ABSTRACT
OBJECTIVE：To stu dy the regulation effects of Huanglian jiedu decoction on M 1 and M 2 macrophage polarization in atherosclerosis （AS）model mice ，and to elucidate its mechanism of AS prevention and treatment. METHODS ：Sixty ApoE －/－ male mice were randomly divided into blank control group ，model group ，simvastatin group [positive control ，5 mg/（kg·d）]， Huanglian jiedu decoction low-dose ，medium-dose and high-dose groups [ 5，10，20 mg/（kg·d），by crude drug] ，with 10 mice in each group. Except for blank control group ，other groups were given high-fat diet to induce AS model. After modeling ， administration groups were given relevant medicine intragastrically ；blank control group and model group were given normal saline intragastrially，once a day ，for consecutive 4 weeks. After medication ，the contents of triglyceride （TG），total cholesterol （TC）， low density lipoprotein cholesterol （LDL-C） and high density lipoprotein cholesterol （HDL-C） in serum were detected by automatic biochemical analyzer. Sirius red staining was used to observe the formation of collagen fibers in the aorta of mice. The serum contents of iNOS and CD 206 were determined by ELISA. mRNA expression levels of IL- 1β，iNOS，TNF-α，YM1 and Fizz1 in the aorta were detected by RT-PCR. RESULTS ：Compared with blank control group ，the serum contents of TC ，TG， LDL-C and iNOS ，mRNA expression levels of IL- 1β，iNOS，TNF-α in the aorta were significantly increased in model group（P＜ 0.05 or P＜0.01），while the serum contents of HDL-C and CD 206 and mRNA expression levels of IL- 10，YM1，Fizz1 in the aorta were significantly decreased （P＜0.01）. There was a thick layer of collagen fibers under the endothelium of aorta. Compared with model group ，above serum indexes of mice were improved significantly in administration groups （P＜0.05 or P＜0.01）；mRNA expression levels of IL- 1 ，iNOS and TNF-α in the aorta in Huanglian jiedu decoction medium-dose and high-dose groups were decreased significantly （P＜0.01），while mRNA expression levels of IL- 10，YM1 and Fizz 1 were increased significantly （P＜0.05 or P＜0.01）. The vascular endothelium was relatively smooth. CONCLUSIONS ：Huanglian jiedu decoction can inhibit the polarization of M 1 macrophages and promote the polarization of M 2 macrophages，reduce the inflammatory reaction ，maintain the stability of atherosclerotic plaque in artery ，so as to play the role of anti-AS.
ABSTRACT
OBJECTIVE：To study the mechanism of H uanglian jiedu decoction （HJD）regulating macrophage polarization in order to explore its anti-inflammatory mechanism. METHODS ：The active components and predicted targets of HJD were screened through TCMSP and Swiss Target Prediction database ；the related targets of macrophage polarization were obtained by GeneCards and OMIM database ，and the network diagram of active ingredient-macrophage polarization target of HJD was drawn by using Cytoscape 3.6.0 software；protein interaction network was constructed by String database and core targets were extracted. Gene ontology（GO）enrichment analysis and Kyoto Encyclopedia of genes and genomes （KEGG）pathway enrichment analysis were carried out by using Cytoscape 3.6.0 software and DAVID website. Combined with the results of network pharmacology analysis ， RAW264.7 macrophage cells were divided into blank control group ，model group ，simvastatin group （10 μmol/L）and serum containing HJD group （obtained from the blood after the rats were given HJD at the dose of 10 g/kg）. Except the blank control group and model group were added culture medium ， the other groups were added with 100 μ L of relevant drug solution or serum containing drug. After 2 h of culture ，except for the blank control group ，LPS solution （100 μg/L）was added to the other groups for 24 h to induce inflammation. Western blot assay was used to detect the expression of AMPK. mRNA expression of M 1 type polarization factor （IL-1 β，iNOS）and M 2 type polarization factor （IL-10，Fizz1）were detected by RT-PCR. RESULTS ：A total of 50 active components of HJD （such as acacetin ，wogonin，quercetin，β-sitosterol）were screened ， which could regulate macrophage polarization through 12 GO items （such as anoikis ，astrocyte activation ），and 20 KEGG pathways（such as estrogen signaling pathway ，bladder cancer pathway ，AMPK signaling pathway ）. The results of cell test showed that compared with blank control group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in model group were significantly decreased （P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly increased（P＜0.01）； compared with model group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in serum containing HJD group and simvastatin group were significantly increased （P＜0.05 or P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly decreased （P＜0.01）. CONCLUSIONS ：HJD can regulate macrophage polarization through multiple targets and pathways；it can up regulate the expression of M 2-type polarization factors and down-regulate the expression of M 1-type polarization factors through AMPK signaling pathway ，regulate macrophage polarization and play an anti-inflammatory role.
ABSTRACT
OBJECTIVE：To study the effects of berberine on mic e macrophage polarization based on TLR 4-MyD88-NF-κB signaling pathway. METHODS ：Using mice RAW 264.7 macrophage as the object ，atorvastatin calcium as positive control ， inflammatory cell model was induced by lipopolysaccharide （LPS）；ELISA method was used to detect the contents of TNF-α，IL-6 and NF-κB in cell culture medium after treated with low，medium and high doses of berberine （5，10，20 μmol/L）for 24 h. The real-time fluorescence quantitative PCR was conducted to determine the mRNA expression of TLR 4 and MyD 88 in cells. Western blotting assay was used to detect the protein expression of TLR 4，MyD88，iNOS and CD 206 in cells. RESULTS ：Compared with blank control group ，the contents of TNF-α，IL-6 and NF-κB in cell culture medium，mRNA expression of TLR 4 and MyD 88， protein expression of TLR 4，MyD88 and iNOS in cells were increased significantly in LPS induction group （P＜0.05）. Compared with LPS induction group ，the contents of TNF-α and IL-6，mRNA and protein expression of TLR 4 and MyD 88 in atorvastatin calcium group ，berberine medium-dose and high-dose groupsas well as the content of NF-κ B and protein expression of iNOS in administration groups were decreased significantly ， while the content of NF-κB in berberine high-dose group was significantly lower than atorvastatin calcium group （P＜0.05）. The protein expressions of CD206 in atorvastatin calcium group and berberine high-dose group were increased significantly ，while the protein expression of CD 206 in berberine high-dose group was significantly higher than atorvastatin calcium group （P＜0.05）. CONCLUSIONS ：Different doses of berberine can intervene in mice macrophage polarization to different extents ，the mechanism of which may be associated with the regulation of TLR4/MyD88/NF-κB signaling pathway.