mRNA Expression Levels Of TNF-α Research Articles

Role of glucagon-like peptide 1 (GLP-1) and its association with inflammatory markers in the pathogenesis of type 2 diabetes mellitus

Background: Diabetes mellitus is a multifactorial disease associated with hyperglycemia and increased risk of progression of vascular complications. Stimulation of insulin secretion by the incretin hormone glucagon-like peptide 1 (GLP-1) has been found to be diminished in hyperglycemia. We hypothesized that this impairment is due to defect at the receptor level induced by the diabetic state. Inflammatory markers like TNF-α and IL-6 plays a potential role in the pathogenesis of T2DM. Therefore, the present study aims to evaluate whether GLP-1 plays a role in the development of T2DM by modulating the balance between pro and anti-inflammatory markers. Material and methods: A total of 60 subjects were recruited in this study among them 30 were T2DM cases and 30 were healthy controls. m-RNA expression and protein level of GLP-1 receptor, TNF-α and IL-6 in peripheral blood lymphocytes were determined by real time PCR and ELISA respectively. Results: We observed plasma level of GLP-1 was significantly lower in diabetic subjects while serum level of IL-6 and TNF-α were significantly higher level in diabetic subjects (p < 0.05). We found significant down regulation of GLP-1 receptor m-RNA expression in diabetic subjects while expression level of IL-6 and TNF-α were 5.8 and 4 folds respectively higher in diabetic subjects. We found significant negative correlation of m-RNA expression of GLP-1 with protein level while IL-6 and TNF-α showed significant positive correlation. Conclusion: Inflammation plays an important role in the pathogenesis of diabetes mellitus and low GLP-1 levels may promote expression of inflammatory markers due to lack of anti-inflammatory effects of GLP-1

POLYDATIN, A HERBAL BIOFLAVONOID, IS PROTECTIVE AGAINST CEREBRAL ISCHEMIA-REPERFUSION INJURY: MOLECULAR, BIOCHEMICAL AND HISTOLOGICAL DATA

This study aims to research the protective effects Polydatin have against cerebral ischemia/reperfusion damage. Polydatin is a natural polyphenic phytoalexin and which has strong antioxidant properties. In the present study, 5 groups were prepared as control, sham, ischemia/reperfusion (IR), Polydatin 30 (Pol 30), and Polydatin 60 (Pol 60). The four-vessel occlusion model was used to induce ischemia. Polydatin was injected intraperitoneally 30 minutes before ischemia. Hematoxylin Eosin staining were applied for histopathological study, SOD, CAT, and MDA levels determined, and TNF-α mRNA expression levels were measured by the RT-qPCR technique in brain tissue. According to the results, a serious loss of neurons in the CA 1 region of the hippocampus was observed in the IR group. Neuronal damage in the hippocampus decreased and the number of neurons increased significantly in the Pol 60 group compared to the IR group. CAT and SOD levels were reduced, and the MDA level rose in the IR group. In Pol 60 and Pol 30 groups, an increase was observed in the CAT and SOD levels, a decrease was observed in the MDA and total protein levels compared to the IR group. The amount of TNF-α mRNA expression in the brain tissues of the IR group was significantly higher compared to the control group. In the Pol 60 group, mRNA expression level decreased significantly compared to the IR group. In conclusion, the increase in MDA, decrease in SOD and CAT values, increase in TNF-α gene mRNA expression, and histological damage in the brain because of cerebral ischemia/reperfusion in rats were restored to normal levels with 30 and 60 mg/kg polydatin administration as protective before ischemia. Especially at 60 mg/kg polydatin supplementation with antioxidant properties has a neuroprotective effect against oxidative stress damage caused by cerebral ischemia/reperfusion.

M2 macrophage-derived exosomal lncRNA NR_028113.1 promotes macrophage polarization possibly by activating the JAK2/STAT3 signaling pathway

To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism. Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured in vitro. After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 μg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α were detected using qRT-PCR and Western blotting. The percentage of CD206+ cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α, CD206+ cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting. LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages (P < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 (P < 0.05), lowered the expressions of iNOS and TNF-α (P < 0.05), and increased CD206+ cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs (P < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF-α mRNA (P < 0.05), resulting also in signi-ficantly reduced CD206+ cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM (P < 0.05). M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.

