mRNA Expression Of IL-1β Research Articles

Suppression of inflammation-induced lung cancer cells proliferation and metastasis by exiguaflavanone A and exiguaflavanone B from Sophora exigua root extract through NLRP3 inflammasome pathway inhibition.

Objective: Non-small cell lung cancer (NSCLC) is recognized for its aggressive nature and propensity for high rates of metastasis. The NLRP3 inflammasome pathway plays a vital role in the progression of NSCLC. This study aimed to investigate the effects of S. exigua extract and its active compounds on NLRP3 regulation in NSCLC using an in vitro model. Methods: S. exigua was extracted using hexane, ethyl acetate and ethanol to obtain S. exigua hexane fraction (SE-Hex), S. exigua ethyl acetate fraction (SE-EA), and S. exigua ethanol fraction (SE-EtOH) respectively. The active compounds were identified using column chromatography and NMR analysis. A549 cells were primed with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) for activated NLRP3 inflammasome. The anti-inflammatory properties were determined using ELISA assay. The anti-proliferation and anti-metastasis properties against LPS-ATP-induced A549 cells were determined by colony formation, cell cycle, wound healing, and trans-well migration and invasion assays. The inflammatory gene expressions and molecular mechanism were determined using RT-qPCR and Western blot analysis, respectively. Results: SE-EA exhibited the greatest anti-inflammation properties compared with other two fractions as evidenced by the significant inhibition of IL-1β, IL-18, and IL-6, cytokine productions from LPS-ATP-induced A549 cells in a dose-dependent manner (p < 0.05). The analysis of active compounds revealed exiguaflavanone A (EGF-A) and exiguaflavanone B (EGF-B) as the major compounds present in SE-EA. Then, SE-EA and its major compound were investigated for the anti-proliferation and anti-metastasis properties. It was found that SE-EA, EGF-A, and EGF-B could inhibit the proliferation of LPS-ATP-induced A549 cells through cell cycle arrest induction at the G0/G1 phase and reducing the expression of cell cycle regulator proteins. Furthermore, SE-EA and its major compounds dose-dependently suppressed migration and invasion of LPS-ATP-induced A549 cells. At the molecular level, SE-EA, EGF-A, and EGF-B significantly downregulated the mRNA expression of IL-1β, IL-18, IL-6, and NLRP3 in LPS-ATP-induced A549 cells. Regarding the mechanistic study, SE-EA, EGF-A, and EGF-B inhibited NLRP3 inflammasome activation through suppressing NLRP3, ASC, pro-caspase-1(p50 form), and cleaved-caspase-1(p20 form) expressions. Conclusion: Targeting NLRP3 inflammasome pathway holds promise as a therapeutic approach to counteract pro-tumorigenic inflammation and develop novel treatments for NSCLC.

PSIX-11 Testing the Anti-Inflammatory Effects of brewer’s Yeast Extract on Macrophage Cell Lines in Vitro

Abstract Recent studies have demonstrated that brewer’s yeast may modulate gastrointestinal and immune health. Yeasthas been reported to contain bioactive components that possess anti-inflammatory properties in vitro and in animal models, but source of yeast and processing conditions are unique to each product and require testing. The objective of this study was to evaluate the anti-inflammatory effects of a novel brewer’s yeast product in response to a lipopolysaccharide (LPS) challenge using two macrophage cell lines (Raw 264.7 cells; J774A.1 cells) in vitro. Once cells were stable, they were exposed to one of the following treatments for 24 h: sterile phosphate-buffered saline (placebo), brewer’s yeast extract (low dose or high dose), LPS only (positive control), and LPS + brewer’s yeast extract (low dose or high dose). Quantitative real time PCR (qPCR) was used to analyze relative mRNA production of pro-inflammatory cytokines (IL-6, TNF-α, IL-1α and IL-1β) in response to the treatments. Statistical analysis was performed using the PROC MIXED procedure of SAS, testing for the main effect of treatment. Significance was declared as P ≤ 0.05. As expected, LPS increased expression of IL-6, TNF-α, IL-1α and IL-1β to greater (P &amp;lt; 0.05) concentrations than that of the placebo and yeast treatments in both cell lines. The results also showed that brewer’s yeast treatment in RAW 264.7 cells led to greater (P ≤ 0.01) mRNA expression of IL-6, IL-1α and IL-1β during LPS challenge compared with control (LPS only) cells. In J774A.1 cells, brewer’s yeast treatments reduced (P ≤ 0.01) the mRNA expression of IL-1α and IL-1β during LPS challenge in a dose-dependent manner. However, no differences were observed in the mRNA abundance of TNF-α or IL-6 in J774A.1 cells. These findings suggest that the brewer’s yeast extract tested may possess anti-inflammatory effects in macrophage cell lines in vitro, although the responses varied across cell lines and yeast extract concentrations. Further research is needed to explore the potential applications of brewer’s yeast as an anti-inflammatory agent in the target animals of interest.

