In the present study, we investigated whether baicalin could reduce the damage caused to RAW264.7 cells following infection with H6N6 avian influenza virus. In addition, we studied the expression of autophagy-related genes. The morphological changes in cells were observed by hematoxylin and eosin (H&E) staining, and the inflammatory factors in the cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to detect the levels of RAW264.7 autophagosomes, and western blotting and immunofluorescence were used to detect the protein expression of autophagy marker LC3. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the mRNA transcription levels of autophagy key factors. The results showed that different doses of baicalin significantly reduced the H6N6 virus-induced damage of RAW264.7 cells. The contents of interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF)-α in the cell supernatant significantly decreased. In addition, the protein expression of LC3 and Beclin-1, ATG12, ATG5 the mRNA levels were significantly decreased. This study showed that baicalin can reduce cell damage and affect the H6N6-induced autophagy level of RAW264.7 cells.

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including Group IVA cytosolic PLA2 (cPLA2), Group V secreted PLA2 (sPLA2), Group VIA calcium independent PLA2 (iPLA2) and Group VIIA lipoprotein-associated PLA2, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2\'s associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2, and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

Baicalin, a flavonoid extracted from traditional Chinese medicine, Scutellaria baicalensis has significant anti-inflammatory effects. Microsponges are drug delivery systems that improve drug stability and slow the release rate. The combination of baicalin and the microsponges produced a new and stable system for its delivery, resulting in a novel formulation of baicalin. Baicalin microsponges (BM) were prepared using the quasi-emulsion solvent diffusion method. Effects of the mass ratio of the polymer (ethylcellulose) to baicalin, the concentration of the emulsifier polyvinyl alcohol (PVA), the stirring speed on the encapsulation efficiency (EE), and yield of the microsponges were investigated by combining the one-factor test and Box-Behnken design (BBD). The preparation process was standardised using 2.61:1 mass ratio of ethyl cellulose to baicalin, 2.17% concentration of PVA, with stirring at 794 rpm. Optimised BM formulations were evaluated for the parameters of EE (54.06 ± 3.02)% and yield of (70.37 ± 2.41)%, transmission electron microscopy (TEM), and in vitro cell evaluation. Results of the in vitro anti-inflammatory assay showed that baicalin microsponges-pretreated-lipopolysaccharide (LPS)-induced RAW264.7, mouse macrophages showed reduced inflammatory response, similar to that seen in baicalin-treated macrophages.

UNASSIGNED: Previous studies have revealed dexmedetomidine have potential protective effects on vital organs by inhibiting the release of inflammatory cytokines. To investigate the effects of dexmedetomidine on sepsis, especially in the initial inflammatory stage of sepsis. RAW264.7 cells were used as the cell model in this study to elucidate the underlying mechanisms.
UNASSIGNED: In this study, we conducted several assays to investigate the mechanisms of dexmedetomidine and HOTAIR in sepsis. Cell viability was assessed using the CCK-8 kit, while inflammation responses were measured using ELISA for IL-1β, IL-6, and TNF-α. Additionally, we employed qPCR, MeRIP, and RIP to further explore the underlying mechanisms.
UNASSIGNED: Our findings indicate that dexmedetomidine treatment enhanced cell viability and reduced the production of inflammatory cytokines in LPS-treated RAW264.7 cells. Furthermore, we observed that the expression of HOTAIR was increased in LPS-treated RAW264.7 cells, which was then decreased upon dexmedetomidine pre-treatment. Further investigation demonstrated that HOTAIR could counteract the beneficial effects of dexmedetomidine on cell viability and cytokine production. Interestingly, we discovered that YTHDF1 targeted HOTAIR and was upregulated in LPS-treated RAW264.7 cells, but reduced in dexmedetomidine treatment. We also found that YTHDF1 increased HOTAIR and HOTAIR m6A levels.
UNASSIGNED: Collectively, our results suggest that dexmedetomidine downregulates HOTAIR and YTHDF1 expression, which in turn inhibits the biological behavior of LPS-treated RAW264.7 cells. This finding has potential implications for the prevention and treatment of sepsis-induced kidney injury.