Potential Anti-Inflammatory Components of Rhododendron molle G. Don Leaf Extracts in LPS-Induced RAW 264.7.

As a traditional Chinese medicine, Rhododendron molle G. Don has a long history of treating rheumatoid arthritis. In this study, RAW 264.7 cells induced by lipopolysaccharide (LPS) were established as cell inflammatory model to evaluate the anti-inflammatory activity of chloroform extract from R. molle leaves (CERL), ethyl acetate extract from R. molle leaves (EERL) and butanol extract from R. molle leaves (BERL) and analyze the potential anti-inflammatory components of R. molle. Potential anti-inflammatory components analysis of CERL were performed by HPLC and UHPLC-Q-TOF-MS. Prediction of potential anti-inflammatory components by molecular docking experiments. Compared with negative control group, 25 μg/mL CERL could reduce the release level of NO by 62 %, and the mRNA expression levels of COX-2, IL-6, IL-1β and TNF-α were reduced by 69.74 %, 86.25 %, 77.94 % and 56.80 %, respectively. Western-Blot showed similar results. CERL, EERL and BERL exerted their inhibitory activity in dose-dependent manner. All results showed that the higher the concentration, the better the anti-inflammatory activity. CERL showed the best inhibitory activity, the second was EERL, and then was BERL. 21 terpenoids and 4 flavonoids were identified in CERL by UHPLC-Q-TOF-MS. Molecular docking results showed that triterpenoids in CERL had better interaction with target proteins (TNF-α, IL-1β). It indicated that triterpenoids may be potential anti-inflammatory components of R. molle leaves. This study explored the anti-inflammatory activities of CERL, EERL, BERL, which laid a foundation for further promoting the clinical application of R. molle.

A54 THE IMPACT OF NON-INVASIVE TRANSCUTANEOUS AURICULAR VAGUS NERVE STIMULATION (NITAVNS) ON THE DEVELOPMENT OF EXPERIMENTAL COLITIS IN MICE

Abstract Background The anti-inflammatory impacts of stimulating the vagus nerve as a part of the gut-brain axis on intestinal macrophage activity and inflammatory cytokine production have been documented. While vagus nerve stimulation until now has been chiefly invasive, non-invasive auricular vagus nerve stimulation (NitaVNS) has no adverse effects associated with invasive stimulation. The NitaVNS-mediated activation of brainstem nuclei can exert anti-inflammatory effects directly on the colon or indirectly through the spleen via a macrophage-acetylcholine-dependent pathway. Purpose To examine the effects of NitaVNS on the development of acute colitis in mice using a preclinical model. Method Thirty C57BL/6 mice aged 11 to 12 weeks were divided into four groups: control (CTRL)-NitaVNS, CTRL-Sham, dextran sulphate sodium (DSS)-NitaVNS and DSS-Sham. Pre-set stimulation parameters (10 minutes duration, 10 V voltage, 500 s pulse width, and 20 Hz frequency) were applied for NitaVNS or sham-paw stimulation. 5% DSS was administered to induce colitis for five consecutive days, whereas CTRL mice received regular water. Weight loss, stool consistency, blood in the feces, and disease activity index were evaluated daily. Rectal bleeding, colon bleeding, and fecal consistency were determined as a composite macroscopic score upon sacrifice. Collected colon and blood samples were assessed for pro-inflammatory parameters such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, C-reactive protein (CRP), matrix metalloproteinase (MMP)-2, macrophage inflammatory protein (Mip)-1α and β, and anti-inflammatory cytokine tumor growth factor (TGF)-β using qRT-PCR and ELISA. Result(s) Compared to colitic-sham mice, colitic NitaVNS mice demonstrated a delayed onset and a significantly lower clinical disease severity. Colitis induced significant weight loss, rectal bleeding, and stool consistency loss, and NitaVNS significantly abolished these effects. In colitic conditions, NitaVNS decreased the disease activity index significantly compared to the colitic sham group. Colitis increased the macroscopic scores, and NitaVNS decreased them significantly. Conversely, colon length was significantly reduced in colitic conditions, but NitaVNS abrogated that deleterious effect. mRNA expression levels of IL-1β and TNF-α were significantly increased in colitic mice and NitaVNS decreased them significantly. Whereas Mip-1α and 1β were not modified in colitic conditions, NitaVNS significantly decreased these markers. MMP2 mRNA expression and colonic IL-6 and IL-1β protein levels were not altered in all conditions. However, colitis induced a significant decrease in colonic TGF-β levels, but NitaVNS did not amplify that effect. Interestingly, in the serum of colitic mice, NitaVNS significantly increased and decreased TGF-β and CRP levels, respectively. Conclusion(s) These findings suggest that NitaVNS possess some spatial anti-inflammatory effects in acute colitis through the up or downregulation of anti- or pro-inflammatory markers. Disclosure of Interest F. Hesampour: None Declared, D. Tshikudi: None Declared, A. Veysel Özden Employee of: Company providing the stimulator, C. N. Bernstein: None Declared, J.-E. Ghia: None Declared