PSVIII-26 Baicalin Alleviates Lincomycin-Induced Intestinal Injury By Regulating Gut Microbiota in Weaned Pigs

Abstract Lincomycin and related antibiotics are widely used in swine clinics, but its side effects including diarrhea and inflammatory bowel disease are difficult to detect and controlled. The purpose of this study was to investigate the effect and mechanism of baicalin, a flavonoid extracted from scutellaria baicalensis, on alleviating lincomycin-related intestinal injury. Forty-eight 21-day-old weaned pigs were selected and randomly divided into three groups: control group, lincomycin group (LC), and baicalin treatment group (LC + BL). Pigs in the LC and LC + BL groups were administered with 1 g/kg lincomycin in feed for 1 week. In the second week, pigs in the LC + BL group were administered 500 mg/kg baicalin in feed. After 2 weeks, 6 piglets in each group were selected to obtain intestinal samples for subsequent analysis, and the body weight of the remaining piglets was recorded on day 28. The results showed that lincomycin significantly reduced the body weight of the pigs on day 28 (P &amp;lt; 0.05), but baicalin treatment restored the body weight of piglets on day 28 to the control level. As for intestinal morphology, lincomycin damaged intestinal morphology and reduced villus height in the jejunum of piglets, but these indexes recovered to the control level after baicalin treatment (P &amp;lt; 0.05). In addition, lincomycin decreased claudin-1, TNF-α, and IL-1β mRNA expression in the jejunum (P &amp;lt; 0.05), but these indexes recovered to the control level after baicalin treatment. Then 16S rDNA sequencing was used to examine the microbial composition in the jejunum. Lincomycin significantly increased the abundance of Proteobacteria, Chloroflexi, and Deinococcota (P &amp;lt; 0.05), but baicalin decreased the abundance of Chloroflexi, Acidobacteriota, Patescibacteria, Myxococcota, and Gemmatimonadota in phylum level (P &amp;lt; 0.05). At genus level, lincomycin significantly increased the abundance of Citrobacter, Corynebacterium, Delftia, Agathobacter, Trichococcus, Tissierellales, Enterococcus, Paracoccus, Shewanella, unclassified_c__Clostridia, Alistipes, Sphingomonas, and Massilia, etc (P &amp;lt; 0.05). However, these results were not observed in the LC + BL group, and baicalin treatment increased the abundance of beneficial bacteria such as norank_f__Muribaculaceae (P &amp;lt; 0.05). Fecal microbiota transplantation was employed to further investigate whether these results were relevant to gut microbes. The body weight, villus height, and crypt depth of mice receiving LC group microbiota were significantly lessthan that of the control group, while the body weight of mice receiving LC + BL group microbiota was not significantly different from that of the control group. Overall, these results suggest that baicalin may alleviate lincomycin-induced intestinal damage and inflammation by inhibiting the growth of harmful bacteria. Our findings expand our knowledge of antibiotic-induced injury and provide new avenues for identifying potential therapeutic targets for the treatment of antibiotic-induced injury and its complications.

The effects of preosteoblast-derived exosomes on macrophages and bone in mice.

The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated invitro, and bone formation studies were performed invivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1β, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For invivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.

JAK2-V617F Promotes up-Regulation of Pro-Inflammatory Cytokines Including IL-1 upon Adhesion-Induced Activation of β1/β2 Integrins

