Supercritical CO2 fluid extract from Stellariae Radix ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis by inhibit M1 macrophages polarization via AMPK activation.

Yin chai hu (Radix Stellariae) is a root medicine that is frequently used in Chinese traditional medicine to treat fever and malnutrition. In modern medicine, it has been discovered to have anti-inflammatory, anti-allergic, and anticancer properties. In a previous study, we were able to extract lipids from Stellariae Radix using supercritical CO2 extraction (SRE), and these sterol lipids accounted for up to 88.29% of the extract. However, the impact of SRE on the development of atopic dermatitis (AD) has not yet been investigated. This study investigates the inhibitory effects of SRE on AD development using a 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model. Treatment with SRE significantly reduced the dermatitis score and histopathological changes compared with the DNCB group. The study found that treatment with SRE resulted in a decrease of pro-inflammatory cytokines TNF-α, CXC-10, IL-12, and IL-1ß in skin lesions. Additionally, immunohistochemical analysis revealed that SRE effectively suppressed M1 macrophage infiltration into the AD lesion. Furthermore, the anti-inflammatory effect of SRE was evaluated in LPS + INF-γ induced bone marrow-derived macrophages (BMDMs) M1 polarization, SRE inhibited the production of TNF-α, CXC-10, IL-12, and IL-1ß and decreased the expression of NLRP3. Additionally, SRE was found to increase p-AMPKT172, but had no effect on total AMPK expression, after administration of the AMPK inhibitor Compound C, the inhibitory effect of SRE on M1 macrophages was partially reversed. The results indicate that SRE has an inhibitory effect on AD, making it a potential therapeutic agent for this atopic disorder.

Paeoniflorin Coordinates Macrophage Polarization and Mitigates Liver Inflammation and Fibrogenesis by Targeting the NF-[Formula: see text]B/HIF-1α Pathway in CCl4-Induced Liver Fibrosis.

Liver fibrosis is a disease largely driven by resident and recruited macrophages. The phenotypic switch of hepatic macrophages can be achieved by chemo-attractants and cytokines. During a screening of plants traditionally used to treat liver diseases in China, paeoniflorin was identified as a potential drug that affects the polarization of macrophages. The aim of this study was to evaluate the therapeutic effects of paeoniflorin in an animal model of liver fibrosis and explore its underlying mechanisms. Liver fibrosis was induced in Wistar rats via an intraperitoneal injection of CCl4. In addition, the RAW264.7 macrophages were cultured in the presence of CoCl2 to simulate a hypoxic microenvironment of fibrotic livers in vitro. The modeled rats were treated daily with either paeoniflorin (100, 150, and 200[Formula: see text]mg/kg) or YC-1 (2[Formula: see text]mg/kg) for 8 weeks. Hepatic function, inflammation and fibrosis, activation of hepatic stellate cells (HSC), and extracellular matrix (ECM) deposition were assessed in the in vivo and in vitro models. The expression levels of M1 and M2 macrophage markers and the NF-[Formula: see text]B/HIF-1[Formula: see text] pathway factors were measured using standard assays. Paeoniflorin significantly alleviated hepatic inflammation and fibrosis, as well as hepatocyte necrosis in the CCl4-induced fibrosis model. Furthermore, paeoniflorin also inhibited HSC activation and reduced ECM deposition both in vivo and in vitro. Mechanistically, paeoniflorin restrained M1 macrophage polarization and induced M2 polarization in the fibrotic liver tissues as well as in the RAW264.7 cells grown under hypoxic conditions by inactivating the NF-[Formula: see text]B/HIF-1[Formula: see text] signaling pathway. In conclusion, paeoniflorin exerts its anti-inflammatory and anti-fibrotic effects in the liver by coordinating macrophage polarization through the NF-[Formula: see text]B/HIF-1[Formula: see text] pathway.

Unraveling the mechanism of ethyl acetate extract from Prismatomeris connata Y. Z. Ruan root in treating pulmonary fibrosis: insights from bioinformatics, network pharmacology, and experimental validation.

