Migration Of RAW264 Research Articles

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. Invitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. Invivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1β, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

The effect of Jian Gan powder on the proliferation, migration and polarization of macrophages and relative mechanism

Context Jian Gan powder (JGP) is a Chinese medicine compound comprised ginseng, Radix Paeoniae Alba, Radix Astragali, Salvia miltiorrhiza, Yujin, Rhizoma Cyperi, Fructus aurantii, Sophora flavescens, Yinchen, Bupleurum and licorice. Objective This study explored the inhibitory effects, polarization and potential mechanisms associated with JGP in macrophages. Materials and methods RAW264.7 cells were randomly divided into six groups for 24 h: control, lipopolysaccharide (LPS), overexpression, 1% JGP, 2% JGP, 4% JGP, 8% JGP and 16% JGP. The effects of JGP on RAW264.7 cell proliferation were assessed using colony formation assays and cell counting kit-8 (CCK-8) assays. The Transwell assay was used to evaluate its impact on RAW264.7 cell migration. Moreover, we analysed the interleukin-6 (IL-6)/signal transducer and activator of the transcription 3 (IL-6/STAT3) signaling pathway using quantitative real-time PCR and Western blotting. Furthermore, we examined the M1/M2 polarization levels. Results Unlike LPS stimulation, JGP serum treatment markedly suppressed macrophage proliferation and migration capacity, while STAT3 overexpression enhanced RAW264.7 cell proliferation and migration. JGP inhibited the proliferation and migration of RAW264.7 cells by attenuating the IL-6/STAT3 signaling pathway. Furthermore, it inhibited macrophage M1 polarization, promoting M2 polarization. Discussion and conclusions JGP effectively suppressed the cellular function of RAW264.7 cells by down-regulating the IL-6/STAT3 signaling pathway and modulating macrophage M1/M2 polarization. These findings provide valuable theoretical and experimental basis for considering the potential clinical application of JGP in the treatment of immune-mediated liver injury in clinical practice.

(+)-Catechin Alleviates CCI-Induced Neuropathic Pain in Rats by Modulating the IL34/CSFIR Axis and Attenuating the Schwann Cell-Macrophage Cascade Response in the DRG.

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats.

Cinnamaldehyde inhibits Enterococcus faecalis biofilm formation and promotes clearance of its colonization by modulation of phagocytes in vitro

The nosocomial pathogen, Enterococcus faecalis plays a crucial role in the pathogenesis of variety of infections including endocarditis, urinary tract, and recurrent root canal infections. Primary virulence factors of E. faecalis such as biofilm formation, gelatinase production and suppression of host innate immune response can severely harm host tissue. Thus, novel treatments are needed to prevent E. faecalis biofilm development and pathogenicity due to the worrisome rise in enterococcal resistance to antibiotics. The primary phytochemical in cinnamon essential oils, cinnamaldehyde, has shown promising efficacy against a variety of infections. Here, we looked into how cinnamaldehyde affected the growth of biofilms, the activity of the enzyme gelatinase, and gene expression in E. faecalis. In addition, we looked at the influence of cinnamaldehyde on RAW264.7 macrophages' interaction with biofilm and planktonic E. faecalis in terms of intracellular bacterial clearance, NO generation, and macrophage migration in vitro. According to our research, cinnamaldehyde attenuated the biofilm formation potential of planktonic E. faecalis and gelatinase activity of the biofilm at non-lethal concentrations. The expression of the quorum sensing fsr locus and its downstream gene gelE in biofilms were also found to be significantly downregulated by cinnamaldehyde. Results also demonstrated that cinnamaldehyde treatment increased NO production, intracellular bacterial clearance, and migration of RAW264.7 macrophages in presence of both biofilm and planktonic E. faecalis. Overall these results suggest that cinnamaldehyde has the ability to inhibit E. faecalis biofilm formation and modulate host innate immune response for better clearance of bacterial colonization.

