IL-4 mRNA Levels Research Articles

Effects of Lactiplantibacillus plantarum 19-2 on immunomodulatory function and gut microbiota in mice.

This study aims to evaluate the effects of Lactiplantibacillus plantarum 19-2 (L. plantarum 19-2) on mice treated with the alkylating agent cyclophosphamide (CTX). Our findings show that L. plantarum 19-2 restored the spleen and thymus index and the number of white blood cells and lymphocytes% in CTX treated mice. Serum immunoglobulin levels in CTX-treated mice were increased by L. plantarum 19-2. In addition, as compared to the model group, L. plantarum 19-2 upregulated the content of SIgA, while L. plantarum 19-2 regulates the mRNA and protein expression levels of GATA-3, T-bet, IFN-γ, and IL-4 in small intestinal tissues, which adjusted mucosal barriers, structural status, and the balance of Helper T-cell 1 and Helper T-cell 2. Lactiplantibacillus plantarum 19-2 regulated the distribution of intestinal flora in mice, promoting the growth of Bacteroides and Proteobacteria. In addition, L. plantarum 19-2 inhibited the growth of several harmful bacteria, including Actinobacteria and Firmicutes.

Glaesserella parasuis induces IL-17 production might through PKC-ERK/MAPK and IκB/NF-κB signaling pathways

In our previous study, the outer membrane protein P2 of Glaesserella parasuis (G. parasuis) induced the production of interleukin-17A (IL-17A) in host cells. However, how the G. parasuis infection produces IL-17A in the host is unknown. In this study, at 48 hpi in piglets, the proportion of Th17 cells in the peripheral blood significantly increased as determined by flow cytometry, but the overall proportion was low. IL-17 mRNA and protein levels significantly increased in the lungs and spleens of infected piglets. Histopathological examination staining, immunohistochemistry and viability counts, revealed that IL-17 expression might be positively correlated with bacterial content and the degree of pathological injury in lung and spleen tissues. Furthermore, when infected with the G. parasuis Nagasaki strain compared with avirulent strain C5, the primary porcine alveolar macrophages (PAMs) significantly produced IL-17 in a time-dose dependent manner. In addition, we demonstrated that PKC, MEK and NF-κB signaling pathways were essential for G. parasuis-induced IL-17 production because the addition of corresponding inhibitors dramatically reduced IL-17 mRNA production. The phosphorylation levels of PKC, ERK1/2 and IκB in G. parasuis-infected primary PAMs also increased significantly as the infection dose increased as assessed by western blotting. Thus, these finding imply that IL-17 induced by G. parasuis infection is dependent on activation of PKC-ERK/MAPK and IκB/NF-κB signaling pathways.

E.faecium QH06 alleviates TNBS-induced colonic mucosal injury in rats

To investigate the effect of Enterococcus faecium QH06 on TNBS-induced ulcerative colitis (UC) in rats and explore the mechanisms in light of intestinal flora and intestinal immunity. Thirty-six male Wistar rats were randomized equally into control group, UC model group, and E.faecium QH06 intervention group. The rats in the latter two groups were subjected to colonic enema with 5% TNBS/ethanol to induce UC, followed by treatment with intragastric administration of distilled water or E.faecium QH06 at the dose of 0.21 g/kg. After 14 days of treatment, the rats were examined for colon pathologies with HE staining. The mRNA and protein expression levels of IL-4, IL-10, IL-12, and IFN-γ in the colon tissues were detected using RT-qPCR and ELISA, and the expression of TLR2 protein was detected with immunohistochemistry and ELISA. Illumina Miseq platform was used for sequencing analysis of the intestinal flora of the rats with bioinformatics analysis. The correlations of the parameters of the intestinal flora with the expression levels of TLR2 and cytokines were analyzed. The rats with TNBS- induced UC showed obvious weight loss (P < 0.01) and severe colon tissue injury with high pathological scores (P < 0.01). The protein expression levels of IFN-γ, IL-12, and TLR2 (P < 0.01) and the mRNA expression levels of IFN-γ, IL-12 and IL-10 (P < 0.05) were significantly increased in the colon tissues of the rats with UC. Illumina Miseq sequence analysis showed that in UC rats, the Shannon index (P < 0.05) ACE (P < 0.01)and Chao (P < 0.05) index for the diversity of intestinal flora both decreased with a significantly increased abundance of Enterobacteriaceae (P < 0.01) and a lowered abundance of Burkholderiaceae (P < 0.05). Compared with the UC rats, the rats treated with E. faecium QH06 showed obvious body weight gain (P < 0.05), lessened colon injuries, lowered pathological score of the colon tissue (P < 0.05), decreased protein expressions of IFN- γ, IL- 12, and TLR2 and mRNA expressions of IFN- γ and IL-12 (P < 0.01 or 0.05), and increased protein expressions of IL- 4 (P < 0.05). The Shannon index ACE (P < 0.05) and Chao (P < 0.05) index of intestinal microflora were significantly increased, the abundance of Enterobacteriaceae was lowered and that of Burkholderiaceae and Rikenellaceae was increased in E.faecium QH06- treated rats (P < 0.01 or 0.05). Correlation analysis showed that IFN-γ was positively correlated with the abundance of Enterobacteriaceae, and IFN-γ was negatively correlated with the abundance of Prevotellaceae, Desulfovibrionaceae, norank_o_Mollicutes_RF39 and Clostridiales_vadinBB60_group; TLR2 was negatively correlated with Clostridiales_vadinBB60_group, norank_o_Mollicutes_RF39 and Prevotellaceae. E.faecium QH06 can alleviate TNBS-induced colonic mucosal injury in rats, and its effect is mediated possibly by increasing the abundance of SCFA-producing bacteria such as Prevotellaceae and inhibiting abnormal immune responses mediated by TLR2.