Introduction In JAK2-V617F (JAK2-VF) mutated cells, over-activation of β1/β2 integrin inside-out signaling has been described (Edelmann-Stephan et al., 2017, JCI). Integrins play a fundamental role in cell interaction and cell invasion during inflammatory processes. In patients suffering from Myeloproliferative Neoplasms (MPNs), highly elevated pro-inflammatory cytokine levels including IL-1β play an important role in pathophysiology. Importantly, Rai et al. (2023, BioRχ) have shown that IL-1β promotes initiation of MPN. In this study, we investigated a potential link between integrin over-activation and elevated pro-inflammatory cytokines including IL-1α and IL-1β in JAK2-VF induced disease. Methods We employed (1) murine myeloid progenitor 32D cells co-transfected with EPO-R and JAK2-WT or JAK2-VF, (2) granulocytes and hematopoietic progenitor cell populations from total bone marrow of Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice (described by Mullally et al. 2010), and (3) human granulocytes isolated from PB of JAK2-VF positive patients and healthy volunteers. Cells were allowed to adhere on immobilized VCAM-1/ICAM-1 or on BSA (control) and subsequently subjected to further analysis including (1) qPCR analysis and ELISA of a panel of inflammatory cytokines (IL-1β, IL-1α, TNF-α, IL-6, CXCL10), (2) Western-blotting monitoring integrin outside-in signaling pathways, (3) FLICA assays assessing adhesion-induced active caspase-1, (4) RNAseq of mouse and human granulocytes (3 JAK2-VF patients, 2 controls). Results In the 32D cell model, adhesion on mVCAM-1/mICAM-1 strongly induced mRNA expression of IL-1α, IL1-β, IL-6, TNF-α and CXCL10. Interestingly, cytokine RNA induction was significantly higher in 32D JAK2-VF cells as compared to the 32D JAK2-WT cells (Figure 1). By applying a panel of integrin signaling inhibitors we could show an essential involvement of pivotal signaling nodes such as PI3K, Syk, FAK and NFκB, respectively. Next, cytokine proteins were investigated by ELISA. Adhesion on mVCAM-1/mICAM-1 increased intracellular IL-1α and IL-1β cytokine levels. Again, this effect was strongly amplified by the JAK2-VF mutation (Figure 2). Importantly, both cytokines were found in their mature form, indicating intact protein processing. RNAseq of bone marrow (BM) derived mouse granulocytes (including immature pro-and pre-neutrophils) also showed significant upregulation of a number of pro-inflammatory genes including Nlrp3, Il1a, Il1b, Tnf, Cxcl2 and Cxcl10 upon adhesion on mVCAM-1/mICAM-1. However, no significant differences between WT and VF cells were observed. In contrast, RNAseq of VCAM-1/ICAM-1 stimulated human JAK2-VF positive PB granulocytes (which represent terminally differentiated cells) revealed no cytokine up-regulation. Based on this finding, we hypothesize that the adhesion-induced cytokine phenotype is restricted to more immature cell populations as observed in the 32D cell model. Therefore, we are currently investigating (by qPCR and intracellular IL-1 immunophenotyping) hematopoietic progenitor cell populations from Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice. Since active IL-1β requires processing by the inflammasome, FLICA assays for active caspase-1 were performed using mouse hematopoietic progenitor cells. Importantly, CD41+ cells and megakaryocyte progenitors (MKP) showed an increased FLICA signal upon adhesion on mVCAM-1/mICAM-1. This indicates that caspase 1 is activated upon adhesion in distinct progenitor cell populations. Finally, to support our hypothesis that JAK2-VF augments induction of pro-inflammatory cytokines upon adhesion to VCAM-1/ICAM-1, Vav1-Cre x JAK2 VF/+ mice (n=8) were treated in vivo with anti-β1/β2-integrin antibodies for 1 week. As expected we found a strong reduction of IL-1α concentration in serum of antibody treated mice (528.7 ± 82.2 pg/ml) as compared to PBS-treated control mice (1302.3 ± 370.4 pg/ml, p = 0.0541) Conclusion Here, we show a functional link between elevated cytokine levels and integrin activation in JAK2-VF induced disease. Adhesion to VCAM-1/ICAM-1 promoted the expression of a number of pro-inflammatory cytokines which are known to play important roles in initiation, clinical symptoms and prognosis of MPNs. Further studies aimed at precisely defining the primary cell populations involved in the mouse MPN disease models as well as in humans are currently ongoing.

Adipose tissue-derived mesenchymal stromal cells attenuate acute lung injury induced by trauma and haemorrhagic shock

BackgroundMesenchymal stromal cells (MSCs) have shown promising therapeutic options for acute lung injury (ALI) caused by multiple factors. Here, we evaluated the therapeutic potential of adipose tissue-derived mesenchymal stromal cells (ADSCs) in trauma and hemorrhagic shock (THS)-induced ALI. MethodsALI model induced by THS was constructed by fractures plus abdominal trauma plus acute hemorrhage plus fluid resuscitation. The ADSCs group rats were generated by injecting 2 × 106 ADSCs at 0 and 1 h after THS. The sham, ALI, and ADSCs group rats were sacrificed at 24 h after resuscitation. The changes in lung histopathology, total protein in bronchoalveolar lavage fluid (BALF), mRNA expression of pro-inflammatory/anti-inflammatory cytokines, antioxidant, and anti-apoptotic indicator, and the activity of Toll-like receptor 4 (TLR4) signaling in lung tissues were evaluated. ResultsAdministration of the ADSCs reversed ALI induced by THS, including lung histopathological changes/scores, and BALF total protein concentration. Additionally, ADSCs therapy also significantly down-regulated mRNA expression of pro-inflammatory TNF-α, IL-1β, and IL-6, up-regulated mRNA expression of anti-inflammatory IL-10, anti-apoptotic molecule Bcl-2, and anti-oxidative molecule HO-1 in THS rats. Furthermore, ADSCs suppressed the expression of TLR4 in lung tissue. ConclusionOur data show that ADSCs administration can exert therapeutic effects on THS-induced ALI in rats and may provide beneficial in preventative strategies for ALI.

