mRNA Expression Of Inflammatory Cytokines Research Articles

Total glucosides of paeony (TGP) alleviates Sjogren's syndrome through inhibiting inflammatory responses in mice

BackgroundSjogren's syndrome (SS) is an inflammatory autoimmune disease whose etiology is complicated. Total glucosides of paeony (TGP) has a variety of pharmacological effects. PurposeTo evaluate the therapeutic effects of TGP on SS in mice and anti-inflammatory mechanism. Study designSS animal model was developed from C57BL/6J mice through immunological induction (SS mice) and NOD/ShiltJNju (NOD) mice. Inflammatory cytokines and other related indicators were measured. MethodsTGP (720, 360, 180 mg/kg) was intragastrically administered for 6 or 16 weeks for SS mice and NOD mice, respectively. Average food and water intake, average body weight, saliva flow, submandibular gland (SMG) and spleen index, and SMG pathology were measured. ELISA was used to evaluate serum inflammatory cytokines in SS mice and autoantigens in NOD mice. Real-time PCR, Western blot and Luminex liquid suspension chip assay were applied to analyze SMG inflammatory cytokines mRNA and protein expression of NOD mice. ResultsCompared with SS mice, TGP treatment improved SMG pathological damage. TGP (720 mg/kg) treatment increased saliva flow, and reduced organ indexes and serum IL-6 and IFN-γ concentration. TGP (360 mg/kg) treatment decreased serum IFN-γ concentration. TGP (180 mg/kg) treatment for 6 weeks decreased average body weight.Compared with NOD mice, TGP treatment increased saliva flow from 9 to 15 weeks, decreased body weight, and alleviated pathological damage of SMG after 2 and 16 weeks. After 2 weeks of administration, TGP treatment inhibited serum concentration of SSB/La, SSA/Ro and α-fodrin, decreased TNF-α, IL-1β and IFN-γ in SMG, and down-regulated protein expressions of BAFF and IL-17A and mRNA expressions of BAFF, TNF-α, IL-17A, CXCL9 and CXCL13 in SMG. After 8 weeks of administration, TGP treatment decreased the concentration of α-fodrin in serum, TNF-α and IL-6 in SMG, and down-regulated mRNA expressions of IL-17A, TNF-α, CXCL9, CXCL13 and BAFF and protein expressions of IL-17A and BAFF in SMG. After 16 weeks of administration, TGP treatment reduced serum SSA/Ro, SSB/La and α-fodrin concentration, and decreased BAFF protein expression and TNF-α, CXCL9, CXCL13, IL-17A, and BAFF mRNA expressions. ConclusionTGP has a certain therapeutic effect on SS mice and NOD mice through inhibiting inflammatory responses.

Oral glutathione administration inhibits the oxidative stress and the inflammatory responses in AppNL−G-F/NL−G-F knock-in mice

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by the presence of extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles. Reduced antioxidants and increased oxidative stress and inflammation are responsible for the pathological features characteristic of an AD brain. We observed decreased levels of the reduced form of glutathione (GSH), the most abundant brain antioxidant, and decreased GSH/glutathione disulfide (GSSG) ratios in AppNL−G-F/NL−G-F knock-in (NL-G-F) mouse brains. Repeated oral GSH administration for 3 weeks dose-dependently increased GSH levels and restored the GSH/GSSH ratio. Consistent with the restoration of GSH levels, the levels of 4-hydroxy-2-nonenal (4-HNE), a marker of oxidative stress, were significantly decreased in the hippocampus of NL-G-F mice. Additionally, inflammatory responses, such as microgliosis and increased mRNA expression of inflammatory cytokines, were also inhibited. Moreover, behavioral deficits including cognitive decline, depressive-like behaviors, and anxiety-related behaviors observed in NL-G-F mice were significantly improved by oral and chronic GSH administration. Taken together, our data suggest that oral GSH administration is an attractive therapeutic strategy to reduce the excessive oxidative stress and inflammatory responses in the AD brain.