Introduction: Pulmonary fibrosis is a terminal lung disease characterized by fibroblast proliferation, extracellular matrix accumulation, inflammatory damage, and tissue structure destruction. The pathogenesis of this disease, particularly idiopathic pulmonary fibrosis (IPF), remains unknown. Macrophages play major roles in organ fibrosis diseases, including pulmonary fibrosis. The phenotype and polarization of macrophages are closely associated with pulmonary fibrosis. A new direction in research on anti-pulmonary fibrosis is focused on developing drugs that maintain the stability of the pulmonary microenvironment. Methods: We obtained gene sequencing data and clinical information for patients with IPF from the GEO datasets GSE110147, GSE15197, GSE24988, GSE31934, GSE32537, GSE35145, GSE53845, GSE49072, GSE70864, and GSE90010. We performed GO, KEGG enrichment analysis and GSEA analysis, and conducted weighted gene co-expression network analysis. In addition, we performed proteomic analysis of mouse lung tissue. To verify the results of bioinformatics analysis and proteomic analysis, mice were induced by intratracheal instillation of bleomycin (BLM), and gavaged for 14 days after modeling. Respiratory function of mice in different groups was measured. Lung tissues were retained for histopathological examination, Western Blot and real-time quantitative PCR, etc. In addition, lipopolysaccharide, interferon-γ and interleukin-4 were used to induce RAW264.7 cells for 12h in vitro to establish macrophage inflammation and polarization model. At the same time, HG2 intervention was given. The phenotype transformation and cytokine secretion of macrophages were investigated by Western Blot, RT-qPCR and flow cytometry, etc. Results: Through bioinformatics analysis and experiments involving bleomycin-induced pulmonary fibrosis in mice, we confirmed the importance of macrophage polarization in IPF. The analysis revealed that macrophage polarization in IPF involves a change in the phenotypic spectrum. Furthermore, experiments demonstrated high expression of M2-type macrophage-associated biomarkers and inducible nitric oxide synthase, thus indicating an imbalance in M1/M2 polarization of pulmonary macrophages in mice with pulmonary fibrosis. Discussion: Our investigation revealed that the ethyl acetate extract (HG2) obtained from the roots of Prismatomeris connata Y. Z. Ruan exhibits therapeutic efficacy against bleomycin-induced pulmonary fibrosis. HG2 modulates macrophage polarization, alterations in the TGF-ß/Smad pathway, and downstream protein expression in the context of pulmonary fibrosis. On the basis of our findings, we believe that HG2 has potential as a novel traditional Chinese medicine component for treating pulmonary fibrosis.

Tongxie-Yaofang formula regulated macrophage polarization to ameliorate DSS-induced colitis via NF-κB/NLRP3 signaling pathway.

BACKGROUND: Macrophages infiltration and activation play multiple roles in maintaining intestinal homeostasis and participate in the occurrence and development of UC. Thus, the restoration of immune balance can be achieved by targeting macrophage polarization. Previous studies have reported that TXYF could effectively ameliorate DSS-induced colitis. However, the underlying mechanisms of TXYF for DSS-induced colitis are still ill-defined. METHODOLOGY: This study was designed to explore the therapeutic effect of TXYF and its regulation in macrophages polarization during DSS-induced mice. In C75BL/6 mice, dextran sulfate sodium (DSS) was used to induce colitis and concomitantly TXYF was taken orally to evaluate its curative effect. In vitro experiment was implemented on BMDMs by lipopolysaccharide, IFN- and ATP. RESULTS: Here, we found that TXYF ameliorated clinical features in DSS-induced mice, decreased macrophages M1 polarization but remarkably increased M2 polarization. Mechanically, TXYF treatment effectively inhibited the activities of nuclear transcription factor NF-κB, which further contributed to the decrease of the inflammasome genes of NLRP3, limiting the activation of NLRP3 inflammasome in vivo and in vitro. CONCLUSION: Our findings demonstrated administration of TXYF can interfere with macrophage infiltration and polarization to improve the symptoms of acute colitis, by repressing NF-κB/NLRP3 signaling pathway activation. This enriches the mechanism and provides new prospect for TXYF in the treatment of colitis.

Viola yedoensis Makino formula alleviates DNCB-induced atopic dermatitis by activating JAK2/STAT3 signaling pathway and promoting M2 macrophages polarization.