Deoxynivalenol (DON) as a mycotoxin was commonly found in food and cereals which can affect immune function and inflammatory response. The majority of foods contain DON at levels below the official limit. This study aimed to evaluate the effects of non-cytotoxic concentration of DON on inflammation and its mechanisms using the IL-10 gene-silenced RAW264.7 cell model. The results showed that a non-cytotoxic concentration of DON at 25 ng/ml aggravated IL-10 knockdown-induced inflammation, which was manifested by increasing IL-1β and TNF-α mRNA expression, migration and phagocytosis, decreasing IL-10 mRNA expression, and enhancing JAK2/STAT3 phosphorylation. Adding JAK2 inhibitor AG490 attenuated the aggravating effect of DON on IL-10 knockdown-induced inflammation. In conclusion, a non-cytotoxic concentration of DON enhances the inflammatory response through the JAK2/STAT3 signaling pathway when inflammation occurs in the body. These results indicated that non-cytotoxic concentrations of DON could aggravate inflammation when inflammation was induced by IL-10 knockdown, which increases vigilance against DON contamination at low concentration especially when an animal\'s body has inflammation.

BACKGROUND: Shangkehuangshui (SK) has been traditionally used to treat traumatic injury, soft tissue and bone injury in Foshan hospital of traditional Chinese medicine for more than 60 years, which composed of many Chinese herbs such as Coptis chinensis Franch., Gardenia jasminoides Ellis, Phellodendron chinense Schneid. and etc. SK exhibits heat-clearing and detoxifying, enhancing blood circulation to eliminate blood stasis properties, and demonstrates noteworthy clinical efficacy. Nevertheless, the underlying mechanism remains uncertain.
OBJECTIVE: The early study found that SK had good anti-inflammatory effects in acute soft tissue injury model. This research is to verify the anti-inflammatory properties of SK both in vitro and in vivo via TLR4/TLR2-NF-κB signaling pathway, to clarify the underlying mechanisms responsible for the curative effect of SK.
METHODS: The RAW264.7 cells inflammatory model was established with lipopolysaccharide (LPS) in vitro. NO and TNF-α, IL-6, IL-1β were determined with Griess method and ELISA method respectively. The mRNA and protein expression levels of TLR4/TLR2-NF-κB pathway were evaluated by qPCR and Western blot method. In vivo experiment, chronic soft tissue injury rat models were established by tracking gastrocnemius muscle with electrical stimulation, then local appearance and pathological changes were observed and recorded, the contents of inflammatory factors in serum and tissue were performed. Moreover, we also measured and contrasted the expression of TLR4/TLR2-NF-κB related factors.
RESULTS: SK effectively inhibited the LPS-induced generation of inflammatory cytokines, including NO, TNF-α, IL-6 and IL-1β in RAW264.7 cells, and significantly suppressed the expression of TLR4, TLR2, MyD88, IκB, and NF-κB. In vivo, SK remarkably decreased the damage appearance scores after 4 and 14 days of administration and inhibit the quantity of NO and leukocytes present in the serum. Additionally, the inflammatory infiltration in the pathological section was alleviated, myofibrillar hyperplasia and blood stasis were reduced. SK markedly downregulated NO, TNF-α, IL-6 and IL-1β in injured tissues of rats, also declined the expression of TLR4, TLR2, MyD88, IκB, NF-κB, IL-6, TNF-α and IL-1β.
CONCLUSIONS: This study revealed that SK had obvious effects of anti-inflammatory actions in vivo and vitro, effectively reduced acute and chronic soft tissue injury in clinical, this might be attributed to inhibit the TLR4/TLR2-NF-κB pathway, further inhibit the expression of downstream relevant pro-inflammatory cytokines.