SUN-097 APOA1-MIMETIC PEPTIDES ENHANCE CELLULAR CLEARANCE OF CALCIPROTEIN PARTICLES AND ATTENUATE INFLAMMATORY PRIMING

Calciprotein particles (CPP) - colloidal nanoaggregates of calcium phosphate mineral, proteins and other biomolecules - have emerged as potential mediators of pro-inflammatory mineral stress in CKD. Transformation of CPP to the crystalline state (secondary CPP), as favoured by uraemia, enhances their inflammatory potential due to an enrichment of endogenous and bacteria-derived cargos that are able to ligate the innate immune sensor, Toll-like receptor 4 (TLR4). Unless overwhelmed, rapid removal of particles from the cell surface by scavenger receptor (SR)-A-mediated endocytosis restrains activation of TLR4 and licensing of downstream inflammatory cascades. Strategies aimed at augmenting immunologically-inert clearance of CPP by SR-A may abrogate the reaction to chronic mineral excess and lessen the inflammatory burden in CKD. However, in order to test whether exploiting such pathways may be beneficial, the native ligand for SR-A needed to be identified. We used a combination of genetic and biochemical approaches to elucidate the ligand for SR-A-mediated endocytosis of secondary CPP. Ligand-depleted sera were generated by immunoaffinity chromatography. CPP were synthesized in uraemic human serum supplemented with supersaturating concentrations of calcium and phosphate salts, isolated by gel filtration chromatography and dosed according to concentrations determined by nanoparticle tracking analysis (NTA). We used flow cytometry and live-cell confocal imaging to monitor the binding and internalisation of fluorescently-labelled CPP in BM-derived WT and SR-A-deficient murine macrophages as well as in human lines in which either SR-A or TLR4 had been deleted using CRISPR/Cas9 technology. A peptide mimetic of the ligand was synthesised along with scrambled control peptides. NFkB induction was evaluated using a luciferase reporter and inflammatory cytokine mRNA expression by qPCR. Ripening of CPP crystalline state is associated with an enrichment of Apo-A1. Exposure of human macrophage to labelled CPP synthesised in serum depleted of Apo-A1, resulted in an 80% decrease in cell-associated fluorescence relative to CPP from Apo-A1-replete serum. A similar reduction in uptake of Apo-A1-replete CPP2 was observed in macrophage derived from SR-A-/- mice and SR-A knockout human macrophage relative to their respective wild-type controls. Two patients with genetically confirmed Apo-A1 deficiency were found to have modestly elevated serum CPP levels compared to age- and gender-matched controls (n=20), but a markedly increased CPP-2 to CPP-1 ratio, consistent with an important role for Apo-A1 in CPP2 metabolism. Compared with Apo-A1-replete CPP2, those synthesised from Apo-A1-depleted, uraemic serum induced greater NFkB induction and cytokine mRNA levels (TNF-alpha & IL-6) at equivalent doses, but not in TLR4-knockout cells. Addition of an 18mer Apo-A1 mimetic peptide dose-dependently increased CPP2 uptake in macrophage, but this effect was absent in murine and human cells lacking SR-A. Reconstitution of Apo-A1-deplted CPP2 with mimetic peptide restored uptake and inhibited inflammatory signalling. Therapeutic enhancement of SR-A-dependent CPP uptake using Apo-A1 mimetics may help ameliorate the inflammatory response to chronic mineral dysregulation∖

The role of nitric oxide in small intestine differs between a single and a consecutive administration of methotrexate to rats

The role of nitric oxide (NO) on intestinal mucosal injury induced by single or consecutive administration of methotrexate was investigated in a rodent model. Rats received methotrexate intraperitoneally either as a single administration (50 mg/kg) or as a consecutive administration (12.5 mg/kg/day) for 4 days. NG-nitro-l-arginine methyl ester (L-NAME) was given subcutaneously to inhibit NO synthase (NOS). Ninety-six hours after the first administration of methotrexate, ileal tissues were collected for analysis. Consecutive administration of methotrexate led to decreased body weight and reduced intake of food and water, which were further worsened by L-NAME. Although a slight mucosal injury resulted from single administration of methotrexate, L-NAME had almost no effect. Consecutive administration of methotrexate caused a significant mucosal injury, which was further worsened by L-NAME. Consecutive, but not single, administration of methotrexate induced mRNA expression of inflammatory cytokines in ileal tissue. Consecutive administration of methotrexate significantly induced constitutive NOS expression in ileal tissue. These results suggest that consecutive administration, rather than single administration, of methotrexate aggravates mucosal injury. Potentiation of constitutive NOS expression by consecutive administration might be one of the main reason to antagonize the intestinal mucosal injury as well as lead to a reduction in rat quality of life.