Rac1 inhibition protects the kidney against kidney ischemia/reperfusion through the inhibition of macrophage migration

Kidney ischemia/reperfusion (I/R) injury, a common cause of acute kidney injury (AKI), is associated with the migration of inflammatory cells into the kidney. Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho family of small GTPase, plays an important role in inflammatory cell migration by cytoskeleton rearrangement. Here, we investigated the role of Rac1 on kidney I/R injury and macrophage migration. Male mice were subjected to either 25 min of bilateral ischemia followed by reperfusion (I/R) or a sham operation. Some mice were administrated with either NSC23766, an inhibitor of Rac1, or 0.9% NaCl (vehicle). Kidney damage and Rac1 activity and expression were measured. The migration and lamellipodia formation of RAW264.7 cells, mouse monocyte/macrophage, induced by monocyte chemoattractant protein-1 (MCP-1, a chemokine) were determined using transwell migration assay and phalloidin staining, respectively. In sham-operated kidneys, Rac1 was expressed in tubular cells and interstitial cells. In I/R-injured kidneys, Rac1 expression was decreased in tubule cells in correlation with the damage of tubular cells, whereas Rac1 expression increased in the interstitium in correlation with an increased population of F4/80 cells, monocytes/macrophages. I/R increased Rac1 activity without changing total Rac1 expression in the whole kidney lysates. NSC23766 administration blocked Rac1 activation and protected the kidney against I/R-induced kidney damage and interstitial F4/80 cell increase. NSC23766 suppressed monocyte MCP-1-induced lamellipodia and filopodia formation and migration of RAW 264.7 cells. These results indicate Rac1 inhibition protects the kidney against I/R via inhibition of monocytes/macrophages migration into the kidney.

Abstract P5-17-06: P38 kinase as a therapeutic target to reverse an immune suppressive tumor microenvironment in metastatic breast cancer

Abstract The immune suppressive tumor microenvironment (TME) in metastatic breast cancer (MBC) limits the benefits of immunotherapy with immune checkpoint inhibitors (ICIs). In particular, the primary TME drives the expansion and recruitment of immune suppressive myeloid cell populations, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Treatments targeting these populations can potentially improve the efficacy of ICI therapy. To that end, our published and new findings have revealed that the p38 Mitogen-Activated Protein Kinase (MAPK) contributes to the expansion and mobilization of TAMs and MDSCs. Further, we found that pharmacological blockade of p38 decreased metastasis and increased the levels of CD8+ T cells while decreasing TAMs in the primary TME. Depletion of PMN-MDSCs, a major MDSC subset, was accompanied by reduced TAM infiltration and phenocopied the anti-metastatic effects of p38 blockade. Next, we explored the impact of p38 blockade on the composition and functionality of the immune populations in the primary TME by using single-cell RNA-sequencing. We found that p38 blockade increased levels of Irf8+ monocytic populations, indicating a decrease in immune-suppressive properties of the TME. Notably, p38 blockade increased the expression of factors related to the activation of CD8+ cytotoxic T lymphocytes, i.e., Jchain, Icos, and Cd137. Thus, our data indicate that p38 blockade alters the immune landscape within the primary TME and favors an antitumor immune response. Our data also suggest that the p38 kinase controls the production of tumor-derived factors (TDFs) which facilitate the recruitment of those pro-tumor myeloid populations. Thus, we explored this p38-TDF-myeloid axis by using trans-well migration assays. We tested the migration of the monocyte-like cell line RAW 264.7 in response to tumor-conditioned media prepared from tumor cells treated with or without the p38 inhibitor, Ralimetinib. Our data showed that the migration of RAW 264.7 cells was significantly diminished towards the conditioned media from tumor cells treated with the p38 inhibitor or from tumor cells with a genetic inactivation of p38α by CRISPR/Cas9 compared to the corresponding controls. Altogether, our studies demonstrate that p38 kinase is a potential therapeutic target, which reshapes the immune suppressive contexture of TME in MBC to improve antitumor immunity. Citation Format: Priyanka Rajan, Justin Zonneville, Sean Colligan, Scott Abrams, Andrei Bakin. P38 kinase as a therapeutic target to reverse an immune suppressive tumor microenvironment in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-06.

Herbal medicine AnoSpray suppresses proinflammatory cytokines COX-2 and RANTES in the management of hemorrhoids, acute anal fissures and perineal wounds.

