| Atherosclerosis（AS）is a chronic and progressive disease,which is the pathological basis of cardiovascular diseases.It can be formed in the large and middle arteries.Inflammatory response runs through the development of AS,and anti-inflammatory therapy is an important treatment for AS.Autophagy is a"self-feeding"phenomenon in cells.The stability of intracellular environment and normal physiological function largely depend on the phenomenon of autophagy.Macrophages are the main inflammatory cells in atherosclerotic plaques,and the autophagy function of macrophages plays an important role in the prevention and treatment of AS.Phosphatidylinositol 3-kinase（PI3K）/protein kinase B（PKB/Akt）is the main signaling pathway regulating autophagy,which can affect the progression of AS.Berberine（BBR）,which can be extracted from Coptis chinensis,Phellodendron chinense and other plants,is a kind of isoquinoline alkaloid,which can prevent and treat AS by regulating lipid,anti-inflammation,anti-oxidative stress,and improving vascular endothelial dysfunction.However,whether BBR can promote macrophage autophagy and reduce inflammatory response by regulating PI3K/Akt signaling pathway needs to be further studied.Objective:In this study,lipopolysaccharide（LPS）was used to induce macrophage inflammation to establish a cell model to explore whether BBR can promote macrophage autophagy and reduce inflammation by regulating PI3K/Akt signal pathway,which could offer a potential theoretical basis for BBR prevention and treatment of AS.Methods:1.In this study,RAW264.7 macrophages from Apo E-/-mice were isolated and LPS was used to induce inflammatory reaction of RAW264.7 macrophages to establish cell model.2.The grouping and treatment of the cells were divided into 7 groups:（1）blank control group:only 1640 cell medium was added;（2）LPS group:LPS 200μg·ml-1was added for 48 hours;（3）PI3K-si RNA group:pretreatment with 30 nmol·L-1PI3K-si RNA for 1 h,pretreatment with 100μmol·L-1BBR for 1 h,and then co-cultured with LPS 200μg·ml-1for 48 h;（4）Akt-si RNA group:pretreatment with 30nmol·L-1Akt-si RNA for 1 h,pretreatment with 100μmol·L-1BBR for 1 h,and then co-cultured with LPS 200μg·ml-1for 48 h;（5）BBR low dose group:pretreatment with25μmol·L-1BBR for 1 h followed by LPS 200μg·ml-1for 48 h;（6）BBR medium dose group:pretreatment with 50μmol·L-1BBR for 1 h followed by LPS 200μg·ml-1for48 h;（7）BBR high dose group:pretreatment with 100μmol·L-1BBR for 1 h followed by LPS 200μg·ml-1for 48 h.3.Detection methods and indicators:using CCK-8 to detect the viability of macrophages;immunofluorescence was used to label autophagy;the number of autophagosomes in RAW 264.7 macrophages was observed by transmission electron microscope;the levels of hs-CRP,IL-6 and TNF-αin macrophage culture medium were detected by enzyme-linked immunosorbent assay;the protein expressions of p-PI3K,p-Akt,Beclin-1,LC3-II,IL-6,TNF-αand hs-CRP in macrophages were detected by Western blot.Results:1.The effect of BBR on the viability of macrophages:Compared with the blank control group,the viability of macrophages in the LPS group was significantly decreased（P<0.05）;Compared with the LPS group,the activity of macrophages in the medium dose and high dose BBR groups was significantly increased（P<0.05）,and there was no significant difference in the low dose BBR group（P>0.05）;Compared with the BBR high dose group,the viability of macrophages in the PI3K-si RNA group and Akt-si RNA group was significantly decreased（P<0.05）.2.Effects of BBR on the fluorescence expression of Beclin-1 and LC3-II in macrophages:（1）Comparison of macrophage Beclin-1 fluorescence expression levels among groups:Compared with the blank control group,macrophage Beclin-1fluorescence expression was significantly lower in the LPS group（P<0.05）;Compared with the LPS group,macrophage Beclin-1 fluorescence expression was significantly higher in the BBR medium dose group and BBR high dose group（P<0.05）,and there was no significant difference in the BBR low dose group（P>0.05）;Compared with the BBR high dose group,the fluorescence expression of Beclin-1 in macrophages was significantly higher in the PI3K-si RNA group and Akt-si RNA group（P<0.05）.