PDZK1 upregulates nitric oxide production through the PI3K/ERK2 pathway to inhibit porcine circovirus type 2 replication

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease. Changes in host cell gene expression are induced by PCV2 infection. Here, we showed that porcine PDZ Domain-Containing 1 (PDZK1) expression was enhanced during PCV2 infection and that overexpression of PDZK1 inhibited the expression of PCV2 Cap protein. PCV2 genomic DNA copy number and viral titers were decreased in PDZK1-overexpressing PK-15B6 cells. PDZK1 knockdown enhanced the replication of PCV2. Overexpression of PDZK1 activated the phosphoinositide 3-kinase (PI3K)/ERK2 signaling pathway to enhance nitric oxide (NO) levels, while PDZK1 knockdown had the opposite effects. A PI3K inhibitor (LY294002) and a NO synthase inhibitor (L-NAME hydrochloride) decreased the activity of PDZK1 in restricting PCV2 replication. ERK2 knockdown enhanced the proliferation of PCV2 by decreasing levels of NO. Levels of interleukin (IL)− 4 mRNA were reduced in PDZK1 knockdown and ERK2 knockdown PK-15B6 cells. Increased IL-4 mRNA levels were unable to decrease NO production in PDZK1-overexpressing cells. Thus, we conclude that PDZK1 affected PCV2 replication by regulating NO production via PI3K/ERK2 signaling. PDZK1 affected IL-4 expression through the PI3K/ERK2 pathway, but PDZK1 modulation of PCV2 replication occurred independently of IL-4. Our results contribute to understanding the biological functions of PDZK1 and provide a theoretical basis for the pathogenic mechanisms of PCV2.

β-Sitosterol inhibits ovalbumin-induced asthma-related inflammation by regulating dendritic cells

Aim To investigate the effects of β-sitosterol (B-SIT) and the underlying mechanisms of action in an ovalbumin-induced rat model of asthma. Methods The pathological and morphological changes in lung and tracheal tissues were observed by H&E, PAS, and Masson’s staining. The levels of IgE, TNF-α, and IFN-γ in the bronchoalveolar lavage fluid (BALF) and those of IL-6, TGF-β1, and IL-10 in serum were measured by ELISA. The relative expression levels of IL-5, IL-13, IL-21, CD11c, CD80, and CD86 mRNA in lung tissue were examined by RT-qPCR. Flow cytometry was performed to assess the levels of immune cells, including macrophages and neutrophils in spleen tissue and Th cells, Tc cells, NK cells, and DCs in peripheral blood. The protein expression levels of CD68, MPO, CD11c, CD80, and CD86 were detected by western blotting or immunohistochemistry. Results B-SIT improved the injury in OVA-induced pathology, decreased the levels of inflammatory factors of IgE, TNF-α, IL-6, TGF-β1, IL-5, IL-13, and IL-21 and increased the levels of IFN-γ and IL-10. In addition, B-SIT decreased the number of macrophages and neutrophils and the relative expression levels of CD68 and MPO in the spleen. Moreover, B-SIT increased the number of Th cells, Tc cells, NK cells, and DCs in peripheral blood and upregulated the levels of CD11c, CD80, and CD86 in the spleen and lung. Conclusion B-SIT improved symptoms in a rat model of asthma likely via the inhibition of inflammation by regulating dendritic cells.

