Apoptosis In RAW264 Research Articles

In vitro effects of l-kynurenine and quinolinic acid on adhesion, migration and apoptosis in B16 F10 melanoma cells

IntroductionThe inhibition of melanoma adhesion through adhesion molecules, such as integrins and E-cadherin, may represent a promising strategy for managing melanoma metastasis. Compounds, namely l-kynurenine (L-kyn) and quinolinic acid (Quin), previously displayed anti-cancer effects at half-maximal inhibitory concentration (IC50) against B16 F10 melanoma cells in vitro. However, the role of these compounds in B16 F10 melanoma cell adhesion, migration and apoptosis remain unknown. MethodsPost-exposure to the compounds, flow cytometry was used to analyse the expression of very late antigen-5 (VLA-5), E-cadherin and cleaved caspase-3 in B16 F10 melanoma and RAW 264.7 murine macrophage cells. An adhesion assay was used to quantify the adhesion of both cell lines to vitronectin. A scratch migration assay was used to measure the possible inhibition of cell migration in B16 F10 cells in response to L-kyn and Quin. ResultsIn both B16 F10 and RAW 264.7 cells, neither L-kyn nor Quin induced significant effects on VLA-5 expression or cell adhesion to vitronectin. In B16 F10 cells, both L-kyn and Quin elevated E-cadherin expression and displayed a trend of suppressed migration. However, only L-kyn elevated E-cadherin in RAW 264.7 cells. L-kyn induced apoptosis by elevating cleaved caspase-3 expression in both cell lines. ConclusionL-kyn and Quin demonstrated promising antimetastatic effects in their ability to elevate E-cadherin expression and induce apoptosis in B16 F10 melanoma cells. However, these effects did not occur in response to vitronectin or VLA-5 integrin alterations. Furthermore, it cannot be excluded that L-kyn also induced apoptosis in RAW 264.7 cells. As such, these effects should be confirmed in additional control cell lines and substantiated with in vivo models.

Anti-inflammatory, antioxidant, and anti-apoptotic properties of Thunbergia laurifolia Lindl. aqueous leaf extract on LPS-stimulated RAW 264.7 macrophages

Thunbergia laurifolia Lindl., a Thai medicinal herb, has garnered substantial attention among researchers due to its recognized efficacy as an antidote, antioxidant, and anti-inflammatory agent. Despite its extensive investigation, the specific role of T. laurifolia in apoptotic inhibition remains unexplored. Therefore, this study aimed to explore the protective properties of T. laurifolia leaf extract against lipopolysaccharide (LPS)-induced apoptosis in RAW 264.7 macrophages and determine the extractʼs total phenolic as well as flavonoid content, and its antioxidant. Moreover, the cytotoxicity including anti-inflammatory potential on LPS-stimulated RAW 264.7 macrophages were assessed by measuring the inhibition of nitric oxide (NO). The outcomes of this investigation revealed remarkable similarity between the total phenolic and flavonoid contents and antioxidant capacity of the leaf extract under scrutiny and those documented in prior studies. In addition, the extract demonstrated negligible cytotoxicity at low concentrations. Furthermore, it effectively impeded the LPS-mediated production of NO, inflammatory mediator, in the macrophages. Moreover, pre-treatment with T. laurifolia leaf extract improved LPS-induced apoptosis by reducing rate of apoptotic cell death up to 41 % at the same concentration (1.68 μL/mL) in LPS-stimulated RAW 264.7 macrophages. Collectively, these findings provide compelling initial evidence attesting to the ability of T. laurifolia leaf extract to inhibit LPS-induced apoptosis in RAW 264.7 macrophages, thereby augmenting our understanding of its potential therapeutic applications in inflammatory conditions.