Yinhuapinggan granule ameliorates lung injury caused by multidrug-resistant Acinetobacter baumannii via inhibiting NF-κB/NLRP3 pathway

Yinhuapinggan granule (YHPG) is a traditional Chinese medicine prescription with rich clinical experience for the treatment of colds and coughs. The aim of this study is to investigate the protective effect of YHPG on multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infection in vivo and its potential anti-inflammatory mechanism. BALB/c mice were intranasally inoculated with MDR A. baumannii strain to establish the pneumonia infection model, and received intraperitoneally cyclophosphamide to form immunosuppression before attack. YHPG (6, 12 and 18 g/kg) was administered by gavage once a day for 3 consecutive days after infection. The protective effect of YHPG was evaluated by lung index, spleen index, thymus index, pathological changes of lung tissue and inflammatory factors (IL-1β, IL-6 and TNF-α) in serum. The expression of key targets of NF-κB/NLRP3 signaling pathway in vivo was analyzed by immunohistochemistry, immunofluorescence, reverse transcription quantitative PCR (RT-qPCR) and Western blot. The results showed that YHPG improved the lung index and its inhibition rate, immune organ indexes and lung pathological changes in infected mice, and significantly reduced IL-1β, IL-6 and TNF-α levels in serum. In addition, YHPG significantly down-regulated the mRNA and protein expression of NF-κB p65, NLRP3, ASC, Caspase-1, TNF-α, IL-6 and IL-1β in mice lung tissue. The results of the current study demonstrated that YHPG has significant protective effects on mice infected with MDR A.baumannii, which may be related to the regulation of inflammatory factors and NF-κB/NLRP3 signaling pathway, indicating that YHPG has a wide range of clinical application value and provides a theoretical basis for its treatment of MDR A.baumannii infection.

Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1β mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

Hydralazine alleviates noise-induced hearing loss by scavenging acrolein

Noise-induced hearing loss (NIHL) is the most common cause of hearing loss. This study investigates the therapeutic efficacy of the acrolein scavenger hydralazine for NIHL in rats. NIHL was induced by exposure to a continuous pure tone of 10 kHz. Auditory function was evaluated by auditory brainstem response (ABR) testing and scanning electron microscopy. The expression of acrolein and glial cell markers GFAP and OX42 was assessed by immunofluorescence staining. The protein and mRNA expression of GFAP, OX42, interleukin-18 (IL-18), IL-1β, and fractalkine (FTK) was measured by qRT-PCR and western blotting. A rat model of NIHL was successfully developed, as evidenced by increased ABR thresholds. The results showed that noise exposure increased the expression of acrolein, GFAP, OX42, FTK, IL-1β, and IL-18 in the rat cochlear nucleus. Furthermore, hydralazine alleviates NIHL by reversing the effects of acrolein. These results demonstrate that acrolein is involved in glial cell activation and NIHL, it’s also a therapeutic potential target for NIHL.

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Cordyceps militaris Solid Medium Extract Alleviates Lipoteichoic Acid-Induced MH-S Inflammation by Inhibiting TLR2/NF-κB/NLRP3 Pathways.