Mechanism of Yishen Tonglong Decoction inhibiting TLR4/p38 MAPK/NF-κB signaling pathway against prostate cancer via upregulating miR-145-5p

ObjectiveTo investigate the mechanism of Yishen Tonglong Decoction (益肾通癃汤, YSTLD) inhibiting the toll-like receptor 4/p38 mitogen activated protein kinases/nuclear factor kappa-B (TLR4/p38 MAPK/NF-κB) signaling pathway against prostate cancer by up-regulating miR-145-5p.Methods miRNA microarray technology was used to detect the changes of miRNA expression profile in prostate cancer PC-3 cells treated with YSTLD, and miRNAs with marked differences in miRNA microarray results were screened and validated by real-time polymerase chain reaction (qRT-PCR). Lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, Cell Counting Kit-8 (CCK8) assay, and scratch assay were adopted to detect the effects of miR-145-5p on prostate cancer PC-3 cell proliferation and migration. qRT-PCR and Western blot were employed to detect the effects of miR-145-5p on TLR4/p38 MAPK/NF-κB signaling pathway and the expression levels of apoptosis-related genes caspase3, tumor necrosis factor-α (TNF-α), Bax, and Bcl-2. qRT-PCR and Western blot were used to detect the effects of serum containing YSTLD on miR-145-5p, TLR4/p38 MAPK/NF-κB signaling pathway, and the expression levels of apoptosis-related genes caspase3, TNF-α, Bax, and Bcl-2. ResultsThe expression levels of 35 miRNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group, with miR-145-5p being the most significantly different; qRT-PCR validation revealed that the miR-145-5p levels in prostate cancer PC-3 cells treated with YSTLD were significantly higher than those in the DMSO control group (P < 0.05). After lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, miR-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3 cells. Overexpression of miR-145-5p up-regulated expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulated expression levels of p38 MAPK, p65 NF-κB, and Bcl-2 mRNA in prostate cancer PC-3 cells (P < 0.05), while up-regulated caspase3 protein expression levels in prostate cancer PC-3 cells and down-regulated expression levels of TLR4, p38 MAPK, and p65 NF-κB protein (P < 0.05). Serum containing YSTLD could up-regulate the expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulate the mRNA expression levels of p38 MAPK, p65 NF-κB, Bcl-2, and TNF receptor-associated factor 1 (TRAF1) in prostate cancer PC-3 cells after intervening prostate cancer PC-3 cells (P < 0.05). Simultaneously, it up-regulated the expression levels of caspase3 protein and down-regulated the protein expression levels of TLR4, p38 MARK, p65 NF-κB, and TRAF1 in prostate cancer PC-3 cells (P < 0.05). ConclusionYSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of miR-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway, which may be an important mechanism of YSTLD against prostate cancer.

Protective effects of Pudilan Tablets against osteoarthritis in mice induced by monosodium iodoacetate