Abstract 144: Roles of Aging and Cellular Senescence in Intracranial Aneurysm Rupture

Background: Aging is an independent risk factor for the rupture of intracranial aneurysm. One of the hallmarks of aging is chronic tissue inflammation. Sirtuin-1 keeps inflammation in check through the deacetylation of various proteins. It is well known that the levels of Sirtuin-1 in vascular tissues decrease with aging, resulting in chronic vascular inflammation. Age-dependent decrease in Sirtuin-1 may explain the link between aging and increased risk for aneurysmal rupture. Hypothesis: Reduction of Sirtuin-1 promotes aneurysmal rupture by inducing sustained aneurysmal wall inflammation. Methods: First, we assessed the levels of Sirtuin-1 expression in intracranial aneurysm tissues from patients older than 70 y.o. and compared with those from the younger patients (40 to 50 y.o.). Second, using a mouse model, we tested effects of Sirtuin-1 specific activator SRT1720 (15mg/kg/day) and specific inhibitor EX-527 (2.5mg/kg/day) on the development of aneurysmal rupture. In addition, we assessed the mRNA expression of inflammatory cytokines (IL-6, IL-1beta, MCP-1, and MMP-9) in cerebral arteries and aneurysms in mice treated with vehicle, SRT1720, or EX-527. Results: Sirtuin-1 expression levels in intracranial aneurysm tissues from the older patients were lower than those from the younger patients. The pharmacological inhibition of Sirtuin-1 increased rupture rate in mice (vehicle vs. EX-527: 58% vs. 88%, P <0.05). Moreover, the pharmacological activation of Sirtuin-1 reduced rupture rate in mice (vehicle vs. SRT1720: 80% vs. 50%, P <0.05). Levels of IL-6, MMP-9 mRNAs in cerebral arteries were significantly higher in the inhibitor group than in the vehicle group. On the other hand, both mRNA levels were lower in the activator group than in the vehicle group. Conclusions: Our findings suggest that the reduction of Sirtuin-1 promotes aneurysmal rupture via the induction of inflammation. This may explain the increased risk for aneurysmal subarachnoid hemorrhage in the older population. Our findings may become a basis for future studies to develop new therapies that target Sirtuin-1 for the prevention of aneurysmal rupture, especially in older patients.

Sodium absorption stimulator prostasin (PRSS8) has an anti-inflammatory effect via downregulation of TLR4 signaling in inflammatory bowel disease.

Prostasin (PRSS8) is a stimulator of epithelial sodium transport. In this study, we evaluated alteration of prostasin expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and investigated the role of prostasin in the gut inflammation. Prostasin expression was evaluated by immunohistochemical staining. Dextran sodium sulfate (DSS)-colitis was induced in mice lacking prostasin specifically in intestinal epithelial cells (PRSS8ΔIEC mice). In colonic mucosa of healthy individuals, prostasin was strongly expressed at the apical surfaces of epithelial cells, and this was markedly decreased in active mucosa of both ulcerative colitis and Crohn's disease. DSS-colitis was exacerbated in PRSS8ΔIEC mice compared to control PRSS8lox/lox mice. Toll-like receptor4 (TLR4) expression in colonic epithelial cells was stronger in DSS-treated PRSS8ΔIEC mice than in DSS-treated PRSS8 lox/lox mice. NF-κB activation in colonic epithelial cells was more pronounced in DSS-treated PRSS8ΔIEC mice than in DSS-treated PRSS8lox/lox mice, and the mRNA expression of inflammatory cytokines was significantly higher in DSS-treated PRSS8ΔIEC mice. Broad-spectrum antibiotic treatment completely suppressed the exacerbation of DSS-colitis in PRSS8ΔIEC mice. The mRNA expression of tight junction proteins and mucosal permeability assessed using FITC-dextran were comparable between DSS-treated PRSS8lox/lox and DSS-treated PRSS8ΔIEC mice. Prostasin has an anti-inflammatory effect via downregulation of TLR4 expression in colonic epithelial cells. Reduced prostasin expression in IBD mucosa is linked to the deterioration of local anti-inflammatory activity and may contribute to the persistence of mucosal inflammation.