Bicyclol attenuates pulmonary fibrosis with silicosis via both canonical and non-canonical TGF-β1 signaling pathways

BackgroundSilicosis is an irreversible fibrotic disease of the lung caused by chronic exposure to silica dust, which manifests as infiltration of inflammatory cells, excessive secretion of pro-inflammatory cytokines, and pulmonary diffuse fibrosis. As the disease progresses, lung function further deteriorates, leading to poorer quality of life of patients. Currently, few effective drugs are available for the treatment of silicosis. Bicyclol (BIC) is a compound widely employed to treat chronic viral hepatitis and drug-induced liver injury. While recent studies have demonstrated anti-fibrosis effects of BIC on multiple organs, including liver, lung, and kidney, its therapeutic benefit against silicosis remains unclear. In this study, we established a rat model of silicosis, with the aim of evaluating the potential therapeutic effects of BIC.MethodsWe constructed a silicotic rat model and administered BIC after injury. The FlexiVent instrument with a forced oscillation system was used to detect the pulmonary function of rats. HE and Masson staining were used to assess the effect of BIC on silica-induced rats. Macrophages-inflammatory model of RAW264.7 cells, fibroblast-myofibroblast transition (FMT) model of NIH-3T3 cells, and epithelial-mesenchymal transition (EMT) model of TC-1 cells were established in vitro. And the levels of inflammatory mediators and fibrosis-related proteins were evaluated in vivo and in vitro after BIC treatment by Western Blot analysis, RT-PCR, ELISA, and flow cytometry experiments.ResultsBIC significantly improved static compliance of lung and expiratory and inspiratory capacity of silica-induced rats. Moreover, BIC reduced number of inflammatory cells and cytokines as well as collagen deposition in lungs, leading to delayed fibrosis progression in the silicosis rat model. Further exploration of the underlying molecular mechanisms revealed that BIC suppressed the activation, polarization, and apoptosis of RAW264.7 macrophages induced by SiO2. Additionally, BIC inhibited SiO2-mediated secretion of the inflammatory cytokines IL-1β, IL-6, TNF-α, and TGF-β1 in macrophages. BIC inhibited FMT of NIH-3T3 as well as EMT of TC-1 in the in vitro silicosis model, resulting in reduced proliferation and migration capability of NIH-3T3 cells. Further investigation of the cytokines secreted by macrophages revealed suppression of both FMT and EMT by BIC through targeting of TGF-β1. Notably, BIC blocked the activation of JAK2/STAT3 in NIH-3T3 cells required for FMT while preventing both phosphorylation and nuclear translocation of SMAD2/3 in TC-1 cells necessary for the EMT process.ConclusionThe collective data suggest that BIC prevents both FMT and EMT processes, in turn, reducing aberrant collagen deposition. Our findings demonstrate for the first time that BIC ameliorates inflammatory cytokine secretion, in particular, TGF-β1, and consequently inhibits FMT and EMT via TGF-β1 canonical and non-canonical pathways, ultimately resulting in reduction of aberrant collagen deposition and slower progression of silicosis, supporting its potential as a novel therapeutic agent.

Effect of Ganoderma lucidum on Cisplatin-induced Nephrotoxicity and Gastrointestinal Toxicity.

Cancer remains a major global health challenge, with an estimated 10 million cancer-related deaths in 2020, hindering efforts to extend life expectancy. Cisplatin, an effective platinum-based chemotherapeutic agent used against various malignancies, has numerous side effects. Ganoderma lucidum is a traditional Chinese medicine with extensive historical use and proven biological activity. This study investigated the effects of G. lucidum on cisplatin-induced nephrotoxicity and gastrointestinal toxicity. RAW264.7 cells were treated with cisplatin, G. lucidum, or both. Cytotoxicity and antioxidant capacity were measured. Slc:ICR (ICR) mice were treated with cisplatin, G. lucidum, or both. The survival rate and physiological data were measured. G. lucidum suppressed cisplatin-induced cytotoxicity and apoptosis in RAW264.7 cells. G. lucidum suppressed cisplatin-induced nephrotoxicity and gastrointestinal toxicity via its antioxidant effects in ICR mice. The suppressive mechanism of G. lucidum may be mediated via its antioxidant effects. These findings indicate its potential to reduce the side effects of cisplatin.

Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Icariside II modulates pulmonary fibrosis via PI3K/Akt/β-catenin pathway inhibition of M2 macrophage program

BackgroundIdiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated. ObjectiveTo scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions. MethodsThe effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation. ResultsOur investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/β-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of β-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and β-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice. ConclusionThis study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/β-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Cyclizine induces cytotoxicity and apoptosis in macrophages through the extrinsic and intrinsic apoptotic pathways.