The aim of this study was to investigate the inhibitory effects of Cordyceps militaris solid medium extract (CME) and cordycepin (COR) on LTA-induced inflammation in MH-S cells and their mechanisms of action. In this study, the establishment of an LTA-induced MH-S inflammation model was determined, the CCK-8 method was used to determine the safe concentration range for a drug for COR and CME, the optimal concentration of COR and CME to exert anti-inflammatory effects was further selected, and the expression of inflammatory factors of TNF-α, IL-1β, IL-18, and IL-6 was detected using ELISA. The relative expression of TNF-α, IL-1β, IL-18, IL-6, IL-10, TLR2 and MyD88 mRNA was detected using RT-PCR, and the IL-1β, IL-18, TLR2, MyD88, NF-κB p-p65, NLRP3, pro-caspase-1, Caspase-1 and ASC protein expression in the cells were detected using Western blot; immunofluorescence assay detected the expression of Caspase-1 in MH-S cells. The results revealed that both CME and COR inhibited the levels of IL-1β, IL-18, IL-6, and TNF-α in the supernatants of LTA-induced MH-S cells and the mRNA expression levels of IL-1β, IL-18, IL-6, TNF-α, TLR2 and MyD88, down-regulated the LTA-induced IL-1β, IL-18, TLR2 in MH-S cells, MyD88, NF-κB p-p65/p65, NLRP3, ASC, pro-caspase-1, and caspase-1 protein expression levels, and inhibited LTA-induced caspase-1 activation in MH-S cells. In conclusion, CME can play a therapeutic role in LTA-induced inflammation in MH-S cells via TLR2/NF-κB/NLRP3, and may serve as a potential drug for bacterial pneumonia caused by Gram-positive bacteria.

Sodium butyrate (SB) ameliorated inflammation of COPD induced by cigarette smoke through activating the GPR43 to inhibit NF-κB/MAPKs signaling pathways

Cigarette smoke is recognized as a major trigger for individuals with chronic obstructive pulmonary disease (COPD), leading to an amplified inflammatory response. The onset and progression of COPD are affected by multiple environmental and genetic risk factors, such as inflammatory mechanisms, oxidative stress, and an imbalance between proteinase and antiprotease. As a result, conventional drug therapies often have limited effectiveness. This study aimed to investigate the anti-inflammatory effect of sodium butyrate (SB) in COPD and explore its molecular mechanism, thereby deepening our understanding of the potential application of SB in the treatment of COPD. In our study, we observed an increase in the mRNA and protein expressions of inflammatory factors interleukin-1beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), Matrix metallopeptidase 9 (MMP9) and MMP12 in both NR8383 cell and rat models of COPD. However, these expressions were significantly reduced after SB treatment. Meanwhile, SB treatment effectively decreased the phosphorylation levels of nuclear transcription factor-kappa B (NF-κB) p65, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) and inhibited the nuclear translocation of these proteins in the COPD cells, leading to a reduction in the expression of various inflammatory cytokines. Additionally, SB also inhibited the expression level of the Nod-like receptor pyrin domain 3 (NLRP3) inflammasome, which consists of NLRP3, apoptosis-associated speck-like protein (ASC), and Caspase-1 in the cigeratte smoke extract (CSE)-stimulated cells. Our results showed that CSE down-regulated the mRNA levels of G-protein-coupled receptor 43 (GPR43) and GPR109A, while SB only up-regulated the expression of GPR43 and had no effect on GPR109A. Moreover, additional analysis demonstrated that the knockdown of GPR43 diminishes the anti-inflammatory effects of SB. It is evident that siRNA-mediated knockdown of GPR43 prevented the reduction in mRNA expression of IL-1β, IL-6, TNF-α, MMP9, and MMP12, as well as the expression of phosphorylated proteins NF-κB p65, JNK, and p38 MAPKs with SB treatment. These findings revealed a SB/GPR43 mediated pathway essential for attenuating pulmonary inflammatory responses in COPD, which may offer potential new treatments for COPD.

Macroporous resin purification of phenolics from Irish apple pomace: Chemical characterization, and cellular antioxidant and anti-inflammatory activities

Apple pomace (AP) is a highly prevalent waste product worldwide in the fruit processing sector. This study compared the chemical profile, antioxidant, and anti-inflammatory activities of crude (CE) and an extract purified using XAD-7 resin (PE). The purification process increased the total phenolic content, flavonoids, and tannins by 3.35, 40.31, and 8.87-fold, respectively. The main phenolic compounds identified in PE were phlorizin (20.54 mg/g), chlorogenic acid (10.01 mg/g), and hyperoside (2.77 mg/g). No difference was found between CE and PE in protecting human plasma against oxidation. In human erythrocytes, both CE and PE decreased the reactive oxygen species (ROS) generation and decreased lipoperoxidation. However, PE had stronger anti-inflammatory effects than CE by promoting HO-1 gene expression, suppressing NO production, and inhibiting IL-1β, IL-6, and IL-10 mRNA expression in lipopolysaccharide-challenged RAW.264.7 macrophages. Therefore, purifying apple pomace crude extract is a promising approach to boosting valuable antioxidants and anti-inflammatory phenolics.