Hemorrhoids, anal fistula and fissure are common anorectal complications. Anorectal diseases are associated with severe pain, inflammation, swelling, itching and bleeding. These diseases may be managed with different medical treatments or surgical procedures, depending on their severity. Surgical procedures, however, are highly invasive and are associated with higher costs and the possibility of recurrence. In addition, surgical removal of fistula-in-ano leads to the formation of perineal wounds. Therefore, developing therapeutic interventions that are effective in alleviating inflammation and pain are desirable for the effective management of anorectal diseases. Herbal compounds have previously been indicated to suppress inflammation and pain in different pathological conditions. The aim of the present study was to examine the effects elicited by a polyherbal formulation, AnoSpray®, on the migration of inflammatory cells and on the expression of inflammatory cytokines in anorectal diseases. The effect of AnoSpray on cell viability and migration was studied using MTT and wound-migration assays, respectively. Furthermore, the effects of AnoSpray on the expression of the inflammatory cytokines regulated upon activation, normal T cell expressed and presumably secreted (RANTES) and VEGF, as well as on cyclooxygenase-2 (COX)-2, were investigated using western blot analysis. The expression of RANTES and COX-2 in human hemorrhoid specimens was also analyzed to corroborate the in vitro findings. The results obtained revealed that AnoSpray did not exhibit any cytotoxic effects; however, it did lead to a significant suppression in the migration of RAW 264.7 and BJ cells. Furthermore, the results suggested that AnoSpray suppressed the expression of the inflammatory cytokines RANTES and VEGF, and also the expression of COX-2. In addition, RANTES and COX-2 were significantly downregulated in the clinical specimens of AnoSpray-treated hemorrhoids compared with the controls. Taken together, the results of the present study suggested that AnoSpray may be a potential therapeutic agent in the treatment of bleeding hemorrhoids, anal fissures and perineal wounds.

Preclinical evaluation of JAK1/2 inhibition by ruxolitinib in a murine model of chronic graft-versus-host disease

The objective of this study was to examine the therapeutic effect of ruxolitinib, an orally administered selective Janus kinase (JAK) 1/2 inhibitor, on chronic graft-versus-host disease (cGVHD) using a murine model of sclerodermatous GVHD (scl-GVHD). Compared with scl-GVHD controls, ruxolitinib-treated recipients had scl-GVHD of significantly attenuated clinical and pathological severity in the skin and decreased frequencies of effector cells, CD4+ T cells, and CD11b+ macrophage/monocytes. Regulatory CD4+ Foxp3+ T cells were expanded whereas interferon-γ (IFN-γ)-producing CD4+ T cells were significantly decreased in ruxolitinib-treated recipients. Ruxolitinib suppressed not only the production of IFN-γ from CD4+ T cells and monocyte chemoattractant protein 1 (MCP-1) from CD11b+ macrophage/monocytes, but also the proliferation of these cells in vitro. Levels of both cytokines (IFN-γ and MCP-1) were also reduced in the spleen and skin of ruxolitinib-treated recipients in vivo. IFN-γ-induced MCP-1 production and migration of RAW 264.7 cells, a macrophage cell line, were inhibited by ruxolitinib. However, supplementation with MCP-1 restored this effect of ruxolitinib. In addition, blocking JAK-STAT signaling using ruxolitinib reduced the activation of STAT1 in stimulated immune effector cells. Taken together, these results suggest that ruxolitinib can prevent scl-GVHD by suppressing IFN-γ produced by T cells and MCP-1 expression in macrophage/monocytes via inhibition of JAK-STAT signaling.

Investigation of PPARβ/δ within Human Dental Pulp Cells: A Preliminary In Vitro Study.

Controlling the inflammatory response to restore tissue homeostasis is a crucial step to maintain tooth vitality after pathogen removal from caries-affected dental tissues. The nuclear peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a ligand-activated transcription factor with emerging anti-inflammatory roles in many cells and tissues. However, its expression and functions are poorly understood in human dental pulp cells (hDPCs). Thus, this study evaluated PPARβ/δ expression and assessed the anti-inflammatory effects evoked by activation of PPARβ/δ in lipopolysaccharide- (LPS-) induced hDPCs. Our results showed that hDPCs constitutively expressed PPARβ/δ mRNA/protein, and treatment with LPS increased PPARβ/δ mRNA expression. The selective PPARβ/δ agonist GW0742 significantly decreased inflammation-related mRNA expression in hDPCs (IL6, IL1β, TNFα, MMP1, and MMP2) and RAW264.7 cells (Il6 and Tnfα). Further, PPARβ/δ agonist attenuated MMP2/9 gelatinolytic activity in hDPCs. Previously LPS-conditioned hDPCs increased the migration of RAW264.7 cells through the membrane of a Transwell coculture system. Conversely, pretreatment with GW0742 markedly decreased macrophage recruitment. These findings provide among the first evidence that hDPCs express PPARβ/δ. In addition, they suggest that activation of PPARβ/δ by GW0742 can attenuate some cellular and molecular in vitro aspects related to the inflammatory process, pointing out to investigate its potential target role in dental pulp inflammation.