Cyclizine, an over-the-counter and prescription antihistamine, finds widespread application in the prevention and treatment of motion sickness, encompassing symptoms such as nausea, vomiting, dizziness, along with its effectiveness in managing vertigo. However, the overuse or misuse of cyclizine may lead to hallucinations, confusion, tachycardia, and hypertension. The molecular mechanisms underlying cyclizine-induced cytotoxicity and apoptosis remain unclear. During the 24 h incubation duration, RAW264.7 macrophages were exposed to different concentrations of cyclizine. Cytotoxicity was assessed through the lactate dehydrogenase assay. Flow cytometry employing annexin V-fluorescein isothiocyanate and propidium iodide was utilized to evaluate apoptosis and necrosis. Caspase activity and mitochondrial dysfunction were evaluated through a fluorogenic substrate assay and JC-1 dye, respectively. Flow cytometry employing fluorogenic antibodies was utilized to evaluate the release of cytochrome c and expression of death receptor, including tumor necrosis factor-α receptor and Fas receptor. Western blotting was utilized to evaluate the expression of the Bcl2 and Bad apoptotic regulatory proteins. The findings unveiled from the present study demonstrated that cyclizine exerted a concentration-dependent effect on RAW264.7 macrophages, leading to the induction of cytotoxicity, apoptosis, and necrosis. This compound further activated the intrinsic apoptotic pathway by inducing mitochondrial dysfunction, Bcl2/Bad exchange expression, cytochrome c liberation, and activation of caspases contained caspase 3, 8, and 9. Moreover, the activation of the extrinsic apoptotic pathway was observed as cyclizine induced the upregulation of death receptors and increased caspase activities. Based on our investigations, it can be inferred that cyclizine prompts cytotoxicity and apoptosis in RAW264.7 macrophages in a concentration-dependent manner by triggering both the intrinsic and extrinsic apoptotic pathways.

Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression.

Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55°C and in 1mM of hydrogen peroxide (H2O2) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.

Protective effects of peroxiredoxin 6 in modeling proinflammatory response using RAW 264.7 macrophages

The aim of the work was to study the effects of peroxiredoxin 6 (PRDX6), a recombinant antioxidant protein, on the level of proinflammatory responses caused by endotoxin exposure to RAW 264.7 macrophages. The addition of LPS to the RAW 264.7 cell culture medium expectedly increased the production of TNF-α, and the addition of PRDX6 led to a significant decrease in its production by 15-20%. The level of production of another proinflammatory cytokine, IL-1β, significantly activated by endotoxin, was completely normalized under the PRDX6 action. In addition, the addition of PRDX6 reduced the production of reactive oxygen species (ROS) induced by endotoxin and also prevented overexpression of the iNos gene in RAW 264.7 cells. The results showed that PRDX6 has a suppressive effect on the expression of the Nfe2l2 gene and the production of the transcription factor NRF-2 during the first 6 h of cell culture. The addition of endotoxin causes the activation of the NF-κB and SAPK/JNK signaling cascades, while in the presence of PRDX6, the activity of these signaling cascades decreases. It is known that the proinflammatory response of cells caused by bacterial LPS leads to the activation of apoptosis and the elimination of damaged cells. Our studies confirm this, since LPS leads to the activation of the Trp53 gene, a marker of apoptosis. The addition of peroxiredoxin 6 in the first hours of the development of an acute proinflammatory response leads to the suppression of Trp53 gene expression, which indicates a protective effect of PRDX6 that reduces apoptosis in RAW 264.7 macrophages.

Salmonella Typhimurium secondary infection induced macrophages apoptosis NO-dependent during Mycobacterium neoaurum infection