BACKGROUND: Artemisia mongolica is well known for its use in folk medicine, it is commonly used to alleviate a variety of diseases associated with inflammation, such as laryngitis, tonsillitis, headaches and hepatitis in northwest China. However, its anti-inflammatory mechanism is still unknown.
OBJECTIVE: The most potential anti-inflammatory part (AMPA) was identified by screening individual parts of A. Mongolica. After the network pharmacological analysis, the anti-inflammation effects and molecular mechanisms of AMPA were evaluated in RAW264.7 cells induced by LPS.
METHODS: AMPA was chosen as the most anti-inflammatory of the A. Mongolica, as measured by the effect of each part of the A. Mongolica on NO and COX-2. The chemical composition of AMPA was identified using HPLC-Q-TOF-MS/MS, and targets of bioactive chemicals and targets related to inflammation were found using open-source databases. The \"Compound-targets\" network and PPI network were established by combining compounds and overlapped targets, and targets in the PPI networks were analyzed by GO and KEGG enrichment. The RAW26.7 cells induced by LPS were used as a model of inflammation examination. MTT assay was performed to assess the cytotoxicity of AMPA on LPS-induced RAW264.7 cells. The level of NO was measured by the Griess method while the inflammatory factors were detected by ELISA. The protein expression levels of iNOS, COX-2, MAPK, NF-κB signaling pathway and AMPK/Nrf2-related proteins were determined by Western blot. The results of nuclear translocation of p65 and Nrf2 were analyzed by immunofluorescence assay.
RESULTS: A total of 18 compounds with potential bioactivity were identified, and after intersecting 640 compound-predicted targets and 1608 inflammation targets, the compounds and intersected targets were utilized to structure \"compound-target\" and PPI networks. Among AMPA, AM6, AM7, AM11, AM8 and AM1 compounds were essential in the \"compound-targets\" network, meanwhile, TNF, RELA, MAPK1, NOS2, PRKAG, and PTGS2 targets play important roles in the PPI network. The top 10 terms and pathways were obtained based on GO and KEGG. The cell experiments show that 50 μg/mL was the maximum concentration of AMPA without cytotoxicity in the LPS-induced RAW264.7 cell model. When compared with the LPS group, AMPA treatment not only effectively suppressed the generation of NO, TNF-α, IL-6, PGE2, IL-1β and MCP-1 in LPS-induced RAW264.7 cells, but also down-regulated the expression of COX-2, iNOS and the protein levels p-ERK, p-p38, p-IκB-α and p-p65, inhibited the nuclear translocation of p65. Furthermore, the expression levels of p-LKB1, p-AMPK, Nrf2 and HO-1 proteins were up-regulated and Nrf2 nuclear translocation was promoted.
CONCLUSIONS: AMPA should be considered an anti-inflammatory agent for the results of network pharmacology and in vitro, which could inhibit the MAPK pathway and NF-κB pathway and activate the AMPK/Nrf2 pathway in LPS-stimulated RAW264.7 cells.

U(VI) pollution has already led to serious harm to the environment and human health with the increase of human activities. The viability of RAW264.7 cells was assessed under various U(VI) concentration stress for 24 and 48 h. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD) activities of RAW264.7 cells under U(VI) stress were measured. The results showed that U(VI) decreased cell activity, induced intracellular ROS production, abnormal MMP, and increased SOD activity. The flow cytometry with Annexin-V/PI double labeling demonstrated that the rate of late apoptosis increased with the increase of U(VI) concentration, resulting in decreased Bcl-2 expression and increased Bax expression. The morphology of RAW264.7 cells dramatically changed after 48 h U(VI) exposure, including the evident bubble phenomenon. Besides, U(VI) also increased the proportion of LDH releases and increased GSDMD, and Ras, p38, JNK, and ERK1/2 protein expression, which indicated that the MAPK pathway was also involved. Therefore, U(VI) ultimately led to apoptosis and pyroptosis in RAW264.7 cells. This study offered convincing proof of U(VI) immunotoxicity and established the theoretical framework for further fundamental studies on U(VI) toxicity.