Suppression of macrophage migration by down-regulating Src/FAK/P130Cas activation contributed to the anti-inflammatory activity of sinomenine

A large number of macrophages in inflamed sites not only amplify the severity of inflammatory responses but also contribute to the deleterious progression of many chronic inflammatory diseases, autoimmune diseases and cancers. Macrophage migration is a prerequisite for their entry into inflammatory sites and their participation of macrophages in the pathologic processes. Inhibition of macrophage migration is therefore a potential anti-inflammatory mechanism. Moreover, alleviation of inflammation also prevents the macrophages infiltration. Sinomenine (SIN) is an alkaloid derived from the Chinese medicinal plant Sinomenium acutum. It has multiple pharmacological effects, including anti-inflammation, immunosuppression, and anti-arthritis. However, its anti-inflammatory molecular mechanisms and effect on macrophage migration are not fully understood. The purpose of this research was to investigate the pharmacological effects and the molecular mechanism of SIN on macrophage migration in vivo and in vitro as well as to elucidate its anti-inflammatory mechanisms associated with macrophage migration. Our results showed that SIN reduced the number of RAW264.7 cells migrating into inflammatory paws and blocked lipopolysaccharide (LPS)-induced RAW264.7 cells and bone marrow-derived macrophages (BMDMs) migration in vitro. Furthermore, SIN attenuated the 3D mesenchymal migration of BMDMs. The absence of macrophage migration after circulatory and periphery macrophages depletion led to a reduction in the severity of inflammatory response. In macrophages depleted (macrophages-/-) mice, as inflammatory severity decreased, RAW264.7 cells migration was suppressed. A non-obvious effect of SIN on the inflammatory response was found in macrophages-/- mice, while the inhibitory effect of SIN on RAW264.7 cells migration was still observed. Furthermore, the migration of RAW264.7 cells pre-treated with SIN was suppressed in normal mice. Finally, Src/focal adhesion kinase (FAK)/P130Cas axis activation, which supports macrophages mesenchymal migration, and iNOS expression, NO production, integrin αV and in integrin β3 expressions, which promote Src/FAK/P130Cas activation, were down-regulated by SIN. However, SIN had no obvious effect on the expression of the monocyte chemoattractant protein-1 (MCP-1), which is an important chemokine for macrophage migration. These results indicated that SIN significantly inhibited macrophage mesenchymal migration by down-regulating on Src/FAK/P130Cas axis activation. There was a mutual regulatory correlation between the inflammatory response and macrophage migration, and the effects of SIN on macrophage migration were involved in its anti-inflammatory activity.