Stimulatory effects of seaweed Laminaria digitata polysaccharides additives on growth, immune-antioxidant potency and related genes induction in Rohu carp (Labeo rohita) during Flavobacterium columnare infection

Stimulatory effects of dietary supplementation of large brown alga, Laminaria digitata polysaccharides (LDPs) at doses of 0, 100, 200, and 300 mg/kg diets were assessed on growth, hemato-biochemical, and antioxidant-immune variables as well as related genes expression in Indian rohu carp (Labeo rohita) during Flavobacterium columnare infection. Fish growth indices were significantly (P < 0.05) influenced by dietary LDPs and bacterial infection but not their interaction. However, growth indices of rohu carp in normal unchallenged (NuC) increased pointedly at treatments of 200 or 300 mg/kg diets with no significant difference between them. In normal challenged bacterial (NbC) groups, highest fish growth was observed at the treatment of 300 mg LDPs/kg diets. Feed intake and FCR values were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The highest feed intake accompanied with lowest FCR value was observed in the treatment of 200 mg LDPs/kg diets. No mortality was seen in NuC groups but lowest fish survival was observed in NbC groups fed with 0.0 and 100 mg LDPs/kg diets. Highest values of white blood cell, total protein, albumin, and globulin found in NuC fish groups administrated with 200 or 300 mg/kg diets with no differences between them. Feeding NbC fish groups on 300 mg LDPs/kg diet recovered the adverse effects of bacterial infection. It is noted that levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) were significantly (P < 0.05) affected by dietary LDPs, bacterial infection, and their interaction. The values of antioxidant indices were significantly (P < 0.05) higher in NuC fish groups received 200 or 300 mg LDPs/kg diets with no significant (P > 0.05) differences between them. In NbC fish groups, values of antioxidant indices were significantly (P < 0.05) lower that those in NuC fish groups. Malondialdehyde (MDA) was significantly influenced by dietary LDPs alone and its lowest values were observed in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. Phagocytic activity, alternate complement pathway, lysozyme, respiratory burst, peroxidase, complement C3, and immunoglobulin M values improved significantly (P < 0.05) in both NuC and NbC fish groups fed with 200 or 300 mg/kg diets with no differences between them. The mRNA manifestation of hepatic SOD, CAT, and GPx genes were significantly (P < 0.05) upregulated in both NuC fish groups fed on 200 and 300 mg LDPs/kg diets but feeding NbC fish on 300 mg LDPs/kg diets enhanced their mRNA expression. The HSP70, HSP90, TNF-α, IL-1β, and IL-8 mRNA expression in the NuC fish groups showed no significant (P > 0.05) differences due to dietary supplementation of LDPs to ruho carp, but their expressions were significantly (P < 0.05) higher in NbC fish groups; particularly at the treatment of 100 mg LDPs/kg diets. The mRNA expression of IL-10, IgM, hepcidin, and Mx1 genes were significantly (P < 0.05) upregulated in NuC fish group fed with 200 or 300 mg LDPs/kg diets. The bacterial infection downregulated their expressions but the dietary LDPs recovered the adverse effects of bacterial infection especially at 200 or 300 mg LDPs/kg diets. These results shown that the use of LDPs (200 or 300 mg/kg diets) has a valuable role in enhancing the growth, antioxidant-immune status, and inflammatory responses in rohu carp during F. columnare infection.

Efficacy of Poria cocos and Alismatis rhizoma against diet-induced hyperlipidemia in rats based on transcriptome sequencing analysis

Hyperlipidemia, a common metabolic disease, is a risk factor for cardiovascular diseases, Poria cocos (PC) and Alismatis rhizoma (AR) serve as a potential treatment. A systematic approach based on transcriptome sequencing analysis and bioinformatics methods was developed to explore the synergistic effects of PC–AR and identify major compounds and potential targets. The phenotypic characteristics results indicated that the high dose (4.54 g/kg) of PC–AR reduced total cholesterol (TC), elevated high-density lipoprotein cholesterol (HDL-C) levels, and improved hepatocyte morphology, as assessed via hematoxylin and eosin (H&E) staining. Transcriptomic profiling processing results combined with GO enrichment analysis to identify the overlapping genes were associated with inflammatory responses. The cytokine-cytokine receptor interaction pathway was found as a potential key pathway using geneset enrichment analysis. Core enrichment targets were selected according to the PC–AR's fold change versus the model. Real-time quantitative PCR analysis validated that PC–AR significantly downregulated the expression of Cxcl10, Ccl2, Ccl4, Cd40 and Il-1β mRNA (P < 0.05). Molecular docking analysis revealed the significant compounds of PC–AR and the potential binding patterns of the critical compounds and targets. This study provides further evidence that the therapeutic effects of PC–AR on hyperlipidemia in rats through the regulation of inflammation-related targets.

