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 effect of total flavonoids of Epimedium brevicornu on postmenopausal osteoporosis （PMOP）model rats based on BMP/Runx 2/Osx signaling pathway so as to confirm the mechanism of preventing and treating osteoporosis（OP）. METHODS ：By body mass stratification ，50 rats were randomly divided into sham operation group ，model group，E. brevicornu total flavonoids low-dose and high-dose groups [265，530 mg/（kg·d）]，estradiol group [0.09 mg/（kg·d）]， with 10 rats in each group. Except that sham operation group underwent sham operation ，PMOP model was established by ovariectomy and castration in other groups. After modeling ，they were given normal diet for 2 months and then given relevant medicine intragastrically for consecutive 84 d，once a day ；rats in sham operation group and model group were given equal volume of normal saline. After last medication ，bone mineral density （BMD）of femur and vertebrae of the right lower limb ，the number of trabecular bone （Tb.N），trabecular bone thickness （Tb.Th）and trabecular separation （Tb.Sp）of femur were determined in each group. The serum levels of Ca 2+，OC and P 1NP in serum were detected by ELISA. HE staining was used to observe pathological changes of femur. mRNA and protein expressions of BMP ，Runx2 and OSX in bone tissue were detected by RT-PCR and Western blotting. RESULTS ：Compared with sham operation group ，BMD of femur and vertebrae ，serum levels of Ca 2 +，OC and P 1NP， Tb.N and Tb.Th of femur ，mRNA and protein expression of BMP ，Runx2 and Osx in femur were decreased significantly in model group，while Tb.Sp of femur was increased significantly （P＜0.01）；the structure of trabecular bone was disordered and the fracture was obvious. Compared with model group ，above indexes of rats in administration groups were improved significantly （P＜ 0.05 or P＜0.01），and the effect of E. brevicornu total flavonoids was dose-dependent （P＜0.05）；the number of trabecular bone increased， arranged orderly and the structure was more brevicornu total flavonoids can improve OP ，the mechanism of which may be associated with com promoting the activity of BMP/Runx 2/Osx signaling pathway.