Palmatine Protects Against MSU-Induced Gouty Arthritis via Regulating the NF-κB/NLRP3 and Nrf2 Pathways.

PurposeGouty arthritis could be triggered by the deposition of monosodium uric acid (MSU) crystals. Palmatine (PAL), a protoberberine alkaloid, has been proven to possess compelling health-beneficial activities. In this study, we aimed to explore the effect of PAL on LPS plus MSU crystal-stimulated gouty arthritis in vitro and in vivo.MethodsPMA-differentiated THP-1 macrophages were primed with LPS and then stimulated with MSU crystal in the presence or absence of PAL. The expression of pro-inflammatory cytokines and oxidative stress-related biomarkers and signal pathway key targets were determined by ELISA kit, Western blot, immunohistochemistry and qRT-PCR, respectively. In addition, the anti-inflammatory and antioxidant activities of PAL on MSU-induced arthritis mice were also evaluated.ResultsThe results indicated that PAL (20, 40 and 80 μM) dose-dependently decreased the mRNA expression and levels of pro-inflammatory cytokines (interleukin-1beta (IL-1β), IL-6, IL-18 and tumor necrosis factor alpha (TNF-α)). The levels of superoxide dismutase (SOD) and glutathione (GSH) were remarkably enhanced, while the level of malondialdehyde (MDA) was reduced. Western blot analysis revealed that PAL appreciably inhibited NF-κB/NLRP3 signaling pathways through inhibiting the phosphorylation of p-65 and IκBα, blocking the expression of NLRP3, ASC, IL-1β and Caspase-1, as well as enhancing the antioxidant protein expression of Nrf2 and HO-1. In vivo, PAL attenuated MSU-induced inflammation in gouty arthritis, as evidenced by mitigating the joint swelling, and decreasing the productions of IL-1β, IL-6, IL-18, TNF-α and MDA, while enhancing the levels of SOD and GSH. Moreover, PAL further attenuated the infiltration of neutrophils into joint synovitis.ConclusionPAL protected against MSU-induced inflammation and oxidative stress via regulating the NF-κB/NLRP3 and Nrf2 pathways. PAL may represent a potential candidate for the treatment of gouty arthritis.

Dissolvable Microneedle Patch Increases the Therapeutic Effect of Jawoongo on DNCB-Induced Atopic Dermatitis in Mice

Background: Jawoongo (JW) is a topical herbal ointment that has been used as an alternative treatment option for atopic dermatitis. Topical ointments are known to have less bioavailability because the stratum corneum allows only lipophilic and low molecular weight drugs to pass across it. This study aimed to investigate whether applying microneedle patches (MNP) increases the therapeutic effect of 2,4-dinitrochlorobenzene (DNCB)+JW for atopic dermatitis by enhancing transdermal delivery. Methods: Atopic dermatitis was induced by DNCB in BALB/c mice. The combination treatment of JW and MNP was estimated to study the effect of MNP in improving transdermal delivery. Histological analysis, quantitative real-time PCR (qPCR), and immunofluorescence were performed to verify the effect of MNP in enhancing the therapeutic effects of DNCB+JW on atopic dermatitis in mice. Results: Both combination treatment and DNCB+JW treatment ameliorated histological alterations and reduced skin thickness and infiltration of CD4+ T cells in atopic dermatitis-like skin lesions in DNCB-exposed BALB/c mice. However, the improvement of histological alterations was better in the combination treatment, which was almost normal. Furthermore, the combination treatment exhibited a larger decrease in mRNA levels of IL-4, IL-6, IL-13, iNOS, and TNF-α, compared to DNCB+JW only. In addition, skin thickness and infiltration of CD4+ T cells in the sensitized skin were significantly lower using the combination treatment than using DNCB+JW only. Conclusion: Combination treatment with JW and MNP further decreased skin thickness and several inflammatory cytokines in atopic dermatitis like skin lesions compared to treatment using JW alone. These findings suggest that applying a dissolvable MNP after JW application could be useful for treating atopic dermatitis.