Inhibition of the PI3K-AKT-mTOR pathway suppresses the adipocyte-mediated proliferation and migration of breast cancer cells.

Objective: Although it is well known that adipocyte significantly affects breast cancer progression, its mechanism has not been fully understood. Here, we analyzed the effect of adipocytes on breast cancer progression including cell proliferation and migration.Materials and Methods: We treated the conditioned media obtained from mouse 3T3-L1-derived or human adipose tissue-derived mesenchymal stem cells (hAMSC)-derived adipocytes to breast cancer cells, MCF-7 and MDA-MB-231. And then, cells viability and proliferation were analyzed using MTT assays and colony forming assays, respectively. Also mRNA expression of inflammatory cytokines and proteins expression in main signal pathway were analyzed by RT-qPCR and immunoblotting, respectively.Results: Adipocyte-derived conditioned media increased the proliferation and migration of MCF-7 and MDA-MB-231 cells while little effects in a human normal immortalized mammary epithelial cell line MCF10A. In addition, adipocyte-derived conditioned media induced phosphorylation of AKT and mTOR and upregulated the expression of target genes of the PI3K-AKT-mTOR pathway including IL6, IL1β, IL1α and TNFα in breast cancer cells. Furthermore, BEZ235 a dual inhibitor of PI3K and mTOR significantly decreased the adipocyte-mediated the proliferation and migration of breast cancer cells.Conclusion: Adipocyte-derived conditioned media enhance the proliferation and migration of breast cancer cells through the PI3K-AKT-mTOR pathway, supporting the importance of heterotypic interactions between breast cancer cells and adipocytes in the tumor microenvironment.

Anti-inflammatory Effects of Extracts of Sweet Basil (Ocimum basilicum L.) on a Co-culture of 3T3-L1 Adipocytes and RAW264.7 Macrophages.

Obesity, a lifestyle disease resulting from excessive caloric intake and insufficient physical activity, results in a state of chronic inflammation. A food ingredient that suppresses chronic inflammation could help prevent associated diseases. Sweet basil (Ocimum basilicum L.) is a herb from the Lamiaceae family with some reported anti-inflammatory effects. Via this in vitro study, we aimed to investigate whether sweet basil exerts anti-inflammatory effects in obese patients. Fresh sweet basil leaves were freeze-dried and powered. After that, this was extracted with 80% methanol. After 3T3-L1 adipocytes were cultured with sweet basil extracts at final concentrations of either 5 or 25 μg/mL for 24h, RAW264.7 macrophages were seeded onto this adipocytes and co-cultured for 12h. We determined the effects of sweet basil extracts on inflammatory cytokine expression by real-time PCR or western blotting. Sweet basil extracts reduced the expression of inflammatory cytokine mRNA induced by co-culture, including that of IL-6 (Il6), IL-1β (Il1b), TNF-α (Tnf), and CCL2 (Ccl2). In addition, sweet basil extracts suppressed the mRNA expression of NF-κB (Nfκb1), a transcription factor of inflammatory cytokines. In an investigation of costimulatory CD137 (Tnfrsf9)/CD137L inflammatory signaling, a member of the TNF super-family, sweet basil extracts inhibited Tnfrsf9 expression induced by the co-culture. Therefore, the results of this study indicated that sweet basil extracts have an anti-inflammatory effect against adipocyte-induced inflammation, possibly through suppression of Tnfrsf9 expression.