Osteoarthritis (OA) is a complicated disorder that is the most prevalent chronic degenerative joint disease nowadays. Pudilan Tablets (PDL) is a prominent traditional Chinese medicine formula used in clinical settings to treat chronic inflammatory illnesses. However, there is currently minimal fundamental research on PDL in the therapy of joint diseases. As a result, this study looked at the anti-inflammatory and anti-OA properties of PDL in vitro and in vivo, as well as the mechanism of PDL in the treatment of OA. We investigated the anti-OA properties of PDL in OA mice that were generated by monosodium iodoacetate (MIA). All animals were administered PDL (2 g/kg or 4 g/kg) or the positive control drug, indomethacin (150 mg/kg), once daily for a total of 28 days starting on the day of MIA injection. The CCK-8 assay was used to test the vitality of PDL-treated RAW264.7 cells in vitro. RAW264.7 cells that had been activated with lipopolysaccharide (LPS) were used to assess the anti-inflammatory properties of PDL. In the MIA-induced OA model mice, PDL reduced pain, decreased OA-induced cartilage damages and degradation, decreased production of pro-inflammatory cytokines in serum, and suppressed IL-1β, IL-6, and TNF-α mRNA expression levels in tibiofemoral joint. In RAW264.7 cells, PDL treatment prevented LPS-induced activation of the ERK/Akt signaling pathway and significantly decreased the levels of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. In conclusion, these results suggest that PDL is involved in combating the development and progression of OA, exerts a powerful anti-inflammatory effect on the knee joint, and may be a promising candidate for the treatment of OA.

The Prevention Effect of Lactobacillus plantarum 17-5 on Escherichia coli-Induced Mastitis in Mice.

Mastitis is the most economically important disease affecting the dairy industry worldwide. Lactobacillus plantarum, an important probiotic with a wide range of applications, has potential anti-inflammatory properties and has become a currently strong candidate for mastitis therapies. In the current study, we evaluated the prevention effect ofLactobacillus plantarum 17-5 on Escherichia coli-induced mastitis in mice. The results showed that pretreatment with L. plantarum 17-5 maintained the integrity of tight junctions; improved inflammatory injury; decreased MPO activity and the mRNA expression levels of IL1β, IL6, and TNFα; and inhibited the NF-κB and MAPK signaling pathways in mice mammary tissue. The results indicated that Lactobacillus plantarum 17-5 had excellent anti-inflammatory activities and could be developed into microecological preparation for clinical use to prevent mastitis.

Perfluorocarbon restrains inflammation and cell apoptosis in rats with lung ischemia reperfusion injury via down regulation of TLR4/NF κB signaling pathway

Purpose: To exaluate the effect of perfluorocarbon on lung ischemia-reperfusion injury in rats, and to unravel the potential underlying mechanism.Methods: A total of 36 Sprague-Dawley (SD) rats were randomly assigned to sham group, model group, and perfluorocarbon group (12 rats per group). The levels of inflammatory factors (TNF-α and IL1β) were determined using enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, while Western blotting was conducted to determine the protein expressions of TLR4 and NF-κB.Results: The levels of inflammatory factors in the model and perfluorocarbon groups were significantly higher than those in operation group (p &lt; 0.05), while their levels in perfluorocarbon group were significantly lower than in model group (p &lt; 0.05). The mRNA expression levels of TNF-α and IL-1β in lung tissues rose significantly in both model and perfluorocarbon groups when compared with those in sham group (p &lt; 0.05), but declined significantly in the perfluorocarbon group in comparison with those in model group (p &lt; 0.05). Furthermore, the perfluorocarbon group exhibited a significantly lower cell apoptosis than model group (p &lt; 0.05). The relative protein expression levels of TLR4 and NF-κBdeclined significantly in perfluorocarbon group than in model group.Conclusions: Perfluorocarbon down-regulates the TLR4/NF-κB signaling pathway, and inhibits inflammation an

Combined Black Phosphorus Nanosheets with ICG/aPDT is an Effective Anti-Inflammatory Treatment for Periodontal Disorders.

Antibacterial photodynamic treatment (aPDT) has indispensable significance as a means of treating periodontal disorders because of its extraordinary potential for killing pathogenic bacteria by generating an overpowering amount of reactive oxygen species (ROS). The elevated ROS that may result from the antibacterial treatment procedure, however, could exert oxidative pressure inside periodontal pockets, causing irreparable damage to surrounding tissue, an issue that has severely restricted its medicinal applications. Accordingly, herein, we report the use of black phosphorus nanosheets (BPNSs) that can eliminate the side effects of ROS-based aPDT as well as scavenge ROS to produce an antibacterial effect. The antibacterial effect of ICG/aPDT was observed by direct microscopic colony counting. A microplate reader and confocal microscope enabled measurements of cell viability and the quantification of ROS fluorescence. BPNS administration regulated the oxidative environment. IL-1β, IL-6, TNF-α, IL-10, TGF-β, and Arg-1 mRNA expression levels were used to assess the inflammatory response after BPNS treatment. In vivo, the efficacy of the combination of BPNSs and ICG/aPDT was evaluated in rats with periodontal disease by histomorphometric and immunohistochemical analyses. The CFU assay results verified the antibacterial effect of ICG/aPDT treatment, and ROS fluorescence quantification by CLSM indicated the antioxidative ability of the BPNSs. IL-1β, IL-6, TNF-α, IL-10, TGF-β, and Arg-1 mRNA expression levels were significantly decreased after BPNS treatment, confirming the in vitro anti-inflammatory effect of this nanomaterial. The histomorphometric and immunohistochemical analyses showed that the levels of proinflammatory factors decreased, suggesting that the BPNSs had anti-inflammatory effects in vivo. Treatment with antioxidative BPNSs gives new insights into future anti-inflammatory therapies for periodontal disease and other infection-related inflammatory illnesses and provides an approach to combat the flaws of aPDT.