Livin promotes Th2-type immune response in airway allergic diseases

ObjectivesTo investigate the effects of livin on the Th2 immune response in airway allergic diseases (AAD) and explore the interaction among livin, GATA3, IL-4 in peripheral blood CD4+ T cells of AAD patients.MethodsWT mice and livin KO mice were developed for model of AAD. Th2 cell levels in the lung tissues and spleen were assessed by flow cytometry. Also, it was assessed in the culture after exposing to livin inhibitor (Lp-15); the protein and mRNA levels of livin, GATA3 and IL-4 in peripheral blood CD4+ T cells isolated from patients with or without AAD were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Finally, Co-immunoprecipitation (Co-IP) was employed to identify the interaction between livin and GATA3.ResultsCompared with WT mouse, Th2 cell frequency in lung tissues and spleen was significantly decreased in livin KO mouse; after adding Lp-15, the differentiation from Naive CD4+T cells in spleen to Th2 cells was blocked; the protein and mRNA levels of livin, GATA3 and IL-4 in AAD group were higher than that in control group. The levels of livin were positively correlated with IL-4, and GATA3 was also positively correlated with IL-4 and livin. GATA3 was detected in the protein complex co-precipitated with livin antibody, and livin was also detected in the protein complex co-precipitated by GATA3 antibody.ConclusionLivin increases the expression of IL-4 and facilitates naive CD4+ T cells to differentiate into Th2 cells, which triggers airway allergy.

Tim-3/Galectin-9 signaling pathway is involved in the cytokine changes in mice with alveolar echinococcosis.

Tim-3/Galectin-9 is involved in the immune escape of many pathogens. However, the role of Tim-3/Galectin-9 in persistent infection of Echinococcus multilocularis (Em), which is related to immune escape, is still unclear. To investigate the role of Tim-3/Galectin-9 and related cytokines in mice with persistent infection of Em. Em infection model was established by injecting the protoscoleces. Serum was collected at days 2, 8, 30, 60, 90, 180 and 270 after infection. Lymphocytes were isolated from liver tissue samples with Ficoll. Tim-3 + CD4 + T percentage was analyzed by flow cytometry. CD4 + T cells were isolated from liver tissues of Em infected mice and cultured in vitro. The mRNA levels of Tim-3, Galectin-9, IFN-γ and IL-4 were detected by qRT-PCR. Cytokine levels in serum and culture supernatant (IFN-γ and IL-4) were analyzed by cytometric bead array. The expression of Tim-3 and Galectin-9 mRNA significantly increased after 30 days of infection, reached peak on day 90, and then decreased slightly on days 180-270. The expression of IFN-γ mRNA, increased on day 2 and 8 after infection, slightly decreased on days 30-60, and obvious decreased on days 90-270, but were still higher than those of the control group. The expression of IL-4 mRNA gradually increased along with the time of infection. In serum of Em infected mice, level of IFN-γ peaked at day 30 and then gradually decreased; whereas IL-4 level peaked at day 90 and then gradually decreased. In vitro experiment found that Tim-3/Galectin-9 directly caused the changes in the levels of IFN-γ and IL-4. Tim-3/Galectin-9 signaling pathway may be involved in the development of persistent infection of Em by regulating the production of Th1 and Th2 cytokines.

Excessive Immune Activation and the Correlation with Decreased Expression of PD-1 at the Maternal-Fetal Interface in Preeclampsia.

The etiology of preeclampsia (PE) is still unknown, and excessive immune activation is an important component of its pathogenesis. Programmed cell death protein 1 (PD-1) is one of immune checkpoints which may prevent overactivated immune attack and lead to a tolerant immune microenvironment. Little is known about the involvement of PD-1-mediated immunoregulation at the maternal-fetal interface in PE. To investigate the inflammatory pattern and the involvement of PD-1 in the decidua of women with PE, decidual tissues were obtained from PE and control pregnant women. Quantitative RT-PCR analysis of the mRNA levels of the inflammatory cytokines was performed. PD-1 expression was detected by immunohistochemistry, western blot analysis, and flow cytometry. To prove the role of PD-1, decidual immune cells were incubated with blocking antibodies, and the inflammatory cytokines were detected by ELISA. We observed that the mRNA levels of IL-1β, IL-6, TNF-α, and IFN-γ were higher in the decidua of the PE group than in the decidua of the control group. The mRNA levels of IL-4 and IL-10 were lower in PE. The expression level of PD-1 was significantly downregulated, and the proportion (%) of PD-1 + CD45 + cells was significantly lower in PE. There was a significant linear correlation between PD-1 expression and common proinflammatory cytokines in the decidua. Anti-PD-1 blocking antibody significantly increased the secretion of proinflammatory cytokines. Our data suggested that the inflammatory pattern and decreased PD-1 expression in the decidua might play an active role in the local immunoregulatory mechanisms of PE. The PD-1 pathway in the maternal-fetal interface possibly function to break the tolerant immune microenvironment in PE via inflammatory cytokines.

Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway.

The immunomodulatory function of mesenchymal stem cells (MSCs) has been considered to be vital for MSC-based therapies. Many works have been devoted to excavate effective strategies for enhancing the immunomodulation effect of MSCs. Nonetheless, canine MSC-mediated immunomodulation is still poorly understood. The inflammatory microenvironment was simulated through the employment of interferon-γ (IFN-γ) in a culture system. Compared with unstimulated cBMSCs, IFN-γ stimulation increased the mRNA levels of Toll-like receptor 3 (TLR3) and indoleamine 2, 3-dioxygenase 1 (IDO-1), and simultaneously enhanced the secretion of immunosuppressive molecules, including interleukin (IL)-10, hepatocyte growth factor (HGF), and kynurenine in cBMSCs. IFN-γ stimulation significantly enhanced the ability of cBMSCs and their supernatant to suppress the proliferation of murine spleen lymphocytes. Lymphocyte subtyping evaluation revealed that cBMSCs and their supernatant diminished the percentage of CD3+CD4+ and CD3+CD8+ lymphocytes compared with the control group, with a decreasing CD4+/CD8+ ratio. Notably, exposure to IFN-γ decreased the CD4+/CD8+ ratio more effectively than unstimulated cells or supernatant. Additionally, IFN-γ-stimulation increased the mRNA levels of the Th1 cytokines TNF-α, and remarkably decreased the mRNA level of the Th2 cytokine IL-4 and IL-10. Our findings substantiate that IFN-γ stimulation can enhance the immunomodulatory properties of cBMSCs by promoting TLR3-dependent activation of the IDO/kynurenine pathway, increasing the secretion of immunoregulatory molecules and strengthening interactions with T lymphocytes, which may provide a meaningful strategy for the clinical application of cBMSCs in immune-related diseases.

OX40 Expression in Eosinophils Aggravates OVA-Induced Eosinophilic Gastroenteritis.

Background & AimsEosinophils are the main inflammatory effector cells that damage gastrointestinal tissue in eosinophilic gastrointestinal diseases (EGIDs). Activation of the OX40 pathway aggravates allergic diseases, such as asthma, but it is not clear whether OX40 is expressed in eosinophils to regulate inflammation in EGIDs. In this study, we assessed the expression and effect of OX40 on eosinophils in WT and Ox40-/- eosinophilic gastroenteritis (EGE) mice.MethodsEosinophil infiltration, ovalbumin (OVA)-specific Ig production, OX40 expression and inflammatory factor levels in the intestine and bone marrow (BM) were investigated to evaluate inflammation.ResultsWe confirmed that OVA-challenged mice produced high levels of Ox40, Mbp, Ccl11, Il5, Il4, Il13, and Il6 mRNA and a low level of Ifng mRNA in the intestine. Increased eosinophils were observed in intestinal and lymph tissues, accompanied by significantly upregulated OX40 and Type 2 cytokine production in eosinophils of EGE mice. Ox40 deficiency ameliorated OVA-induced inflammation, eosinophil infiltration, and cytokine production in the intestine. Consistently, Ox40-/ - eosinophils exhibited decreased proliferation and proinflammatory function. The stimulation of the agonistic anti-OX40 antibody, OX86, promoted the effect of OX40 on eosinophils. The present study also showed that Ox40 deficiency dampened the Traf2/6-related NF-κB signaling pathway in eosinophils.ConclusionsOX40 may play a critical role in the progress of OVA-induced EGE by promoting the maturation and function of eosinophils via the Traf2/6-related NF-κB signaling pathway.

Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells

To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells. C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment. The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05). Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.