（2）Comparison of LC3-II fluorescence expression levels of macrophages in each group:Compared with the blank control group,LC3-II fluorescence expression of macrophages in the LPS group was significantly lower（P<0.05）;Compared with the LPS group,LC3-II fluorescence expression of macrophages in the BBR medium dose group and BBR high dose group was significantly higher（P<0.05）,but there was no significant difference in the BBR low dose group（P>0.05）;Compared with the BBR high dose group,the fluorescence expression of LC3-II in macrophages was significantly higher in the PI3K-si RNA group and Akt-si RNA group（P<0.05）.3.The effect of BBR on the number of macrophage autophagosomes:Compared with the blank control group,the number of macrophage autophagosomes was significantly reduced in the LPS group（P<0.05）;Compared with LPS group,the number of macrophage autophagosomes in medium dose and high dose BBR groups increased significantly（P<0.05）,while there was no significant difference in the BBR low dose group（P>0.05）;Compared with the BBR high dose group,the number of autophagosomes in the PI3K-si RNA group and Akt-si RNA group was significantly increased（P<0.05）.4.The effect of BBR on the levels of IL-6,TNF-αand hs-CRP in the culture medium of macrophages:Compared with the blank control group,the levels of IL-6,TNF-αand hs-CRP in the culture medium of macrophages in the LPS group was significantly increased（P<0.05）;Compared with LPS group,the levels of IL-6,TNF-αand hs-CRP in medium dose BBR group and high dose BBR group was significantly decreased（P<0.05）,and there was no significant difference in low dose BBR group（P>0.05）;Compared with the BBR high dose group,the PI3K-si RNA group and Akt-si RNA group had significant reductions in the levels of IL-6,TNF-αand hs-CRP in the culture medium of macrophages（P<0.05）.5.Effects of BBR on macrophage p-PI3K,p-Akt,Beclin-1,LC3-II,IL-6,TNF-α,hs-CRP protein expression:（1）Comparison of macrophage p-PI3K and p-Akt protein expression levels in each group:Compared with the blank control group,macrophage p-PI3K and p-Akt protein expression was significantly increased in the LPS group（P<0.05）;Compared with the LPS group,macrophage p-PI3K and p-Akt protein expression was significantly lower in the BBR medium dose group and BBR high dose group（P<0.05）,and there was no significant difference in the BBR low dose group（P>0.05）;Compared with the BBR high dose group,the PI3K-si RNA group and Akt-si RNA group had significant reductions in the protein expression of p-PI3K and p-Akt in macrophages（P<0.05）.（2）Comparison of macrophage Beclin-1 and LC3-II protein expression levels among groups:Compared with the blank control group,macrophage Beclin-1 and LC3-II protein expression was significantly decreased in the LPS group（P<0.05）;Compared with the LPS group,macrophage Beclin-1 and LC3-II protein expression was significantly increased in the BBR medium dose group and BBR high dose group（P<0.05）,and there was no significant difference in the BBR low dose BBR group（P>0.05）;Compared with the BBR high dose group,macrophage Beclin-1 and LC3II protein expression was significantly increased in the PI3K-si RNA and Akt-si RNA group（P<0.05）.（3）Comparison of macrophage IL-6,TNF-α,and hs-CRP protein expression levels in each group:Compared with the blank control group,macrophage IL-6,TNF-α,and hs-CRP protein expression was significantly increased in the LPS group（P<0.05）;Compared with the LPS group,macrophage IL-6,TNF-α,hs-CRP protein expression was significantly decreased in the BBR medium dose group and BBR high dose group（P<0.05）,and there was no significant difference in the BBR low dose group（P>0.05）;Compared with the BBR high dose group,the expression of IL-6,TNF-αand hs-CRP protein in the PI3K-si RNA group and Akt-si RNA group was significantly decreased（P<0.05）.Conclusions:1.BBR could significantly reduce the inhibitory effect of LPS on the viability of macrophages,and significantly reduce the levels of IL-6,TNF-αand hs-CRP secreted by macrophages induced by LPS and the protein expression of IL-6,TNF-αand hs-CRP in macrophages,so as to alleviate the inflammatory response.2.BBR could significantly increase the fluorescence and protein expression of Beclin-1 and LC3-II in macrophages induced by LPS,and significantly increase the number of autophagosomes in macrophages,and promote macrophage autophagy.3.BBR can significantly reduce the protein expression of p-PI3K and p-Akt in macrophages,inhibit PI3K/Akt signaling pathway,promote macrophage autophagy,and inhibit inflammatory response.4.BBR can inhibit PI3K/Akt signaling in a dose-dependent manner,promote autophagy of macrophages,and reduce inflammatory response. |
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