Effect of electroacupuncture on myocardial fibrosis in spontaneously hypertensive rats based on cholinergic anti-inflammatory pathway

To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on myocardial fibrosis in spontaneously hypertensive rats (SHR), and explore preliminarily the mediating role of cholinergic anti-inflammatory pathway (CAP) and its downstream nuclear factor κB (NF-κB) signaling pathway. Six 12-week-old WKY male rats were employed as the normal group. Eighteen 12-week-old SHR were randomly divided into 3 groups, i.e. a model group, an EA group and a blocking group (EA after blocking α7 nicotinic acetylcholine receptor [α7nAchR]), with 6 rats in each one. In the EA group, EA was delivered at "Neiguan"(PC 6) and the site 0.5 cm from its left side, with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity. One intervention took 30 min and was given once every 2 days, lasting 8 weeks. In the blocking group, prior to each EA, the α7nAchR specific blocker, α-bungartoxin was injected intravenously in the tails of the rats. After EA intervention, the systolic blood pressure (SBP), the diastolic blood pressure (DBP) and the mean arterial pressure (MAP) were measured with non-invasive blood pressure monitor. Using echocardiogram, the left ventricular (LV) anterior wall end-diastolic thickness (LVAWd) , LV posterior wall end-diastolic thickness (LVPWd) and the LV end-diastolic internal diameter (LVIDd) were measured. The level of hydroxyproline (Hyp) in the myocardial tissue was determined by using alkaline hydrolysis, and that of acetylcholine (Ach) was detected by ELISA. With the real-time PCR adopted, the mRNA expression of NF-κB p65, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were determined. Compared with the normal group, SBP, DBP, MAP, LVAWd and LVPWd were increased (P<0.01), and LVIDd was decreased (P<0.01) in the rats of the model group. SBP, DBP, MAP and LVAWd were dropped (P<0.01, P<0.05), and LVIDd rose (P<0.01) in the EA group when compared with those in the model group. The differences in the above indexes were not statistically significant between the blocking group and the model group (P>0.05). Compared with the normal group, Hyp level and the mRNA expression of NF-κB p65, TNF-α, IL-1β and IL-6 in the myocardial tissue increased (P<0.01, P<0.05) and Ach level decreased (P<0.01) in the model group. Hyp level, the mRNA expression of NF-κB p65, TNF-α, IL-1β and IL-6 in the myocardial tissue were reduced (P<0.05, P<0.01) and Ach level rose (P<0.01) in the EA group when compared with those in the model group. These indexes were not different statistically between the blocking group and the model group (P>0.05). CAP may be involved in ameliorating the pathological damage of myocardial fibrosis during EA at "Neiguan"(PC 6). The underlying effect mechanism is associated with up-regulating the neurotransmitter, Ach and down-regulating mRNA expression of NF-κB p65 and pro-inflammatory factors such as TNF-α, IL-1β and IL-6 in myocardial tissue.

Cyclohexylethanoid derivative and rearranged abietane diterpenoids with anti-inflammatory activities from Clerodendrum bungei and C. inerme

One new dimeric cyclohexylethanoid derivative (1) and one new rearranged abietane diterpenoid (2) were isolated from Clerodendrum bungei and C. inerme, respectively, together with 11 known compounds. The structures of the isolated compounds were elucidatedby detailed spectroscopic and ECD analyses. The absolute configuration of 2 was unequivocally determined using single-crystal X-ray diffraction analysis. Compound 1 showed the greatest inhibitory effect on NO production in RAW 264.7 murine macrophages induced by lipopolysaccharide (LPS) with the IC50 value of 6.328 µM. Compound 1 also suppressed the proinflammatory cytokines IFN-γ, TNF-α, IL-1β, iNOS and IL-6 mRNA expression, while increased the mRNA expression of the anti-inflammatory cytokines IL-4, IL-10 and Arg-1 through NF-κB signaling pathway. The further experiments confirmed that compound 1 demonstrated the strong in vivo anti-inflammatory effect by reducing mortality, the serum TNF-α, IL-6 and IFN-γ levels, and tissue toxicity in CLP-induced septic mice. Collectively, these findings suggested that compound 1might serve as a potential anti-inflammatory agent candidate for the sepsis treatment.