The effects of dietary supplementation of α-lipoic acid on the growth performance, antioxidant capacity, immune response, and disease resistance of northern snakehead, Channa argus

The study was the first time to explore the positive effects of α-LA on growth performance, antioxidant capability, immunity, and disease resistance of northern snakehead (Channa argus). Five hundred and forty northern snakehead fish (initial body weight: 8.74 ± 0.12 g (mean ± SE)) were randomly allocated into six groups with three replicates each. Six diets supplemented with α-LA at doses of 0 (CON), 300 (LA300), 600 (LA600), 900 (LA900), 1200 (LA1200), and 1500 (LA1500) mg/kg were fed to northern snakehead for 8 weeks. The results demonstrated that, when compared with the control group, optimal dietary α-LA increased the weight gain (WG), protein efficiency ratio (PER), and specific growth rate (SGR) and reduced the feed conversion ratio (FCR) of the fish (P < 0.05). Also, optimal dietary α-LA enhanced the immune-related parameters and antioxidant enzyme parameters levels in the head kidney, spleen, and liver of northern snakehead (P < 0.05). Dietary α-LA upregulated the mRNA expression levels of anti-inflammatory cytokines (il10 and tgfβ) and antioxidant related genes (gst, gsh-px, gr and Cu/Zn sod), down-regulated the pro-inflammatory cytokines (il1β, il8, il12 and tnfα) mRNA levels in the liver, spleen and head kidney of the northern snakehead (P < 0.05). The above results demonstrated that optimal dietary α-LA showed enhancement effects on the growth, antioxidant and anti-inflammatory capability, and immune response of northern snakehead. The survival rates in all α-LA treatments were significantly raised after the challenge with Aeromonas veronii (P < 0.05). Based on the quadratic regression analysis of WG, GSH-Px, LYS, and il1β, the optimal dietary α-LA levels were estimated to be 737.0, 775.0, 890.0, and 916.7 mg/kg, respectively. Considering the overall responses in growth performance, antioxidant status, immune response, and inflammatory factors, the recommended dose of α‐LA in the diet of fish is 737.0–916.7 mg/kg.

High-Temperature Soup Foods in Plastic Packaging Are Associated with Phthalate Body Burden and Expression of Inflammatory mRNAs: A Dietary Intervention Study.

Plastic packaging material is widely used to package high-temperature soup food in China, but this combination might lead to increased exposure to phthalates. The health effects and potential biological mechanisms have not been well studied. This study aimed to examine urinary phthalate metabolites and the expression of inflammatory cytokines in the blood before, during, and after a "plastic-packaged high-temperature soup food" dietary intervention in healthy adults. The results showed that compared with those in the preintervention period, urinary creatinine-adjusted levels of monomethyl phthalate (MMP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MIBP), and total phthalate metabolites in the intervention period were significantly higher, with increases of 71.6, 41.8, 38.8, and 29.8% for MMP, MBP, MIBP, and the total phthalate metabolites, respectively. After intervention, the mean levels of IL-1β, IL-4, and TNF-α mRNA increased by 19.0, 21.5, and 25.0%, respectively, while IL-6 and IFN-γ mRNA decreased by 24.2 and 32.9%, respectively, when compared with the preintervention period. We also observed that several phthalates were associated with the mRNA or protein expression of IL-8, TNF-α, and IL-10. Therefore, consumption of plastic-packaged high-temperature soup food was linked to increased phthalate exposure and might result in significant changes in mRNA expression of several inflammatory cytokines.

Tanshinone IIA down-regulates -transforming growth factor beta 1 to relieve renal tubular epithelial cell inflammation and pyroptosis caused by high glucose

ABSTRACT Diabetic nephropathy (DN) is a microvascular disease caused by diabetes. Tanshinone IIA has been indicated to ameliorate streptozotocin-induced DN. This study explores the effect of tanshinone IIA on high glucose-induced renal tubular epithelial cell pyroptosis and inflammation. High glucose-stimulated HK-2 cells were used as the in-vitro model of DN and were treated with tanshinone IIA at concentrations of 1, 5, 10 μM for 24 h with the same doses of tolbutamide as the control. After tanshinone IIA treatment, HK-2 cells were transfected with pcDNA-transforming growth factor beta 1 (TGFB1) or sh-TGFB1 for 48 h. RT-qPCR was used to detect the mRNA levels of TNF-α, IL-6, IL-1β, and IL-18. Cell apoptosis and pyroptosis were detected by flow cytometry and cell immunofluorescence. Bioinformatics screening predicted that tanshinone IIA might be an effective component of Salvia miltiorrhiza Bunge (Labiatae) for the treatment of DN. Tanshinone IIA exerted a protective effect in the in-vitro model of DN by suppressing inflammation and pyroptosis via the TGFB1-dependent pathway. Tanshinone IIA inhibited high glucose-induced renal tubular epithelial cell inflammation and cell death through pyroptosis by regulating TGFB1, indicating the therapeutic potential of tanshinone IIA for DN treatment.