Gambogic acid affects high glucose-induced apoptosis and inflammation of retinal endothelial cells through the NOX4/NLRP3 pathway.

This study aimed to investigate the effect and mechanism of gambogic acid (GA) on the apoptosis and inflammation of human retinal endothelial cells (HRECs) under high glucose conditions. HRECs were cultured in a high glucose medium to simulate retinal endothelial cell injury induced by diabetic retinopathy. Flow cytometry was used to analyze the apoptosis level of HRECs. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Western blotting was applied to detect the intracellular apoptosis-related proteins and expression levels of NADPH oxidase 4 (NOX4), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and interleukin (IL)-1β. Enzyme linked immunosorbent assay (ELISA) was utilized to detect the expression of IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) in the cell supernatants. The messenger RNA (mRNA) levels of IL-6, IL-8, IL-10, and TNF-α were detected by reverse transcription-polymerase chain reaction (RT-qPCR). We observed that high glucose induced the apoptosis and inflammation of HRECs. In addition, the high glucose environment promoted NOX/NLRP3 pathway activation. The activity of HRECs was not significantly affected by the presence of 20 μM or less of GA, and 15 μM of GA could restore the diminished activity of HRECs induced by high glucose. The apoptosis of HRECs cultured under high glucose conditions was significantly inhibited (P<0.05), the levels of IL-6, IL-8, and TNF-α in the cell supernatant were significantly decreased (P<0.05), and the levels of IL-10 were significantly increased (P<0.05). Meanwhile, the relative mRNA expression levels of IL-6, IL-8, and TNF-α in HRECs were significantly decreased (P<0.05), while those of IL-10 were significantly increased (P<0.05). The activity of the high glucose-induced NOX4/NLRP3 pathway in HRECs was significantly inhibited after treatment with 15 μM of GA (P<0.05). Following activation of the NOX4/NLRP3 pathway in HRECs, the apoptosis level was significantly increased (P<0.05), and the inflammatory response was aggravated (P<0.05). Inhibiting the activity of the intracellular NOX4/NLRP3 pathway markedly inhibited cell apoptosis and the inflammatory response (P<0.05). GA can inhibit the apoptosis and inflammation of HRECs under high glucose conditions by inhibiting the activity of the NOX4/NLRP3 pathway. This has a significant inhibitory effect on diabetic retinopathy, which is worthy of further study.

Extracellular vesicles contribute to EMT in adenomyosis by inducing macrophage polarization†.

Adenomyosis is a benign disease, but it exhibits a metastatic property similar to tumors. Its pathogenesis is still unclear. One theory is that adenomyosis is the result of epithelial-mesenchymal transition (EMT) in displaced embryonic Muller cells. Macrophages accumulate in the eutopic endometrium of adenomyosis and play an important role in EMT and the pathogenesis of adenomyosis. Extracellular vesicles (EVs) are considered an important mechanism of intercellular communication, but few studies have shown the role of EVs between endometrial epithelial cells and macrophages. In this study, we collected the eutopic endometrium of adenomyosis, and acquired the primary endometrial cells, then isolated EVs from the culture supernatants. We identified the characteristics of EVs by transmission electron microscopy, nanoparticle tracking, and western blot, and then detected the mRNA expression levels of CD163, IL-10, iNOS, and TNF-α in macrophages by qRT-PCR after co-cultured with EVs; the expression levels of E-cadherin, CK7, N-cadherin, and Vimentin by Western blot, and the migration abilities of epithelial cells by Transwell assay. The results showed that macrophages were highly expressed in the mRNA levels of CD163, IL10, and TNF-α after treated by EVs from adenomyosis patients; endometrial epithelial cells expressed lower protein levels of E-cadherin and CK7, higher levels of N-cadherin and Vimentin after co-cultured with the above polarized macrophages; and the migration abilities of epithelial cells were enhanced. In conclusion, EVs derived from adenomyosis can induce macrophages to polarize toward M2b, and the polarized macrophages could, in turn, induce EMT process in endometrial epithelial cells.