Mycobacterium neoaurum (M. neoaurum) and Salmonella Typhimurium (S. Typhimurium) are important zoonotic pathogens, and both are intracellular bacteria, which can induce cellular immunity. Coinfection is prevalent worldwide, even more prevalent than single infection. This study aimed to investigate the effect of M. neoaurum/S. Typhimurium coinfection on the percentage property of C57BL/6 mice regulatory T cells (Tregs) and the immune activity of RAW264.7 cells. The secretion of TNF-α, IFN-γ, IL-1β, IL-12 and iNOS in RAW264.7 cells was determined by ELISA. The expression of CD40+ , CD80+ and CD86+ costimulatory molecules on the surface of macrophages was analyzed by flow cytometry. A Nitric oxide (NO) assay was used to detect the production of NO in RAW264.7 cells. The apoptosis of RAW264.7 cells was detected by flow cytometry. The results showed that macrophage expressed a large number of cytokines and surface costimulatory molecules to enhance immune activity. S. Typhimurium secondary infection significantly increased the expression of iNOS and generation of NO, and M. neoaurum/S. Typhimurium-induced apoptosis was NO-dependent. Our data provide a theoretical basis for secondary infections by other pathogens after Nontuberculous Mycobacterium (NTM) infection, and lay a foundation for further research.

BDE-47 Induces Immunotoxicity in RAW264.7 Macrophages through the Reactive Oxygen Species-Mediated Mitochondrial Apoptotic Pathway

Polybrominated diphenyl ethers (PBDEs) are classic and emerging pollutants that are potentially harmful to the human immune system. Research on their immunotoxicity and mechanisms suggests that they play an important role in the resulting pernicious effects of PBDEs. 2,2',4,4'-Tetrabrominated biphenyl ether (BDE-47) is the most biotoxic PBDE congener, and, in this study, we evaluated its toxicity toward RAW264.7 cells of mouse macrophages. The results show that exposure to BDE-47 led to a significant decrease in cell viability and a prominent increase in apoptosis. A decrease in mitochondrial membrane potential (MMP) and an increase in cytochrome C release and caspase cascade activation thus demonstrate that cell apoptosis induced by BDE-47 occurs via the mitochondrial pathway. In addition, BDE-47 inhibits phagocytosis in RAW264.7 cells, changes the related immune factor index, and causes immune function damage. Furthermore, we discovered a significant increase in the level of cellular reactive oxygen species (ROS), and the regulation of genes linked to oxidative stress was also demonstrated using transcriptome sequencing. The degree of apoptosis and immune function impairment caused by BDE-47 could be reversed after treatment with the antioxidant NAC and, conversely, exacerbated by treatment with the ROS-inducer BSO. These findings indicate that oxidative damage caused by BDE-47 is a critical event that leads to mitochondrial apoptosis in RAW264.7 macrophages, ultimately resulting in the suppression of immune function.

Visfatin is a multifaceted molecule that exerts regulation effects on inflammation and apoptosis in RAW264.7 cells and mice immune organs.

Visfatin, a multifunctional adipocytokine, is particularly important in the regulation of apoptosis and inflammation through an unidentified mechanism. Clarifying the control mechanisms of visfatin on inflammation and apoptosis in RAW264.7 cells and mice immunological organs was the goal of the current investigation. In order to create a pathophysiological model, the RAW264.7 cells were stimulated with 200 ng/mL visfatin and 20 μg/mL lipopolysaccharide (LPS), either separately or combined. The effects of exogenous visfatin on inflammation and apoptosis in RAW264.7 cells were investigated by flow cytometry assay, RNA-seq analysis and fluorescence quantitative PCR. According to the findings, exogenous visfatin exhibits dual effects on inflammation by modulating the expression of IL-1α, TNFRSF1B, and LIF as well as taking part in various signaling pathways, including the MAPK and Rap1 signaling pathways. By controlling the expression levels of Bcl2l1, Bcl2a1a, and Fas and primarily participating in the PI3K/AKT signaling pathway and Hippo signaling pathway, exogenous visfatin can inhibit apoptosis in RAW264.7 cells. The visfatin inhibitor FK866 was used to further confirm the effects of visfatin on inflammation and apoptosis in mice immune organs. Subsequently, mice spleen and thymus were collected. It is interesting to note that in LPS-treated mice, suppression of endogenous visfatin might worsen the immune system's inflammatory response and even result in rapid mortality. Additionally, endogenous visfatin promotes the apoptosis in mice immune organs by regulating the expression levels of Bcl2l1, Fas, Caspase 3, Bcl2a1a, and Bax. Together, these results imply that visfatin is a multifaceted molecule that regulates inflammation and apoptosis in RAW264.7 cells and mice immunological organs by taking part in a variety of biological processes and regulating the amounts of associated cytokines expression. Our findings offer additional understandings of how visfatin affects apoptosis and inflammation.