Abstract PD15-02: Cx3cl1-cx3cr1 axis in liver metastasis of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a breast cancer subtype with increased risk of distant metastasis, especially to visceral organs. However, the detailed molecular mechanism underlying the visceral metastasis of TNBC is unknown. We used three patient-derived xenograft models derived from TNBC tumors with different metastatic capacities. PDX1 and PDX2 model developed distant metastasis in only lung and liver, respectively. In contrast, PDX3 tumor metastasized to both lung and liver. RNA sequencing of the primary tumor and distant metastatic sites of these three PDX models was performed. The gene expression profiles of the primary tumors of the three models showed minimally overlapping genes. However, the microenvironment gene expression profiles, measured by using the sequencing reads aligned to mouse genome, showed distinct difference between the models. Among the differentially expressed genes, we identified Cx3cr1 as a significantly upregulated gene in the liver microenvironment of the PDX models that metastasized to liver. Next, we used 4T1 syngeneic mouse mammary carcinoma model to validate the Cx3cr1 in the mouse liver tissues. Interestingly, the Cx3cr1 up-regulation occurred during the pre-metastatic period, and Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis suggesting the Cx3cr1 regulates the formation of pre-metastatic niche. Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis. These data suggest that Cx3cl1 and Cx3cr1-mediated tumor-microenvironment interaction is critical in developing liver metastasis in TNBC. Cx3cl1 increased the invasion and migration of Raw264.7 monocyte/macrophage cells and this effect was abrogated by the Cx3cr1 silencing in Raw264.7 cells or treating Cx3cl1 neutralizing antibody. Pathway analysis of the RNA sequencing data showed that genes involved in extracellular matrix remodeling was significantly dysregulated in the liver tissues including Lysyl Oxidase (Lox). Cx3cl1 treatment in Raw264.7 cells resulted in increased expression of Lox and MMP9 mRNA in three-dimensional culture system. The Raw264.7 cells caused increased invasion of 4T1 cells in vitro. In conclusion, our data suggest that Cx3cl1-Cx3cr1 axis plays critical role during the liver metastasis of TNBC. Citation Format: Woohang Heo, Hye-Youn Son, Ju hee Kim, Kyu Jin Lee, Myeonghee Jeon, Songbin Li, Jeehee Jang, Dong-Young Noh, Wonsik Han, Hyeong-Gon Moon. Cx3cl1-cx3cr1 axis in liver metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-02.

RASSF10 regulates bone invasion of growth hormone-secreting adenomas via exosomes

BackgroundInvasion of pituitary growth hormone-secreting adenoma into surrounding tissues poses a challenge for complete resection in surgery, which is the main reason for recurrence of this type of cancer. Studies have shown that abnormal methylation of RASSF10 can promote the expression of MDM2 and regulate the tumor microenvironment by affecting the secretion of exosomes. In the present study, we aim to uncover the specific underlying mechanism of this effect. MethodTranswell co-culture assays was performed using GT1.1 cells or exosomes and RAW264.7 cells. RAW264.7 cells were collected for invasion, proliferation and apoptosis assays, RT-qPCR and western blotting. RNA-seq was performed and used to assess the potential molecular pathways of the effect of GT1.1 cell-exosomes on RAW264.7 cells. ResultsGT1.1 cells with reduced RASSF10 expression could promote the proliferation and migration of RAW264.7 cells, and promote their expression of osteoclast markers TRAP and CK. The effect of GT1.1 cell exosomes on the RAW264.7-cell phenotype was shown to be achieved through the RASSF10-MDM2 pathway. RNA-seq allowed the identification of PI3K-AKT, MAPK, and calcium signaling as important in this regulation system of RASSF10-MDM2. ConclusionOur results indicate that GT1.1 cells activate PI3K-AKT, MAPK and calcium signaling via the RASSF10-MDM2 pathway, and promote the differentiation of RAW264.7 cells into osteoclasts through exosomes. This study may provide new ideas to aid in early diagnosis, prognostic assessment and treatment of aggressive pituitary adenomas.

Effects of TGF‑β1 on the migration and morphology of RAW264.7 cells in vitro.

Osteoclasts (OCs) differentiate from monocyte/macrophage‑lineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)‑β1 on OCP migration in vitro. Pre‑osteoclastic RAW264.7 cells were cultured with and without TGF‑β1 (2, 5 or 20 ng/ml), receptor activator of NF‑κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF‑β1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF‑β1 for 3 days, and the effect was reversed by SB431542. Tartrate‑resistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGF‑β1 (2 ng/ml) and RANKL. When TGF‑β1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF‑β1 accelerates RANKL‑induced OC differentiation, but does not act in a dose‑dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL‑induced osteoclast differentiation in the presence of TGF‑β1. These results were accompanied with the increased expression of small G‑proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF‑β1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGF‑β1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.

High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and β-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1β MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.

Tanshinone IIA suppresses the progression of atherosclerosis by inhibiting the apoptosis of vascular smooth muscle cells and the proliferation and migration of macrophages induced by ox-LDL.