Augmented Urinary Excretion of Angiotensinogen Precedes Albuminuria in Human Young Adults with Overweight and Mice with High Fat Diet‐Induced Type 2 Diabetes

Obesity is linked to the development of hypertension and type 2 diabetes (T2D) and both causing chronic kidney disease (CKD). Increased body mass index (BMI) and high fat diet (HFD) contribute to activation systemic renin angiotensin system (RAS). In addition, activation of intrarenal and intratubular RAS has been associated with increased pressure (BP), CKD, and augmentation of urinary excretion of angiotensinogen (uAGT) during T2D. However, whether augmentation of uAGT reflect intrarenal/intratububular RAS activation in subjects with overweight, remains unclear. Here, we tested the hypothesis that increases in uAGT precedes renal dysfunction in mice and human subjects with overweight,MethodsC57BL/6 mice (N = 10) were fed either a normal fat diet (NFD) or HFD (45% Kcal from fat) for 28 weeks. During this period, metabolic phenotype along with urinary albumin, creatinine, reactive oxygen species (ROS) and AGT, were measured every 4 weeks. In addition, for evidence in humans (age 19‐22 years‐old), 11 individuals with overweight and 17 individuals with normal weight, were studied. In these subjects, anthropometric parameters, systolic and diastolic BP, fasting blood glucose (FBG), and urinary mRNA levels of AGT and kidney injury markers IL‐18 and connecting tissue growth factor, were determined. Patients with history of cardiac or cerebrovascular events and endocrine diseases, anti‐hypertensive treatment (ACE inhibitors, ARBs) and/or anti‐diabetic drugs, were excludedResultsIn mice with metabolic alterations overtime, systolic BP significantly increased after 18 weeks in HFD but not NFD mice. Intrarenal ROS increased in HFD mice. These changes paralleled the augmentation uAGT excretion, which occurred in the absence of overt albuminuria. In human subjects with overweight, systolic and diastolic BP, urinary transcripts levels of AGT, renin, and injury markers IL‐18 and CTGF were increased as compared to normal weight individuals. However, no significant changes were observed in proteinuria or albuminuria.ConclusionsOur results indicate that augmentation of uAGT precedes albuminuria in young human adults with overweight and mice during the development of HFD‐induced T2D. These findings emphasize the relevance of uAGT and indicator of intrarenal/intratubular RAS activations and its potential as early biomarker of kidney injury during metabolic diseases as obesity and diabetes.

Divergent Anabolic Deficits and Pro‐inflammatory Effectors of Muscle Wasting in Xenograft and Metastatic Tumor Models of Colorectal Cancer

Colorectal cancer (CRC) is the second deadliest cancer in the United States, with ~70% of CRC cases developing liver metastases and cachexia. Here we investigated two tumor types (HCT116 and C26) as xenograft (x) or metastatic (m) models. Male Nod SCID gamma and CD2F1 mice were injected subcutaneously or intrasplenically with HCT116 or C26 tumor cells, respectively. CRC tumor growth was investigated at 30 days (xHCT116), 24 days (mHCT116), and 14 days (xC26 and mC26), and resulted in a significant gastrocnemius muscle depletion regardless of tumor model or type (xHCT116: 14.2%, mHCT116: 29.0%, xC26: 20.4%, mC26: 17.1%; P &lt; 0.05). mHCT116 tumors caused a reduction in ribosomal (r)RNA content (21.2%, P &lt; 0.05) and rDNA transcription (40%, P &lt; 0.001), but the mC26 tumor showed no loss of rRNA and the upregulation of rDNA transcription (&gt;2‐fold, P &lt; 0.001). All tumor‐bearing mice experienced a reduction in γ4E‐BP1 phosphorylation (xHCT116: 57.8%, mHCT116: 73.4%, xC26: 50.9%, mC26: 99.2%; P &lt; 0.05) suggesting impaired translational control. Muscle IL‐6 mRNA was elevated in all tumor‐bearing mice except xHCT116 (mHCT116: 1.8‐fold, xC26: 3.6‐fold, mC26: 4.1‐fold; P &lt; 0.05). TNF‐α mRNA was lower in xHCT116 (52.9%, P &lt; 0.05) and was not induced in the other tumor‐bearing mice. Expression of the NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3) was elevated only in mC26 muscle (5‐fold, P &lt; 0.0001). Neither IL‐18 nor IL‐1β mRNA levels were consistent with changes in NLRP3, however IL‐1β mRNA increased in both tumor models and types (xHCT116: 5.5‐fold, mHCT116: 10‐fold, xC26: 2.5‐fold, mC26: 3.5‐fold; P &lt; 0.05) with a greater expression in each metastatic relative to the xenograft model within each type of tumor. Our findings indicate that the mHCT116 tumor results in more drastic muscle depletion and anabolic deficits, while the mC26 exhibited a divergent pro‐inflammatory phenotype than the xenograft model. Our data highlight potentially important divergence in the pathogenesis of muscle wasting among the HCT116 and C26 pre‐clinical models of CRC.

