Objective·To investigate the role and mechanism of CaMKKβ in RAW264.7 macrophage M2 polarization induced by interleukin-4 (IL-4).Methods·IL-4 induced RAW264.7 polarization was measured by RT-PCR and ELISA.Western blotting was used to analyze the total and phosphorylated protein levels of CaMKKβ,AMPK,JAK2 and STAT3 during polarization.A lentiviral vector carrying an shRNA targeting the CaMKKβ gene was successfully constructed.The expression of M1 and M2 markers and the expression of phosphorylated AMPK,JAK2,STAT3 were detected by RT-PCR and Western blotting respectively.The protein activity ofAMPK,JAK2 and STAT3 were selectively blocked,and then the effect of IL-4 on macrophage polarization was detected by RT-PCR.Results·IL-4 significantly polarized RAW264.7 cells towards M2 macrophages (P＜0.05),and the phosphorylated protein expression of CaMKKβ,AMPK,JAK2,STAT3 was significantly increased (all P＜0.05).The expression of M2 markers induced by IL-4 was decreased and the phosphorylation ofAMPK,JAK2 and STAT3 was also decreased after knockdown of CaMKKβ by shRNA (all P＜0.05).The polarization of macrophages to M2 was decreased after the activities of AMPK,JAK2 and STAT3 proteins were blocked respectively (all P＜0.05).Conclusion·CaMKKβ promotes IL-4 induced M2 macrophage polarization via activation of AMPK/JAK2/STAT3 pathway.

Objective To observe the effects of Jianpi Qutan Huayu Prescription on oxidative stress and inflammatory response in mini-pigs with atherosclerosis(AS);To explore its mechanism based on the NOX5-ERK1/2 signaling pathway.Methods Twelve Bama mini-pigs were randomly divided into control group,model group,and Jianpi Qutan Huayu Prescription low-and high-dosage groups,with 3 pigs in each group.A high-fat diet was used to feed for 24 weeks to construct an AS model,and the treatment group was also supplemented with Jianpi Qutan Huayu Prescription in the feed.The general condition of mini-pigs(body length,abdominal circumference,body mass,food intake,and fecal water content)was measured at week 0,16,and 24 of administration,HE staining was used to observe the morphology of aortic tissue,while oil red O staining was used to observe lipid deposition in aortic and myocardial tissue,transmission electron microscopy was used to observe the ultrastructure of aortic tissue,and a fully automated biochemical analyzer was used to detect serum contents of TC,HDL-C,and LDL-C.ELISA was used to detect the contents of serum reactive oxygen species(ROS),interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-α,hypersensitivity-C-reactive protein(hs-CRP),vascular cell adhesion molecule-1(VCAM-1),and intercellular adhesion molecule-1(ICAM-1).Western blot was used to detect the expressions of NADPH oxidase 5(NOX5),extracellular signal regulated kinase 1/2(ERK1/2),p-ERK1/2,VCAM-1,and proliferating cell nuclear antigen(PCNA)proteins.Results Compared with the control group,the abdominal circumference,body mass,and food intake of mini-pigs in the model group increased at 16 and 24 weeks(P<0.01),there was significant thickening of the inner membrane of aorta,destruction of endothelial cells,lipid deposition,edema of smooth muscle cells,and significant swelling of mitochondria,serum TC,LDL-C contents and the contents of ROS,IL-6,IL-10,TNF-α,hs-CRP,VCAM-1,and ICAM-1 increased,while the content of HDL-C decreased(P<0.01);the expressions of NOX5,p-ERK1/2,VCAM-1,and PCNA proteins in aortic tissue increased(P<0.01).Compared with the model group,Jianpi Qutan Huayu Prescription low-and high-dosage groups showed a decrease in abdominal circumference,body mass,and food intake at 16 and 24 weeks(P<0.05,P<0.01),the plaque area and lipid deposition were reduced,and the damage to endothelial cells was alleviated,serum TC,LDL-C contents and the contents of ROS,IL-6,IL-10,TNF-α,hs-CRP,ICAM-1,and VCAM-1 decreased,and the content of HDL-C increased(P<0.01,P<0.05);the expressiond of NOX5,p-ERK1/2,VCAM-1,and PCNA proteins in aortic tissue decreased(P<0.01,P<0.05).Conclusion Jianpi Qutan Huayu Prescription can effectively alleviate AS in mini-pigs,and its mechanism may be related to inhibiting the activation of the NOX5-ERK1/2 signaling pathway and alleviating oxidative stress-induced inflammatory response.