ABSTRACTThe profound inhibitory effect of Tanshinone IIA (Tan IIA) on atherosclerosis has been demonstrated, whereas the latent mechanism is not completely cleared. This study aimed to investigate the cellular and molecular mechanisms underlying Tan IIA protecting against atherosclerosis. Oil Red O staining and ELISA assay showed that Tan IIA suppressed the progress of atherosclerosis and reduced the levels of inflammatory cytokines in serum of apolipoprotein E deficient (ApoE–/–) mice. Flow cytometry assay revealed that Tan IIA inhibited oxidized LDL (ox-LDL)-induced apoptosis of VSMCs. MTT and transwell assay indicated that Tan IIA suppressed the ox-LDL-stimulated proliferation and migration of RAW264.7 cells. Moreover, Tan IIA ameliorated inflammatory cytokine upregulation elicited by ox-LDL in RAW264.7 cells. Additionally, Tan IIA inhibited the apoptosis of VSMCs and decreased the levels of MMP-2, MMP-9 in ApoE–/– mice. In conclusion, our study demonstrated Tan IIA suppressed the progression of atherosclerosis by inhibiting vascular inflammation, apoptosis of VSMCs and proliferation and migration of macrophages induced by ox-LDL.

Flow-Induced Migration of Osteoclasts and Regulations of Calcium Signaling Pathways

Osteoclasts are large multinucleate cells originating from the fusion of monocytes that are differentiated from hematopoietic stem cells. Although activated osteoclasts preferentially move to the area of microcrack by chemotaxis, whether such mechanical cues as fluid shear stress (FSS) regulate the migration of osteoclasts remains unknown. This study focuses on the effect of FSS on the migration of RAW264.7 monocytes and differentiated osteoclasts, as well as the roles of calcium signaling pathways in cell migration behaviors. We study five calcium signaling pathways, namely, mechanosensitive cation-selective channels (MSCC), phospholipase C, endoplasmic reticulum (ER), adenosine triphosphate, and extracellular calcium. Results show that FSS induces the migration of RAW264.7 cells along flow direction, and the directionality, alignment along the flow direction, and speed of cells are significantly enhanced with the increase in FSS levels. Blocking the pathways of MSCC, ER, or extracellular calcium significantly reduces the migration of RAW264.7 cells along the flow direction. However, the inhibition of calcium signaling pathways does not affect the migration behaviors after inducing RAW264.7 cells for 4 or 8 days with the conditioned medium, Therefore, this study indicates that both undifferentiated monocytes and differentiated osteoclasts tend to migrate along flow direction, furthermore the FSS-induced directional migration of the monocytes is regulated by calcium signaling pathways, but that of differentiated osteoclasts is unaffected.

Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-κB and signal transducer and activator of transcription 3 pathways

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs)-mediated regulation of macrophages is critical for inflammation response and tissue injury repair. However, the underlying mechanism is not well understood. In this study, we investigated the effect of mouse bone marrow-derived MSCs on macrophages under normal and inflammatory conditions. Co-culture with MSCs or treatment with MSC-conditioned medium (MSC-CM) reduced the expression of tumor necrosis factor-α while inducing the expression of interleukin 10 (IL-10) and arginase 1 in lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cells and splenic CD11b(+) cells. MSC-CM treatment increased the expression of CD206, a marker of alternatively activated M2 macrophages, in RAW264.7 cells. In addition, MSC-CM promoted the proliferation and migration of RAW264.7 cells. MSC-CM treatment activated signal transducer and activator of transcription 3 (STAT3) but inhibited nuclear factor-κB (NF-κB) pathways in LPS-stimulated RAW264.7 cells. Moreover, STAT3 inhibitor S3I-201 antagonized the induction of IL-10, arginase 1, and CD206 by MSC-CM in RAW264.7 cells. Conclusively, our findings suggest that mouse MSCs induce macrophage M2 activation through the NF-κB and STAT3 pathways.