Untargeted metabolomic profiling identifies disease-specific and outcome-related signatures in chronic rhinosinusitis

Although metabolomics provides novel insights into disease mechanisms and biomarkers, the metabolic alterations in local tissues affected by chronic rhinosinusitis (CRS) are unknown. This study aimed to determine the metabolomic profiles of sinonasal tissues associated with different types of CRS and their treatment outcomes. Untargeted metabolomic profiling was performed on sinonasal tissues obtained from patients with eosinophilic CRS with nasal polyps (CRSwNP), noneosinophilic CRSwNP or CRS without nasal polyps (CRSsNP), and controls. The messenger RNA (mRNA) levels of inflammatory cytokines in nasal tissues were detected by quantitative real-time reverse transcriptase PCR. Nasal polyp tissues were cultured exvivo and treated with glutathione. Distinct metabolomic profiles were observed for the CRS subtypes. Eosinophilic CRSwNP had profoundly enhanced unsaturated fatty acid oxidization, which correlated with mucosal eosinophil numbers and IL-5 mRNA levels. Noneosinophilic CRSwNP was characterized by uric acid accumulation. Increased uric acid levels were positively correlated with mucosal neutrophil numbers and IFN-γ, IL-17A, IL-1β, and IL-8 mRNA levels. Disrupted purine metabolism was specifically detected in CRSsNP. Reduced levels of amino acid metabolites were found in eosinophilic CRSwNP and CRSsNP, and were inversely associated with mucosal total inflammatory cell numbers and inflammatory cytokines. Compared to non-difficult-to-treat CRS, difficult-to-treat CRS had higher glutathione disulfide levels, which were positively correlated with IL-8 mRNA levels. Glutathione treatment reduced IL-8 mRNA expression in cultured nasal polyp tissues. Specific metabolic signatures are associated with different types of CRS, inflammatory patterns, and disease outcomes, which may provide novel insights into pathophysiologic mechanisms, subtype-specific biomarkers, and treatment targets of CRS.

Involvement of NLRP3/Caspase-1/GSDMD-Dependent pyroptosis in BPA-Induced apoptosis of human neuroblastoma cells

Bisphenol A (BPA) induces neurotoxicity via enhancing cell apoptosis and inflammation potently (effective at nanomolar concentrations), but its mechanisms remain unidentified. In this study, human neuroblastoma cell lines, IMR-32 and SK-N-SH cells, isolated from a male and a female subject, respectively, were exposed to BPA at various concentrations, with epigallocatechin gallate (EGCG, an antioxidant from green tea), Z-YVAD-FMK (a caspase-1 inhibitor), and ICI182.780 [an estrogen receptor (ER) inhibitor] as modulators. The results showed that BPA increased the mRNA levels of IL-18, ASC, GSDMD and protein levels of NLRP3, caspase-1 and GSDMD in both cell lines in a nonlinear manner. Noticeably, the direction of changes in the mRNA levels of caspase-1 and IL-1β were opposite, so did each of them in different cell lines: caspase-1 was enhanced in IMR-32 cells but suppressed in SK-N-SH cells, while IL-1β was suppressed in IMR-32 cells but enhanced in SK-N-SH cells. The level of GSDMD in situ increased along with the leakage of IL-1β, IL-18, caspase-1 and lactate dehydrogenase (LDH). Moreover, all the above effects of BPA were reversed by Z-YVAD-FMK, ICI182.780, and EGCG. Besides, BPA significantly increased reactive oxygen species production, LDH leakage and apoptosis, with reduced cell viability and mitochondrial membrane potential, in both cell lines, whereas Z-YVAD-FMK and ICI182.780 significantly alleviated the induction of Bak1, Bax, Bcl-2 and caspase-3 proteins by BPA. In summary, BPA may induce pyroptosis in neuroblastoma cells through NLRP3/caspase-1/GSDMD pathway, as mediated by ER; caspase-1-dependent pyroptosis may also contribute to BPA-induced apoptosis, an effect alleviated by EGCG.