Role of promoting inflammation of Krüppel-like factor 6 in acute kidney injury

Background Krüppel-like factor 6 (KLF6) is a transcription factor that participate in various pathophysiological processes, but its contribution in ischemia acute kidney injury (AKI) is lacking so far. The study aimed to investigate the expression and the role of KLF6 in kidney ischemia–reperfusion (IR) injury. Method Microarray data were collected from GSE58438 and GSE52004. The rat IR model was established to evaluate the mRNA and protein expression of KLF6 and inflammatory cytokines in serum and kidney tissues. SiRNA-KLF6 was transfected with HK-2 cells, and then a cell-based hypoxia-reoxygenation (HR) model was established. Results Bioinformatics showed KLF6 mRNA in kidney tissue is up-regulated in 3 h after IR in rat kidney, which involved in cell activation, leukocyte activation, and response to hydrogen peroxide after IR. The rat IR model results showed that KLF6 expression was peaking at 6 h, and the expression of pro-inflammatory cytokines MCP-1 and TNF-α was increased both in serum and kidney tissues, while anti-inflammatory cytokine IL-10 was decreased after IR. Furthermore, in vitro results showed that KLF6 knock-down reduced the pro-inflammatory cytokines expression. Conclusion These results suggest that (1) KLF6 might be a novel biomarker for early diagnosis of AKI and (2) KLF6 may play a role in promoting inflammation in AKI.

The role of zinc chelate of hydroxy analogue of methionine in cadmium toxicity: effects on cadmium absorption on intestinal health in piglets

Cadmium (Cd) is a toxic heavy metal that poses a threat to the health of humans and animals. It can cause serious damage to the small intestine, which is the main absorption site of Cd and the primary target organ after oral administration. Our previous study found that zinc chelate of hydroxy analogue of methionine (Zn-HMTB), a new type of feed additive, decreased Cd accumulation in the liver and kidneys. The aim of this study was to investigate the effect of Zn-HMTB on Cd absorption and Cd-induced toxicity in the small intestine of piglets. Twenty-four piglets (Landrace × Large White, 13.22 ± 0.58 kg BW) were randomly divided into four dietary treatment groups: basal diet, and diets containing 30 mg/kg Cd from CdCl2 and 0, 100 or 200 mg/kg Zn from Zn-HMTB. The experiment lasted 27 days. The feed intake and final BW of each piglet were recorded at the end of the experiment. Gastrointestinal (GI) tract tissue and samples of liver, kidney, spleen, heart, lung and longissimus muscle tissue and faeces were collected. The concentrations of Cd and metal trace elements in the GI tract and organs were analysed, as was the relative messenger RNA (mRNA) expression of inflammatory cytokines and metal element transporters in the small intestine, and epithelial apoptosis in the small intestine. The results showed that, compared with Cd-treated piglets, piglets in the Zn-HMTB and Cd cotreatment groups had less Cd deposition in the stomach, ileum, caecum, colon, liver, kidneys, spleen, lungs, heart and muscles (P < 0.05), and lower Cd concentrations in faeces (P < 0.05), suggesting that Zn-HMTB increased Cd absorption and the excretion of Cd in other forms (possibly urine). Zinc chelate of hydroxy analogue of methionine increased Zn deposition in the jejunum and the relative mRNA expression of divalent metal transporters 1 and zinc transporter 5 in the duodenum (P < 0.05), indicating that Zn-HMTB may promote the absorption and transportation of Cd and Zn together by upregulating metal element transporters. Competition between Zn and Cd may be responsible for accelerating Cd excretion. Furthermore, Zn-HMTB reduced Cd-induced apoptosis of enterocytes and inflammatory stimuli in the small intestine, suggesting that Zn-HMTB reduced Cd-induced toxicity to the small intestine. These results suggest that Zn-HMTB can be helpful in decreasing Cd accumulation in the GI tract and organs of piglets and relieving Cd-induced toxicity to the small intestine but cannot reduce the absorption of Cd.

Candesartan modulates microglia activation and polarization via NF-κB signaling pathway.