Cucurbitacin IIa induces caspase-3-dependent apoptosis and enhances autophagy in lipopolysaccharide-stimulated RAW 264.7 macrophages

Cucurbitacin IIa (CuIIa), a member of cucurbitacin family, is isolated from the root of Hemsleya amabilis which has been used as an ancient remedy for bacillary dysentery and gastroenteritis. The anti-inflammatory properties of CuIIa have long been recognized but the underlying mechanism is largely unknown. In this study, we investigated the anti-inflammatory effect of CuIIa on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that CuIIa inhibited the proliferation and migration of RAW 264.7 cells in a dose-dependent manner. Whereas CuIIa did not cause apoptosis in unstimulated RAW 264.7 cells, it did induce a significant apoptosis in LPS-stimulated cells, which was caspase-3-dependent and associated with downregulation of survivin. Furthermore, LPS induced autophagy in RAW 264.7 cells and this effect was further enhanced by CuIIa as evidenced by increased levels of LC3-II conjugates and formation of LC3 puncta. In addition, CuIIa disrupted actin cytoskeleton via inducing actin aggregation. However, neither the synthesis of tumor necrosis factor-α, nor the activation of the mitogen-activated protein kinases and NF-κB pathways in LPS-stimulated cells was suppressed by CuIIa treatment. Collectively, these results suggested that induction of apoptosis and enhancement of autophagy contributed to the anti-inflammatory activity of CuIIa against inflammation-related diseases.

Activation of Thromboxane A2 Receptor (TP) Increases the Expression of Monocyte Chemoattractant Protein -1 (MCP-1)/Chemokine (C-C motif) Ligand 2 (CCL2) and Recruits Macrophages to Promote Invasion of Lung Cancer Cells

Thromboxane synthase (TXAS) and thromboxane A2 receptor (TP), two critical components for thromboxane A2 (TXA2) signaling, have been suggested to be involved in cancer invasion and metastasis. However, the mechanisms by which TXA2 promotes these processes are still unclear. Here we show that TXA2 mimetic, I-BOP, induced monocyte chemoattractant protein -1(MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) expression at both mRNA and protein levels in human lung adenocarcinoma A549 cells stably over-expressing TP receptor α isoform (A549-TPα). The induction of MCP-1 was also found in other lung cancer cells H157 and H460 that express relatively high levels of endogenous TP. Using specific inhibitors of several signaling molecules and promoter/luciferase assay, we identified that transcription factor SP1 mediates I-BOP-induced MCP-1 expression. Furthermore, supernatants from I-BOP-treated A549-TPα cells enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Moreover, co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. These findings suggest that TXA2 may stimulate invasion of cancer cells through MCP-1-mediated macrophage recruitment.

Reciprocal activation of macrophages and breast carcinoma cells by nitric oxide and colony-stimulating factor-1

Induction of inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) production and migration of RAW264.7 macrophages by coculture with breast cancer MDA-MB-231 cells or the addition of conditioned medium derived from MDA-MB-231 cells (MDA-CM) was identified. Increased iNOS/NO induction and migration of macrophages by MDA-CM were significantly blocked by adding the c-Jun-N-terminal protein kinase (JNK) inhibitor, SP600125, the nuclear factor-kappa B (NF-κB) inhibitor, BAY117082 and pyrrolidine dithiocarbamic acid and a dominant-negative JNK. The addition of an NO donor, Diethylenetriamine-NONOate, significantly activated expressions of MMP-9 and VEGF-A genes in breast carcinoma MDA-MD-231 cells and invasion of MDA-MB-231 cells in coculture with RAW264.7 macrophages as determined using Transwell systems, but that was inhibited by adding SP600125, BAY117082 and the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester. Induction of heme oxygenase-1 in macrophages reduced MDA-CM-induced iNOS/NO, JNK and NF-κB activations in accordance with inhibiting VEGF-A and MMP-9 gene expressions by MDA-MB-231 cells via Transwell assays. Furthermore, VEGF, sRANKL, TNF-α, IL-1α, TGF-β, CSF-1 and MCP-1 were applied, and CSF-1 showed the most potent stimulation of iNOS/NO production and migration of macrophages. MCF-7 cells with lower CSF-1 expression than MDA-MB-231 cells showed a poor stimulatory effect on iNOS/NO production and migration of macrophages. Neutralization of CSF-1 in MDA-CM using CSF-1 antibody inhibited MDA-CM-induced iNOS protein expression and migration of macrophages, and CSF-1-induced iNOS protein and migration was blocked by adding JNK inhibitor SP and NF-κB inhibitor BAY. The reciprocal activation of breast cancer and macrophages via NO-CSF-1 is first elucidated herein.