UNASSIGNED: Osteoclasts are multinucleated cells formed by macrophage cell fusion that are responsible for bone resorption. Previously, we found that treating osteoclastic progenitor cells with (-)-epigallocatechin gallate (EGCg) increased cell fusion. In this study, we aimed to identify factors involved in the cell fusion induced by EGCg.
UNASSIGNED: We hypothesized that EGCg-induced oxidative stress might be involved in cell fusion, and used macrophage cell line RAW264.7 cells. We evaluated cell fusion activity after adding the antioxidants N-acetyl-l-cysteine (NAC) or catalase in addition to EGCg. The mRNA expressions of genes related to cell fusion and bone resorption were quantified by real-time PCR. Finally, we added hydrogen peroxide and examined its effects on cell fusion and TRAP activity.
UNASSIGNED: EGCg-induced cell fusion was strongly inhibited by the addition of NAC in a dose-dependent manner (EGCg with 5 mM NAC; decreased to 1.5%; p < 0.05), while the inhibitory effect of catalase was limited (EGCg with 500 U/mL catalase; decreased to 27.7%; p < 0.05). DC-STAMP expression was significantly upregulated by EGCg compared with the untreated group, and the upregulation was significantly suppressed by 5 mM NAC. Conversely, Nfatc1 and TRAP expression were not upregulated by EGCg. These results suggest that EGCg induces DC-STAMP expression via reactive oxygen species production, which regulates cell fusion but does not affect the osteoclastic pathway. Although treatment with hydrogen peroxide promoted the formation of multinucleated cells, no increase in TRAP activity was observed, which was similar to EGCg treatment.
UNASSIGNED: This study suggests that the increased cell fusion by EGCg may be induced by oxidative stress due to reactive oxygen species production.

UNASSIGNED: Pterospermum rubiginosum is an evergreen plant in Western Ghats, India, used by traditional tribal healers due to its excellent biological potential for treating inflammation and pain relief procedures. The bark extract is also consumed to relieve the inflammatory changes at the bone fractured site. The traditional medicinal plant in India have to be characterized for its diverse phytochemical moieties, its interactive multiple target sites, and to reveal the hidden molecular mechanism behind the biological potency.
OBJECTIVE: The study focussed on plant material characterization, computational analysis (prediction study), toxicological screening (In vivo), and anti-inflammatory evaluation of P. rubiginosum methanolic bark extracts (PRME) in LPS-induced RAW 264.7 cells.
METHODS: The pure compound isolation of PRME and their biological interactions were used to predict the bioactive components, molecular targets, and molecular pathways of PRME in inhibiting inflammatory mediators. The anti-inflammatory effects of PRME extract were evaluated in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage cell model. The toxicity evaluation of PRME was performed in healthy 30 Sprague-Dawley experimental rats, were randomly divided into five groups for toxicological evaluation for 90 days. The tissue levels of oxidative stress and organ toxicity markers were measured using the ELISA method. Nuclear magnetic resonance spectroscopy (NMR) was performed to characterize the bioactive molecules.
RESULTS: Structural characterization revealed the presence of vanillic acid, 4-O-methyl gallic acid, E-resveratrol, gallocatechin, 4\'-O-methyl gallocatechin, and catechin. Molecular docking of NF-kB exhibited significant interactions with vanillic acid and 4-O-methyl gallic acid with binding energy -351.159 Kcal/Mol and -326.5505 Kcal/Mol, respectively. The PRME-treated animals showed an increase in total GPx and antioxidant levels (SOD and catalase). Histopathological examination revealed no variation in the liver, renal and splenic tissue\'s cellular pattern. PRME inhibited the pro-inflammatory parameters (IL-1β, IL-6, and TNF-α) in LPS-induced RAW 264.7 cells. The protein level of TNF-α and NF-kB protein expression study brought out a notable reduction and exhibited a good correlation with the gene expression study.
CONCLUSIONS: The current study establishes the therapeutic potential of PRME as an effective inhibitory agent against LPS-activated RAW 264.7 cells induced inflammatory mediators. Long-term toxicity evaluation on SD rats confirmed the non-toxic nature of PRME up to 250mg/body weight for 3 months.