BACKGROUND: Atopic dermatitis (AD), a common inflammatory skin disorder, severely affects the life quality of patients and renders heavy financial burden on patient's family. The Chinese medicine Viola yedoensis Makino formula (VYAC) has been widely used for treating various skin disorders. Previous studies have reported that VYAC is effective in relieving DNCB-induced AD and inflammation. However, the anti-inflammatory mechanism of VYAC is still ill-defined and poorly understood. This study aims to investigate the therapeutic effects of VYAC on DNCB-induced AD and to elucidate the underlying anti-inflammatory mechanisms. METHODOLOGY: VYAC were extracted with 70% ethanol and lyophilized for use. AD mice were established by DNCB. The therapeutic effects of VYAC were evaluated by oral administration VYAC (150, 300 and 600 mg/kg) daily in vivo. The histopathological and immunohistochemistry were used to analyze skin lesion and macrophages infiltration, RT-qPCR and Elisa were used to analyze the inflammatory factors in skin tissues and serum. To explore the underlying mechanism of VYAC against AD in vitro. RAW264.7 cells and bone-marrow-derived macrophages (BMDMs) were employed for macrophage polarization analysis. Flow cytometer, immunofluorescence and western blot were used to analyze M2 macrophages markers. STAT3 siRNA were transfected into both cells to validate the effects of VYAC-induced macrophages M2 polarization via JAK2/STAT3 signaling pathway. RESULTS: VYAC ameliorated skin lesion of DNCB-induced AD mice by decreased clinical scores and epidermal thickness, decreased the level of pro-inflammatory factors (IL-1ß, TNF-α and IL-18) and enhanced IL-10 anti-inflammatory factor level, inhibited macrophages infiltration and promoted M2 macrophages polarization in vivo. VYAC significantly promoted M2 macrophages polarization in vitro. It is observed that VYAC not only inhibited the phosphorylation of JAK2 and STAT3 in RAW264.7 cells and BMDMs, but also accelerated the translocation to the nucleus. What's more, VYAC reduced the polarization of M2 macrophage by activating JAK2/STAT3 signaling pathway was observed in both cells. CONCLUSIONS: Our findings demonstrate that VYAC significantly ameliorates skin lesion of DNCB-induced AD mice and reduces the levels of inflammatory factors by activating JAK2/STAT3 signaling pathway and promoting M2 macrophages polarization.

Water extract of Cayratia albifolia C.L.Li root relieves zymosan A-induced inflammation by restraining M1 macrophage polarization.

BACKGROUND: Cayratia albifolia C.L.Li (CAC) is a traditional Chinese herbal medicine used to treat inflammatory diseases. Our laboratory has firstly reported that the water extract from CAC relieved lipopolysaccharide (LPS)-induced inflammation, however stronger evidence is still needed to prove its anti-inflammatory effects and the mechanisms involved are also ambiguous. PURPOSE: This study sought to provide more evidence for the application of CAC in alleviating infectious inflammation and disclose novel pharmacological mechanisms. METHODS: Mice were injected with zymA into their paws or peritoneal cavities, and then treated with CAC. ELISA, immunofluorescence and flow cytometry were performed to detect the cytokines (IL-1ß, IL-6, TNF-α and IL-10) generation, the cell infiltration, and the CD86 or CD206 expression of macrophages. Then in vitro assays were performed on bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) to detect their expression of iNOS, arg-1 and the cytokines above. On mechanisms, western blotting (WB), electrophoretic mobility shift assay (EMSA) and flow cytometry were carried out to measure NF-κB transcriptional activity, mitochondrial bioactivity and the mTORC1 activation when BMDMs were stimulated by zymA and treated with CAC. Finally, the chemical components consisted in the extract were analyzed by LC-MS. RESULTS: 200 mg/kg CAC clearly inhibited zymA induced mouse paw edema and reduced the contents of IL-1ß, IL-6 and TNF-α rather than IL-10 in local tissues. CAC also reduced CD86 but not CD206 in macrophages in situ. Through in vitro experiments, it was discovered that CAC reduced the protein and mRNA levels of IL-1ß, IL-6 and TNF-α, and also inhibited iNOS expression, but showed no influence on IL-10 and arg-1 in macrophages. We found CAC reduced NF-κB transcriptional activity, down-regulated mitochondrial membrane potential and ROS levels, and inhibited mTORC1 activity. Finally, we identified 15 major compounds in the extract, among which 4-guanidinobutyric acid and kynurenic acid were the most abundant. CONCLUSION: This study provides further evidence that CAC significantly reduces zymA induced infectious inflammation. In addition, this novel data revealed that CAC restrained M1 rather than promoting M2 macrophages polarization via multi-target inhibitory effects, based on its potentially active components.

Artesunate attenuates atherosclerosis by inhibiting macrophage M1-like polarization and improving metabolism.