Ozonated oil alleviates dinitrochlorobenzene-induced allergic contact dermatitis via inhibiting the FcεRI/Syk signaling pathway.

Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms. Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions. Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1β, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1β, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05). Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.

An Alkali-extracted polysaccharide from Poria cocos activates RAW264.7 macrophages via NF-κB signaling pathway

An alkali-extracted polysaccharide (PCAPS1) was isolated and purified from the Poria cocos. Our results proved that PCAPS1 was a neutral polysaccharide with a molecular weight of 11.5 kDa. The monosaccharide composition, methylation and NMR analysis results displayed that the polysaccharide was mostly comprised of β-1,3-glucan with 1,4 and 1,6 branches. The Immune activity and mechanism of PCAPS1 were evaluated in RAW264.7 cells. The enzyme-linked immunosorbent assay (ELISA) analysis revealed that PCAPS1 increased the tumor necrosis factor-α (TNF-α) secretion. RNA-sequencing data analysis suggested that PCAPS1 activated macrophages by the classic NF-κB pathway. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed that PCAPS1 enhanced mRNA expression levels of TNF-α and nuclear factor κB (NF-κB) in RAW264.7 cells. Simultaneously, the fluorescence nuclear transport experiment showed that PCAPS1 activated RAW264.7 cells by inducing the NF-κB p65 translocation. Our results indicated that PCAPS1-induced TNF-α expression was mediated via the NF-κB signaling pathway.

Mitogen-activated protein kinase inhibitor PD98059 improves neuroimmune dysfunction in experimental autoimmune encephalomyelitis in SJL/J mice through the inhibition of nuclear factor-kappa B signaling in B cells

Multiple sclerosis (MS) is a severe autoimmune disease leading to demyelination, followed by consequent axonal degeneration, causing sensory, motor, cognitive, and visual symptoms. Experimental autoimmune encephalomyelitis (EAE) is the most well-studied animal model of MS. Most current MS treatments are not completely effective, and severe side effects remain a great challenge. In this study, we report the therapeutic efficacy of PD98059, a potent mitogen-activated protein kinase inhibitor, on proteolipid protein (PLP)139–151-induced EAE in SJL/J mice. Following the induction of EAE, mice were intraperitoneally treated with PD98059 (5 mg/kg for 14 days) daily from day 14 to day 28. This study investigated the effects of PD98059 on C-C motif chemokine receptor 6 (CCR6), CD14, NF-κB p65, IκBα, GM-CSF, iNOS, IL-6, TNF-α in CD45R+ B lymphocytes using flow cytometry. Furthermore, we analyzed the effect of PD98059 on CCR6, CD14, NF-κB p65, GM-CSF, iNOS, IL-6, and TNF-α mRNA and protein expression levels using qRT-PCR analysis in brain tissues. Mechanistic investigations revealed that PD98059-treated in mice with EAE had reduced CD45R+CCR6+, CD45R+CD14+, CD45R+NF-κB p65+, CD45R+GM-CSF+, CD45R+iNOS+, CD45R+IL-6+, and CD45R+TNF-α+ cells and increased CD45R+IκBα+ cells compared with vehicle-treated control mice in the spleen. Moreover, downregulation of CCR6, CD14, NF-κB p65, GM-CSF, iNOS, IL-6, and TNF-α mRNA expression level was observed in PD98059-treated mice with EAE compared with vehicle-treated control mice in the brain tissue. The results of this study demonstrate that PD98059 modulates inflammatory mediators through multiple cellular mechanisms. The results of this study suggest that PD98059 may be pursued as a therapeutic agent for the treatment of MS.

Anti-inflammatory effects and molecular mechanisms of bioactive small molecule garlic polysaccharide.

