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Objective:To study the regulation and mechanism of Asperosaponin Ⅵ on polarization of M1/M2 macrophages.Methods:MTT assay was used to detect the effects of Asperosaponin Ⅵ on RAW264.7 cell viability.The levels of TNF-α and IL-6 in supernatant of RAW264.7 cells induced by lipopolysaccharide(LPS)were determined by ELISA.The content of nitric oxide(NO)in supernatant of RAW264.7 cells induced by LPS was determined by Griess method.The gene expression levels of TNF-α,IL-6,argi-nase-1(Arg-1),heme oxygenase-1(HO-1)and suppressor of cytokine signaling protein-2(SOCS2)were detected by fluorescence quantitative PCR.Western blot was used to detect the expression levels of iNOS and p-p65 protein.Results:In LPS induced RAW264.7 cells,Asperosaponin Ⅵ inhibited protein or gene expression levels of TNF-α,IL-6,iNOS and p-p65,and increased HO-1 gene expression.Asperosaponin Ⅵ inhibited NO secretion in RAW264.7 cells induced by LPS.Asperosaponin Ⅵ increased the gene expression levels of M2 macrophage markers Arg1 and SOCS2 induced by IL-4.Conclusion:Asperosaponin Ⅵ inhibited RAW264.7 macrophage polarization to M1 type and promote it polarization to M2 type,which can play its anti-inflammatory and immunomodulato-ry role by regulating M1/M2 macrophage polarization.
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ObjectiveTo assess the curative effects of Fangji Huangqi detumescence prescription （FHDP） on synovitis and polarization of synovial macrophages of knee osteoarthritis （KOA） model in rats induced by Hulth method. MethodThirty-six rats were randomly divided into sham operation group， model group， high-dose， medium-dose， and low-dose （29.16， 14.58， and 7.29 g·kg-1） FHDP groups， and loxoprofen sodium （16.2 mg·kg-1） group. KOA model in rats was induced by modified Hulth method. Six weeks after the operation， rats were given high， medium， and low concentrations of FHDP， normal saline （NS）， and loxoprofen sodium according to the group to intervene， and sacrificed after 2-week administration. Synovium and cartilage histopathological changes were observed after hematoxylin-eosin （HE） staining. Flow cytometry （FCM） and immunofluorescence （IF） test were used to evaluate the polarization of M1/M2 macrophages. Immunohistochemistry （IMC） and enzyme-linked immunosorbent assay （ELISA） were used to detect the related protein expression levels of macrophage polarization， such as interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， interleukin-10 （IL-10）， and matrix metalloproteinase-13 （MMP-13） in joint tissues and serum. ResultCompared with the sham operation group， Krenn and Mankin scores in the model group were significantly increased （P<0.01）. Compared with the model group， Krenn score was decreased in all administration groups （P<0.05， P<0.01）， but there was no significant difference in Mankin score in any administration groups. Compared with the sham operation group， M1/mø （CD38+） ratio in the model group was significantly increased （P<0.01）， and M2/mø （CD206+） ratio in the model group was decreased （P<0.05）. Compared with the model group， M1/mø ratio in the high， medium， and low-dose FHDP groups was decreased （P<0.05， P<0.01）， but M2/mø ratio was increased in all administration groups （the difference had no statistical significance）. Compared with the sham operation group， M1/M2 ratio in the model group was significantly increased （P<0.01）. Compared with the model group， M1/M2 ratio in all FHDP groups was significantly decreased （P<0.01）， and M1/M2 ratio in the high and medium-dose FHDP groups was lower than that in the loxoprofen sodium group （P<0.05）. Compared with the sham operation group， the levels of TNF-α， IL-1β， and MMP-13 in synovium and cartilage of the model group were significantly increased （P<0.01）， the level of IL-10 was significantly decreased （P<0.01）. Compared with the model group， the levels of TNF-α and IL-1β in synovium were decreased in all administration groups （P<0.05）， but the difference of the levels of MMP-13 and IL-10 in synovium had no statistical significance. The level of inflammatory mediators in cartilage was not affected in all administration groups. Compared with the sham operation group， the levels of TNF-α and IL-β in serum of the model group were significantly increased （P<0.01）， the level of IL-10 was decreased （P<0.05）. Compared with the model group， the level of TNF-α in the high-dose FHDP group was decreased （P<0.05）， and the level of IL-10 was increased in all administration groups （P<0.05， P<0.01）. The difference of the level of IL-β in all administration groups had no statistical significance. ConclusionFHDP attenuated the synovitis of KOA rats. FHDP exert the effect on the releasing of proinflammatory cytokines and MMP by inhibiting the polarization of M1 macrophages in synovium， and had no significant effect on the polarization of M2 macrophages. Modulating the imbalanced polarization of synovial macrophages was a possible mechanism of FHDP on attenuating synovitis and treating KOA.
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Objective To explore the effects of lycium barbarum polysaccharide (LBP) on restraining the mouse pancre?atic cancer cells LTPA by the polarization of macrophages to type 1 macrophages (M1). Methods LTPA tumor model of the subcutaneous CB-17SCID mice was constructed. Model mice were randomly divided into tumor-bearing model group (n=10) and LBP treatment group (n=10). The LBP treatment group was fed 10mg/kg LBP every day, and the tumor-bearing model group was fed the same dose of normal saline. The same amount of macrophages Raw264.7 was randomly divided into the control group and experimental groups (different concentrations of LBP). MTT assay was used to detect the optical density (OD) of Raw264.7 in experimental groups and control group. ELISA was used to detect the levels of the interleukin (IL)-12 and IL-10 in experimental group (LBP was 100 mg/L) and the control group. Flow cytometry was used to test the levels of the membrane protein CD16/32 and CD206 in experimental group (LBP was 100 mg/L) and the control group. The tumor mass was weighted and the volume was calculated after three weeks. The effects of LBP on the growth of subcutaneous tumor were detected. HE staining and KI-67 staining were used to detect the microscopic changes of tumor and the proliferation of the LTPA. Results The dose of 100 mg/L LBP can promote the growth of the macrophages Raw264.7 (P<0.01), and induced the high expression of CD16/32 and low expression of CD206, high secretion of IL-12 and low secretion of IL-10. The weight, volume of the tumor and the expression of KI-67 were significantly lower in experimental group than those in the con?trol group (P<0.01). The microscopic necrosis area range of tumor was larger than that of control group. Conclusion The LBP has the effect of restraining LTPA by the polarization of macrophages to M1.