OBJECT: Atherosclerosis (AS) is caused by chronic inflammation. Artesunate (ART), a sesquiterpene lactone endoperoxide isolated from Chinese herbal medicine, displays excellent anti-inflammatory activity. In this study, we investigated the effects of artesunate on atherosclerosis in ApoE knock-out mice, and used untargeted metabolomics to determine metabolite changes in these mice following ART treatment. METHODS: ApoE knock-out mice were fed a western diet and administered ART for eight weeks. Untargeted metabolomics was used to detect differential metabolites following the administration of ART. Oil Red O was used to assess plaque size, western blot and ELISA were used to detect inflammatory factors, and flow cytometry was used to detect the expression of markers on macrophages. RESULTS: Results of the in vivo experiment suggested that ART reduced atherosclerotic plaques in murine aortic root. In addition both in vivo and vitro experiments suggested that ART reduced the expression levels of inflammating cytokines, but enhanced those of the anti-inflammatory cytokines in macrophages. Untargeted metabolomic analysis demonstrated that multiple metabolic pathways, which were blocked in AS mice, showed different degrees of improvement following ART treatment. Furthermore, bioinformatic analyses showed that the HIF-1α pathway was altered in the AS mice and the ART treatment mice. In vitro experiments confirmed that LPS-induced upregulation of HIF-1α expression and activation of the NF-κB signaling pathways was significantly inhibited by ART treatment. CONCLUSION: These results suggest that ART exerts anti-atherosclerosis effects by inhibiting M1 macrophage polarization. One of the molecular mechanisms is that ART inhibits M1-like macrophage polarization via regulating HIF-1α and NF-κB signaling pathways.

Astragaloside IV Alleviates the Experimental DSS-Induced Colitis by Remodeling Macrophage Polarization Through STAT Signaling.

Inflammatory bowel disease (IBD) is characterized by chronic and relapsing intestinal inflammation, which currently lacks safe and effective medicine. Some previous studies indicated that Astragaloside IV (AS-IV), a natural saponin extracted from the traditional Chinese medicine herb Ligusticum chuanxiong, alleviates the experimental colitis symptoms in vitro and in vivo. However, the mechanism of AS-IV on IBD remains unclear. Accumulating evidence suggests that M2-polarized intestinal macrophages play a pivotal role in IBD progression. Here, we found that AS-IV attenuated clinical activity of DSS-induced colitis that mimics human IBD and resulted in the phenotypic transition of macrophages from immature pro-inflammatory macrophages to mature pro-resolving macrophages. In vitro, the phenotype changes of macrophages were observed by qRT-PCR after bone marrow-derived macrophages (BMDMs) were induced to M1/M2 and incubated with AS-IV, respectively. In addition, AS-IV was effective in inhibiting pro-inflammatory macrophages and promoting the pro-resolving macrophages to ameliorate experimental colitis via the regulation of the STAT signaling pathway. Hence, we propose that AS-IV can ameliorate experimental colitis partially by modulating macrophage phenotype by remodeling the STAT signaling, which seems to have an essential function in the ability of AS-IV to alleviate the pathological progress of IBD.

Eicosapentaenoic acid membrane incorporation stimulates ABCA1-mediated cholesterol efflux from human THP-1 macrophages.

A high intake in polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) (C20:5 n-3), is cardioprotective. Dietary PUFAs incorporate into membrane phospholipids, which may modify the function of membrane proteins. We investigated the consequences of the membrane incorporation of several PUFAs on the key antiatherogenic ABCA1-mediated cholesterol efflux pathway. Human THP-1 macrophages were incubated with EPA, arachidonic acid (AA) (C20:4 n-6) or docosahexaenoic acid (DHA) (C22:6 n-3) for a long time to mimic a chronic exposure. EPA 70 µM, but not AA 50 µM or DHA 15 µM, increased ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 28% without altering aqueous diffusion. No variation in ABCA1 expression or localization was observed after EPA treatment. EPA incorporation did not affect the phenotype of THP-1 macrophages. The membrane phospholipids composition of EPA cells displayed higher levels of both EPA and its elongation product docosapentaenoic acid, which was associated with drastic lower levels of AA. Treatment by EPA increased the ATPase activity of the transporter, likely through a PKA-dependent mechanism. Eicosanoids were not involved in the stimulated ABCA1-mediated cholesterol efflux from EPA-enriched macrophages. In addition, EPA supplementation increased the apo AI binding capacity from macrophages by 38%. Moreover, the increased apo AI binding in EPA-enriched macrophages can be competed. In conclusion, EPA membrane incorporation increased ABCA1 functionality in cholesterol-normal human THP-1 macrophages, likely through a combination of different mechanisms. This beneficial in vitro effect may partly contribute to the cardioprotective effect of a diet enriched with EPA highlighted by several recent clinical trials.