Although garlic polysaccharides have been found to possess anti-inflammatory activities, anti-inflammatory study on small molecule water-soluble garlic polysaccharide (WSGP) is few. In this study, a novel WSGP with a molecular weight of 1853 Da was isolated by DEAE-52 and Sephadex G-100 column and the chemical composition was identified by monosaccharide composition and methylation analysis. Furthermore, the antioxidant effects of WSGP and the potential molecular mechanisms on LPS-induced inflammatory responses in RAW264.7 macrophage cells were investigated. The results showed that WSGP has strong antioxidant activity, such as DPPH, hydroxyl, superoxide anion, ABTS radical scavenging capacity, Fe2+ chelating ability and reducing power. Meanwhile, WSGP could considerably suppress the manufacturing of NO and the mRNA and protein expression degrees of IL-6, TNF-α, and IL-1β in LPS inspired RAW264.7 macrophages WSGP could significantly suppress the production of NO and the mRNA and protein expression levels of IL-1β, IL-6, and TNF-α in LPS stimulated RAW264.7 macrophage cells (p < 0.05). In addition, the phosphorylated IκB-α, p65, and STAT3 proteins were significantly increased in LPS-induced macrophages, while this trend was significantly reversed by WSGP treatment in a concentration-dependent manner (p < 0.05). Consequently, WSGP supplementation might reduce LPS-induced inflammatory responses by suppressing proinflammatory cytokines and NF-κB and STAT3 pathway activation. The finding of this research would give scientific guidelines for the judicious use of small molecular garlic polysaccharide in anti-inflammatory treatments.

Protective effect of bromelain and pineapple extracts on UV-induced damage in human skin cells

In Hawaii, people experience high sunlight exposure, contributing to an increased risk of sunburn. Additionally, most parts of pineapples are discarded, contributing to food waste and microbial spoilage, and we wanted to determine whether bromelain sourced from pineapple could be repurposed as a treatment to protect skin cells from UV damage. Bromelain from pineapple can be used to treat symptoms of sunburn, which occurs when UV rays damage the DNA in cells. Therefore, we hypothesized that bromelain extracted from various parts of the pineapple (crown, flesh, skin, and stem) would protect the skin cells from cell death caused by UV damage. Also, we expected bromelain from the stem, which is known to contain the highest concentration of bromelain, to be the most effective in protecting skin cells from UV-induced cell death. The effect of purified bromelain powder on skin cell survival was analyzed using the PrestoBlue cell viability assay to compare the protective effect of pineapple extracts on UV-induced cell death. We also examined the expression level of tumor necrosis factor-α (TNF-α), which is a pro-inflammatory marker. The results indicated that bromelain protected skin cells from UV-dependent cell death and increased the mRNA expression level of TNF-α. Additionally, bromelain had a more protective effect when it was used as a pre-treatment method than post-treatment. Overall, this study suggests that bromelain may provide some protection to the skin from UV-induced cell death. Thus, further studies may investigate its application in sunscreen products to improve protection.

Coptisine attenuates sepsis lung injury by suppressing LPS-induced lung epithelial cell inflammation and apoptosis.

This study aimed to investigate the functioning and mechanism of coptisine in acute lung injury (ALI). Murine Lung Epithelial 12 (MLE-12) cells were stimulated with lipopolysaccharide (LPS) to construct an in vitro pulmonary injury model to study the functioning of coptisine in sepsis-induced ALI. The viability of MLE-12 cells was assessed by the cell counting kit-8 assay. The cytokine release of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and IL-1β was measured by enzyme-linked-immunosorbent serologic assay. The relative expression levels of TNF-α, IL-6, and IL-1β mRNA were examined by reverse transcription-quantitative polymerase chain reaction. The cell apoptosis of MLE-12 cells was determined by Annexin V/propidium iodide staining and analyzed by flow cytometry. The expressions of apoptosis-related proteins Bax and cleaved Caspase-3 were observed by Western blot analysis. The activation of nuclear factor kappa B (NF-κB) signaling pathway was discovered by the determination of phospho-p65, p65, phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and IκBα through Western blot analysis. Coptisine treatment could significantly restore decrease in MLE-12 cell viability caused by LPS stimulation. The release of TNF-α, IL-6, and IL-1β was significantly inhibited by coptisine treatment. Coptisine treatment inhibited MLE-12 cell apoptosis induced by LPS, and also inhibited the expression levels of Bax and cleaved Caspase-3. Coptisine treatment along with LPS stimulation, significantly reduced the protein level of phospho-IκBα, increased the level of IκBα, and reduced phospho-p65-p65 ratio. These results indicated that coptisine attenuated sepsis lung injury by suppressing lung epithelial cell inflammation and apoptosis through NF-κB pathway. Therefore, coptisine may have potential to treat sepsis-induced ALI.