The exploration of the potential mechanism of oxymatrine-mediated antipruritic effect based on network pharmacology and weighted gene co-expression network analysis.

The treatment of chronic itch is considered to be a challenge for its non-histamine dependence and the search for alternative medicine is still striving. The pathology of the chronic itch is closely related to immune system regulation and inflammatory response. Oxymatrine (OMT) is a traditional Chinese medicine ingredient extracted from the roots of Sophora flavescens Aiton with significant antitumor, analgesic, and anti-inflammatory effects. However, the underlying mechanism of OMT on chronic itch is obscure, which limits clinical application. Hence, this study is aimed to clarify the pruritus alleviation mechanism of OMT by combining network pharmacology analysis, weighted gene co-expression analysis (WGCNA), and molecular docking. We screened 125 common targets of OMT regulating inflammation and pruritus with pharmacology technology, the GO enrichment function analysis and KEGG signaling pathway analysis to demonstrate the close relation to the signaling pathways regulating inflammation such as MAPK signaling pathway and PI3K-AKT signaling pathway. We adopted the most relevant templates for pruritus diseases, combined with network pharmacology to preliminarily screen out 3 OMT functions and regulatory targets, exerting a good connection and correlation with the target at the screened disease targets. Further experiments were conducted to explore the potential mechanism of OMT using the LPS-induced RAW264.7 cell inflammation model. The results showed that pretreatment with different concentrations of OMT (25 μM, 50 μM, and 100 μM) for 24 h, inhibited expression of IL-6, iNOS TLR4 and TGFR-1 as well as apoptosis of Raw264.7 cells induced by LPS. Moreover, OMT effectively inhibited LPS-induced MAPK pathway activation and the expression of related sites MAP2K1, MAPK8 MAP2K4, and MAPKAP-K2 in RAW 264.7 cells. The OMT also reduced the phosphorylation of p-38, associated with site in the activation of MAPK signaling pathway. These results could contribute to a better understanding of the mechanisms underlying how OMT alleviates inflammation to treat chronic pruritic diseases and provide a potential drug for the treatment of chronic itch.

Anti-Inflammatory Effects and Mechanisms of Dandelion in RAW264.7 Macrophages and Zebrafish Larvae.

Dandelions (Taraxacum spp.) play an important role in the treatment of inflammatory diseases. In this study, we investigated the anti-inflammatory effects of Dandelion Extract (DE) in LPS-induced RAW264.7 macrophages and copper sulfate (CuSO4)-induced zebrafish larvae. DE was not toxic to RAW264.7 cells at 75 μg/ml as measured by cell viability, and DE inhibited LPS-induced cell morphological changes as measured by inverted microscopy. In survival experiments, DE at 25 μg/ml had no toxicity to zebrafish larvae. By using an enzymatic standard assay, DE reduced the production of nitric oxide (NO) in LPS-induced RAW264.7 cells. Fluorescence microscopy results show that DE reduced LPS-induced ROS production and apoptosis in RAW264.7 cells. DE also inhibited CuSO4-induced ROS production and neutrophil aggregation in zebrafish larvae. The results of flow cytometry show that DE alleviated the LPS-induced cell cycle arrest. In LPS-induced RAW264.7 cells, RT-PCR revealed that DE decreased the expression of M1 phenotypic genes iNOS, IL-6, and IL-1β while increasing the expression of M2 phenotypic genes IL-10 and CD206. Furthermore, in CuSO4-induced zebrafish larvae, DE reduced the expression of iNOS, TNF-α, IL-6, and IL-10. The findings suggest that DE reduces the LPS-induced inflammatory response in RAW264.7 cells by regulating polarization and apoptosis. DE also reduces the CuSO4-induced inflammatory response in zebrafish larvae.