Qing-Luo-Yin Alleviated Monocytes/Macrophages-Mediated Inflammation in Rats with Adjuvant-Induced Arthritis by Disrupting Their Interaction with (Pre)-Adipocytes Through PPAR-γ Signaling.

BACKGROUND: The Chinese herbal formula Qing-Luo-Yin (QLY) has been successfully used in rheumatoid arthritis treatment for decades. It exhibits notable immune and metabolism regulatory properties. Thereby, we investigated its effects on the interplay between (pre)-adipocytes and monocytes/macrophages under adjuvant-induced arthritis (AIA) circumstances. METHODS: Fat reservoir and histological characteristics of white fat tissues (WAT) in AIA rats receiving QLY treatment were examined upon sacrifice. Metabolic parameters, clinical indicators, and oxidative stress levels were determined using corresponding kits, while mRNA/protein expression was investigated by PCR and immunoblotting methods. M1 macrophage distribution in WAT was assessed by flow cytometry. The effects of QLY on (pre)-adipocytes were further validated by experiments in vitro. RESULTS: Compared with normal healthy controls, body weight and circulating triglyceride were declined in AIA rats, but serological levels of free fatty acids and low-density lipoprotein cholesterol were increased. mRNA IL-1ß and iNOS expression in white blood cells and rheumatoid factor, C-reactive protein, anti-cyclic citrullinated peptide antibody, MCP-1 and IL-1ß production in serum/WAT were up-regulated. Obvious CD86+CD11b+ macrophages were enriched in WAT. Meanwhile, expression of PPAR-γ and SIRT1 and secretion of adiponectin and leptin in these AIA rats were impaired. QLY restored all these pathological changes. Of note, it significantly stimulated PPAR-γ expression in the treated AIA rats. Accordingly, QLY-containing serum promoted SCD-1, PPAR-γ, and SIRT1 expression in pre-adipocytes cultured in vitro. AIA rats-derived peripheral blood mononuclear cells suppressed PPAR-γ and SCD-1 expression in co-cultured pre-adipocytes, but serum from AIA rats receiving QLY treatment did not exhibit this potential. The changes on PPAR-γ expression eventually resulted in varied adipocyte differentiation statuses. PPAR-γ selective inhibitor T0070907 abrogated QLY-induced MCP-1 production decline in LPS-primed pre-adipocytes and reduced adiponectin secretion. CONCLUSION: QLY was potent in promoting PPAR-γ expression and consequently disrupted inflammatory feedback in WAT by altering monocytes/macrophages polarization and adipocytes differentiation.

Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-κB signaling pathway to protect against DSS-induced colitis.

Berberine has been reported to have protective effects in colitis treatment. However, the detailed mechanisms remain unclear. Herein, we demonstrated that berberine could protect against dextran sulfate sodium (DSS)-induced colitis in mice by regulating macrophage polarization. In the colitis mouse model, berberine ameliorated DSS-induced colon shortening and colon tissue injury. Moreover, berberine-treated mice showed significant reduction in the disease activity index (DAI), pro-inflammatory cytokines expression and macrophages infiltration compared with the DSS-treated mice. Notably, berberine significantly reduced the percentage of M1 macrophages. In vitro analysis also confirmed the inhibitory effects of berberine on macrophages M1 polarization in RAW267.4 cells. Further investigation showed that berberine promoted AKT1 expression in mRNA and protein level. Silence of AKT1 abolished the inhibitory effect of berberine on macrophages M1 polarization. The berberine-induced AKT1 expression promoted suppressers of cytokine signaling (SOCS1) activation, which inhibited nuclear factor-kappa B (NF-κB) phosphorylation. In addition, we also found that berberine activated AKT1/SOCS1 signaling pathway but inhibited p65 phosphorylation in macrophages in vivo. Therefore, we concluded that berberine played a regulatory role in macrophages M1 polarization in DSS-induced colitis via AKT1/SOCS1/NF-κB signaling pathway. This unexpected property of berberine may provide a potential explanation for its protective effects in colitis treatment.