Clinical Significance of Decreased TIPE2 Expression in Peripheral Blood Mononuclear Cells of Patients with Psoriasis Vulgaris

Psoriasis is an immune system disorder induced by the interaction of the polygenic background and environmental factors. Tumor necrosis factor‐α (TNF‐α)‐induced protein 8‐like 2 (TNFAIP8L2, TIPE2), a regulator of immunity, is differentially expressed in tumors, inflammation, and autoimmune diseases. However, to our knowledge, no study has evaluated TIPE2 expression in patients with psoriasis vulgaris. The present case‐control study aimed to determine the expression levels of TIPE2 mRNA in the peripheral blood mononuclear cells (PBMCs) of patients with psoriasis vulgaris and analyze the correlation between TIPE2 and other inflammatory cytokine variations in the pathogenesis of psoriasis vulgaris. Thirty‐three patients with psoriasis vulgaris and thirty‐one healthy volunteers were enrolled from October 2021 to March 2022. The mRNA expression levels of TIPE2, RELA (NF‐κb p65), TNF‐α, IL‐10, IL‐6, and IL‐1β in PBMCs were determined using qPCR. The severity of psoriasis vulgaris was determined according to the Psoriasis Area and Severity Index (PASI) scores. TIPE2 mRNA expression significantly decreased, while the expression of RELA, TNF‐α, IL‐10, IL‐6, and IL‐1β increased in patients with psoriasis vulgaris compared with that in the healthy control group. In addition, the expression levels of TIPE2, RELA, TNF‐α, and IL‐1β positively correlated with the PASI score. TIPE2 mRNA expression negatively correlated with IL‐6 and positively correlated with TNF‐α. Moreover, TIPE2 mRNA expression was higher in the active stage than that in the stationary stage. Notably, TNF‐α expression levels were higher in patients with psoriasis vulgaris combined with a history of respiratory infection. TIPE2 may contribute to the pathogenesis of psoriasis and be a potential biomarker of psoriasis vulgaris.

Gene expression profiles for apoptotic and necrotic pathways during Amanita phalloides intoxication in mice

Background and Aims: Amanita phalloides is the deadliest toxic mushroom in the world and causes death from acute liver failure. α-amanitin (α-AMA), the most potent toxin, inhibits RNA polymerase II in hepatocytes, stops protein synthesis, and causes hepatotoxicity. However, the information about the mechanisms underlying hepatotoxicity caused by α-AMA is quite inadequate. This study aims to reveal the complex necrotic and apoptotic mechanisms occurring in mouse hepatocytes depending on A. phalloides exposure time in vivo. Methods: BALB-c male mice were divided into 5 groups (n=7): control, α-AMA-2, α-AMA-12, α-AMA-72, and α-AMA-96 groups. A poisoning model was created by oral administration of A. phalloides mushroom extract containing 10 mg/kg of α-AMA to mice and they were sacrificed after 2, 12, 72, and 96 h. Then, TNF-α, Bax, caspase-3, and Bcl-2 gene expression levels in liver tissues were examined by the RT-qPCR method. Time-dependent damage to liver tissues was also evaluated histopathologically. Results: RT-qPCR results showed that proinflammatory cytokine TNF-α mRNA expression levels increased in mouse liver tissues at 2 and 12 h after A. phalloides administration compared among the groups. Bax mRNA expression levels increased in the 12 and 72 h after A. phalloides ingestion. It was observed that caspase-3 mRNA expression levels increased in the 72 and 96 h groups compared among the groups, while Bcl-2 mRNA expression levels decreased in the 72 and 96 h groups. Conclusion: Our findings showed that necrotic mechanisms develop in the early period after A. phalloides mushroom poisoning, and then apoptotic mechanisms are effective. In conclusion, understanding the mechanisms of A. phalloides-induced hepatotoxicity will provide important information for new treatment strategies to be developed.