Sevoflurane reduces lipopolysaccharide-induced apoptosis and pulmonary fibrosis in the RAW264.7 cells and mice models to ameliorate acute lung injury by eliminating oxidative damages

ABSTRACT Objectives Sevoflurane is identified as an effective candidate drug for acute lung injury (ALI) treatment, but its curing effects and detailed mechanisms have not been fully disclosed. The present study was designed to resolve this academic issue. Methods The ALI mice models were established, and Hematoxylin-eosin staining assay was performed to examine tissue morphologies. Cell viability was determined by CCK-8 assay, and Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The expression levels of proteins were determined by performing Western Blot analysis and immunofluorescence staining assay. ROS levels were examined by using DCFH-DA staining assay. Results In this study, we investigated this issue and the ALI models were respectively established by treating the BALB/c mice and the murine macrophage cell line RAW264.7 with different concentrations of lipopolysaccharide (LPS) in vivo and in vitro, which were subsequently subjected to sevoflurane co-treatment. The results showed that sevoflurane reduced LPS-induced ALI in mice and suppressed LPS-triggered oxidative stress and apoptotic cell death in the RAW264.7 cells. Interestingly, we evidenced that the elimination of reactive oxygen species (ROS) by N-acetyl-L-cysteine (NAC) reversed LPS-induced cell apoptosis in RAW264.7 cells. Then, we verified that sevoflurane suppressed oxidative damages to restrain LPS-induced apoptotic cell death in the RAW264.7 cells through activating the anti-oxidant Keap1/Nrf2 pathway. Mechanistically, sevoflurane down-regulated Keap1 and upregulated Nrf2 in nucleus to activate the downstream anti-oxidant signaling cascades, which further ameliorated LPS-induced cell apoptosis and lung injury by eliminating oxidative damages. Discussion Taken together, our study illustrated that the sevoflurane attenuates LPS-induced ALI by inhibiting oxidative stress-mediated apoptotic cell death and inflammation, and the Keap1/Nrf2 pathway played an important role in this process.

MicroRNA-455-3p regulates proliferation and osteoclast differentiation of RAW264.7 cells by targeting PTEN

BackgroundMacrophages are one of the important cells in immune system. In this article, we aim to explore the regulatory role of miR-455-3p on proliferation and osteoblast differentiation of RAW264.7 cells.MethodsExpression levels of genes and proteins in cells were tested via qRT-PCR and western blot. The targeted correlation between miR-455-3p and PTEN was identified by luciferase analysis. MTT assay and flow cytometry were applied to detect the proliferation and apoptosis of cells. Osteoclastogenesis was completed by stimulating RAW 264.7 cells with RANKL. Tartrate-resistant acid phosphatase (TRAP) activity in different groups of cells were assessed.ResultsFirstly, we determined that up-regulation of miR-455-3p promoted the proliferation and inhibited apoptosis of RAW 264.7 cells. MiR-455-3p deficiency played opposite effect in RAW 264.7 cells. Additionally, osteoclastogenesis-related factors (TRAP, CTSK and NFATc1) expression levels were remarkably up-regulated in miR-455-3p-mimic group of RAW264.7 cells treated with RANKL, but decreased in inhibitor group. Luciferase assay proved that miR-455-3p targeted PTEN. We took a further step and found overexpression of PTEN significantly inhibited the increased proliferation and osteoblast differentiation of RAW264.7 cells induced by miR-455-3p.ConclusionsOur findings supported basic to explore the molecular mechanism of proliferation and osteoblast differentiation of RAW264.7 cells.

Astragaloside IV protects against C/EBP homologous protein-mediated apoptosis in oxidized low-density lipoprotein-treated macrophages by promoting autophagy

Astragaloside Ⅳ (AS-Ⅳ) is one of the main active components extracted from Astragalus membranaceus that exerts an antiatherosclerotic effect. Our study explored the underlying anti-apoptotic effects and the mechanisms of action of AS-Ⅳ in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages and in vulnerable plaques. The results showed that AS-Ⅳ lowered the oxLDL-induced lipid content and reversed the oxLDL-induced reduction in cell viability and elevation in lactate dehydrogenase (LDH) leakage and apoptosis in RAW264.7 macrophages, similar to the effects of 4-phenylbutyric acid (PBA, an ER stress inhibitor). In addition, consistent with the effect exerted by PBA, AS-Ⅳ inhibited oxLDL-triggered ER stress activation by decreasing the level of inositol-requiring enzyme1 phosphorylation and transcription factor 6 nuclear translocation and upregulating the protein and mRNA expression of glucose-regulated protein 78 (GPR78) and C/EBP homologous protein (CHOP). As expected, autophagy activation was induced by AS-IV, evidenced by increased expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ), autophagy-related gene 5, and beclin-1 in macrophages. Furthermore, after pretreatment with 3-methyladenine and beclin-1 small interfering RNA, the inhibitory role played by AS-Ⅳ in oxLDL-induced ER stress-CHOP-mediated macrophage apoptosis was weakened, while its inhibitory effect was further enhanced by rapamycin pretreatment. Moreover, administration of AS-Ⅳ or rapamycin to Apoe−/− mice upregulated LC3-Ⅱ expression and collagen content but decreased CHOP expression, macrophage apoptosis, and lipid areas. Overall, by promoting autophagy, AS-Ⅳ effectively protects macrophages from oxLDL-induced apoptosis mediated by ER stress-CHOP, which may reinforce the stability of atherosclerotic plaques.

