases the stability of atherosclerotic plaques.Carnosic acid(CA)is a phenolic diterpene compound extracted from plants of the genus Rosmarinus,with various pharmacological activities,such as antioxidant,anti-aging,and regulation of lipid metabolism.The Keap1/Nrf2 signaling pathway is an important antioxidant pathway in vivo,which can inhibit the formation of atherosclerotic plaques and increase their stability.This study induced foam cell formation in RAW264.7 cells with sodium palmitate(PA),observed autophagy and polarization changes in RAW264.7 cells after CA intervention,and explored the mechanism by which CA inhibits macrophage foam cell formation based on the Keap1/Nrf2 signaling pathway,providing in vitro experimental basis for the prevention and treatment of atherosclerosis.The methods and main results of this study are as follows:(1)The positive rates of Oil Red O and Bodipy lipid fluorescence staining in the PA+CA group were significantly lower than those in the PA group(p<0.05).(2)Western blot(WB)showed that the expression level of autophagic substrate protein p62 in the PA+CA group was significantly lower than that in the PA group,while the ratio of LC3 II/I was significantly higher than that in the PA group(p<0.05),and there was a dose-dependent relationship.(3)RT-qPCR and WB showed that compared with the PA group,the expression levels of the pro-inflammatory cytokines IL-1β,IL-6,TNF-α,and the M1 polarization markers i NOS m RNA and i NOS protein in the PA+CA group were significantly decreased,while the expression levels of the anti-inflammatory cytokines IL-4,IL-10,TGF-β,and the M2 polarization markers CD206 and Arg1 m RNA,and CD206 protein were significantly increased(p<0.05).Double immunofluorescence showed that compared with the PA group,the relative fluorescence intensity of the M1 polarization marker CD86 in the PA+CA group was significantly decreased,while that of the M2 polarization marker CD206 was significantly increased(p<0.05).Flow cytometry(FC)showed that compared with the PA group,the proportion of M1 macrophages(CD86+/CD206-)in the PA+CA group was significantly decreased,while the proportion of M2 macrophages(CD86-/CD206+)was significantly increased(p<0.05).(4)After inhibiting autophagy in RAW264.7 cells with 3-methyladenine(3-MA),the positive rate of Oil Red O increased significantly,the LC3 II/I ratio decreased significantly,the expression level of i NOS protein increased significantly,the expression level of CD206 protein decreased significantly,the relative fluorescence intensity of CD86 increased,the relative fluorescence intensity of CD206 decreased,the proportion of M1 macrophages(CD86+/CD206-)increased significantly,and the proportion of M2 macrophages(CD86-/CD206+)decreased significantly(p<0.05).When RAW264.7 cells were activated with rapamycin(RAPA),the above situation was reversed.(5)The WB detection of Keap1 and Nrf2 showed that compared with the PA group,the cytoplasmic protein expression of Nrf2 and Keap1 in the 10μM CA group was significantly decreased,while the nuclear protein expression of Nrf2 was significantly increased(p<0.05).(6)After inhibiting Nrf2 expression in RAW264.7 cells with ML385,the Oil Red O positive rate significantly increased,the LC3 II/I ratio significantly decreased,the protein expression level of i NOS significantly increased,the protein expression level of CD206 significantly decreased,the relative fluorescence intensity of CD86 increased,the relative fluorescence intensity of CD206 decreased,the proportion of M1 macrophages(CD86+/CD206-)significantly increased,and the proportion of M2macrophages(CD86-/CD206+)significantly decreased(p<0.05).However,these effects were reversed when Nrf2 expression was activated by Eze.Based on the above results,it can be concluded that CA promotes autophagy in RAW264.7 cells by activating the Keap1/Nrf2 signaling pathway,which increases the proportion of M2 polarized macrophages and inhibits the proportion of M1 polarized macrophages,ultimately suppressing foam cell formation in RAW264.7 cells.These findings provide a theoretical basis and experimental evidence for the intervention of atherosclerosis development using CA. |