Effects of Enteromorpha prolifera polysaccharides on growth performance, intestinal barrier function and cecal microbiota in yellow-feathered broilers under heat stress

BackgroundGlobal warming leading to heat stress (HS) is becoming a major challenge for broiler production. This study aimed to explore the protective effects of seaweed (Enteromorpha prolifera) polysaccharides (EPS) on the intestinal barrier function, microbial ecology, and performance of broilers under HS. A total of 144 yellow-feathered broilers (male, 56 days old) with 682.59 ± 7.38 g were randomly assigned to 3 groups: 1) TN (thermal neutral zone, 23.6 ± 1.8 °C), 2) HS (heat stress, 33.2 ± 1.5 °C for 10 h/d), and 3) HSE (HS + 0.1% EPS). Each group contained 6 replicates with 8 broilers per replicate. The study was conducted for 4 weeks; feed intake and body weights were measured at the end of weeks 2 and 4. At the end of the feeding trial, small intestine samples were collected for histomorphology, antioxidant, secretory immunoglobulin A (sIgA) content, apoptosis, gene and protein expression analysis; cecal contents were also collected for microbiota analysis based on 16S rDNA sequencing.ResultsDietary EPS promoted the average daily gain (ADG) of broilers during 3–4 weeks of HS (P < 0.05). At the end of HS on broilers, the activity of total superoxide dismutase (T-SOD), glutathione S-transferase (GST), and the content of sIgA in jejunum were improved by EPS supplementation (P < 0.05). Besides, dietary EPS reduced the epithelial cell apoptosis of jejunum and ileum in heat-stressed broilers (P < 0.05). Addition of EPS in HS group broilers' diet upregulated the relative mRNA expression of Occludin, ZO-1, γ-GCLc and IL-10 of the jejunum (P < 0.05), whereas downregulated the relative mRNA expression of NF-κB p65, TNF-α and IL-1β of the jejunum (P < 0.05). Dietary EPS increased the protein expression of Occludin and ZO-1, whereas it reduced the protein expression of NF-κB p65 and MLCK (P < 0.01) and tended to decrease the protein expression of TNF-α (P = 0.094) in heat-stressed broilers. Furthermore, the proportions of Bacteroides and Oscillospira among the three groups were positively associated with jejunal apoptosis and pro-inflammatory cytokine expression (P < 0.05) and negatively correlated with jejunal Occludin level (P < 0.05). However, the proportions of Lactobacillus, Barnesiella, Subdoligranulum, Megasphaera, Collinsella, and Blautia among the three groups were positively related to ADG (P < 0.05).ConclusionsEPS can be used as a feed additive in yellow-feathered broilers. It effectively improves growth performance and alleviates HS-induced intestinal injury by relieving inflammatory damage and improving the tight junction proteins expression. These beneficial effects may be related to inhibiting NF-κB/MLCK signaling pathway activation and regulation of cecal microbiota.

Interleukin-1β triggers muscle-derived extracellular superoxide dismutase expression and protects muscles from doxorubicin-induced atrophy.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1β (IL-1β) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1β treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1β (IL-1β) expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

Region-specific remodeling of the enteric nervous system and enteroendocrine cells in the colon of spinal cord injury patients

Patients with spinal cord injury (SCI) suffer from major bowel dysfunction, whose exact pathophysiology, particularly the involvement of the enteric nervous system or epithelial dysfunction is poorly understood. Herein, we aimed to characterize the mucosal biopsies of the right and left colon in SCI patients vs controls (CT): (1) remodeling of key enteric neurotransmitters, (2) remodeling of enteroendocrine cells, and (3) mucosal inflammation compared to those in controls. In SCI, mucosal ACh concentration was lower in the right colon as compared to CT, but no change was observed in the left colon, and AChE expression was lower in both the right and left colons than in CT. While the VIP concentration was similar in the right and left colons, VIP mRNA expression was increased in the right colon and decreased in the left colon, in SCI patients as compared to CT. Interestingly, 5-HT concentration was reduced in the left colon but not in the right colon in SCI patients. Moreover, in SCI patients, as compared to CT, SERT mRNA expression was selectively increased in the left colon while TPH1 mRNA expression was increased in the right and left colons. Although mucosal TNFα and IL-1β mRNA expression did not significantly differ between SCI and CT groups, we identified a significant positive correlation between TNFα and IL-1β mRNA expression and left colon transit time in the SCI group. In conclusion, region-specific changes occur in the enteric neurotransmitter, serotonergic, and inflammatory pathways in the colon of SCI patients. The significant correlations between these pathways and clinical parameters in the left colon further set a scientific basis for designing therapeutic targets to improve colonic motor dysfunction in patients.Biobank information: Spinal cord injury patients: PHRC ConstiCAPE—clinical trial NCT02566746. Controls: Anosain—clinical trial NCT03054415 and biobank of the “Institut des Maladies de l’Appareil Digestif (IMAD)” registered under number DC-2008-402.