Knockdown of GBP1 inhibits BCG-induced apoptosis in macrophage RAW 264.7 cells via p38/JNK pathway.

Alveolar macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb) plays a significant role in mediating the pathogenesis of tuberculosis. There is growing evidence that guanylate-binding proteins (GBPs) are associated with different pathological processes such as microbial infection. However, it remains unclear whether GBPs can regulate the apoptosis of macrophages induced by Mtb. In this study, we investigated the potential effect of GBP1 on RAW 264.7 cell apoptosis during Bacillus Calmette-Guerin (BCG) infection. The results demonstrated that BCG could induce macrophage apoptosis and GBP1 upregulation. In addition, we explored the role of GBP1 in regulating BCG-induced RAW 264.7 cell apoptosis using small interfering RNAs targeting GBP1. The results showed that knockdown of GBP1 could attenuate BCG-induced apoptosis in RAW 264.7 cells. Moreover, we found that GBP1 knockdown decreased the levels of cleaved-Caspase 3 and cleaved-PARP-1, while decreased those of cleaved-Caspase 9, BAX, Cytochrome C and APAF1. These findings imply that GBP1 knockdown can prevent BCG-induced apoptosis through an endogenous apoptosis pathway. In addition, the mitochondrial membrane potential of macrophages was significantly increased after BCG infection, and GBP1 knockdown could alleviate this phenomenon. Furthermore, downregulation of GBP1 also attenuated BCG-induced accumulation of reactive oxygen species in macrophages. Mechanistically, GBP1 suppressed the phosphorylation of the target molecules in p38/JNK pathway, thus regulating the apoptosis of BGC-infected macrophages. Collectively, these findings reveal a significant role of GBP1 in mediating cell apoptosis in macrophages infected with BCG, and the molecular mechanism underlying its suppressive effect on BCG-induced apoptosis.

Semaphorin3A regulates mitochondrial apoptosis in RAW264.7 cells in vitro

Osteoclasts apoptosis plays vital roles in abnormal bone remodeling-associated diseases. It’s reported that Semaphorin3A (Sema3A) may affect osteoarthritis progression through osteoclasts function, while the specific mechanism remains unclear. To determine the effect of Sema3A on osteoclasts, we detected the apoptosis of RAW264.7 cells treated with Sema3A and explored the potential pathways in vitro. Firstly, Cell Counting Kit-8 (CCK8) assay was performed to assess the proliferation to screen out the appropriate concentration of Sema3A. Then, apoptosis was evaluated after cells were incubated with Sema3A for 0, 2, 6, 12, 24 and 48 h, respectively, by Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL) staining and flow cytometry. Finally, phosphorylation of Nrf2 and CREB, mitochondrial apoptosis-related markers and JNK/AKT/ERK pathway activation were examined by qRT-PCR and Western blot. As a result, flow cytometry and TUNEL staining revealed that RAW264.7 cells apoptosis was promoted by Sema3A-treated. Meanwhile, Western blot showed that cleaved-caspase-3 and cleaved-caspase-9 were highly expressed in Sema3A-treated RAW264.7 cells, and the ERK/JNK/AKT pathways were significantly inhibited by Sema3A in RAW264.7 cells. In a word, we demonstrated that Sema3A induces apoptosis of RAW264.7 cells probably by promoting mitochondrial apoptosis, which may provide a potential target for therapeutic strategies.