Microglia are diverse cells that acquire different functional phenotypes in response to microenvironment in which they reside. Several transcriptional regulators have been identified that regulate different microglia phenotypes. They are mainly stimulated into two opposing phenotypes, classically (M1) and alternatively (M2) phenotype. Regulating microglia polarization from M1 to M2 state has been suggested as a potential therapeutic approach in treatment of CNS disorders. Candesartan, an angiotensin II type I receptors antagonist, exerts beneficial effects for antioxidant, anti-inflammation, neurotrophic, and anti-apoptotic function. However, the effect of candesartan on microglia polarization and underlying mechanisms remain unknown. In this study, the resting microglia were stimulated to M1 microglia with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), and then treated with vehicle or candesartan for 24 h. RT-PCR was utilized to detect the mRNA expression of microglia phenotype markers and inflammatory cytokines. Microglia phenotype markers and toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway were determined by western blot. A neuron-microglia co-culture system was used to determine whether candesartan could ameliorate the neurotoxic effect of M1 microglia to oxygen-glucose deprivation (OGD) neuron. Candesartan treatment reduced the expression of M1 markers, and increased M2 markers. Meanwhile, candesartan reduced fluorescence intensity and protein level of M1 marker and enhanced M2 marker. Candesartan also regulated the neuroinflammatory response via reducing the release of pro-inflammatory cytokines and increasing anti-inflammatory cytokines in LPS + IFN-γ stimulated BV2 cells. Candesartan markedly inhibited the protein level of TLR4, the phosphorylation of IKBα and p65, and suppressed nuclear translocation of NF-κB p65. BAY 11-7085, a NF-κB inhibitor, remarkably enlarged the inhibitory effect of candesartan on NF-κB pathway. In addition, M1 phenotype microglia exacerbated post-OGD N2a cells death and LDH release, whereas candesartan reversed such neurotoxic effect. Candesartan treatment may ameliorate stroke-induced neuronal damage through shifting microglia to M2 phenotype in a TLR4/NF-κB-dependent manner.

TRAF6-Mediated Inflammatory Cytokines Secretion in LPS-induced Colorectal Cancer Cells Is Regulated by miR-140.

Colorectal cancer (CRC) cells secrete inflammatory cytokines that affect CRC progression. The aim of the present study was to determine if micro-RNA-140(miR-140) regulates inflammatory cytokine secretion induced by lipopolysaccharide (LPS) in colorectal cancer cells by targeting tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Fifty fresh colon-cancer specimens and normal colorectal tissues were collected from patients with CRC and tested for the expression miR-140. Human CRC cell lines SW480 and HCT116 were treated with various concentrations and times with LPS. miR-140 and mRNA expression of potentially related genes were analyzed by qPCR. Protein expression was analyzed using western blot or ELISA. Overexpression plasmids with pcDNA3.1-TRAF6, pGL4.10-wtTRAF6 and pGL4.10-mutTRAF6 were constructed. miRNA target gene prediction and a dual luciferase assay were used to analyze miR-140-targeted TRAF6. miR-140 expression was up-regulated in CRC tissues. In CRC cells, LPS could increase miR-140 expression in a time- and concentration-dependent manner. LPS increased inflammatory cytokine mRNA expression levels in SW480 and HCT116 human colon-cancer cells. miRNA-140 suppressed TRAF6 expression via targeting the 3'UTR. TRAF6 affected miR-140-mediated inflammatory cytokine expression of SW480 and HCT116 cells under LPS treatment. miR-140 regulates inflammatory cytokine secretion of LPS-induced colorectal cancer cells by targeting TRAF6.

Repeated measures of prenatal thallium exposure and placental inflammatory cytokine mRNA expression: The Ma'anshan birth cohort (MABC) study.

Thallium (Tl), a ubiquitous environmental toxicant, can cross the placental barrier during pregnancy. However, the effects of prenatal Tl exposure on placental function are currently unclear. Based on the Ma'anshan Birth Cohort study, we examined whether long-term prenatal Tl exposure was associated with placental inflammation. Tl concentrations were quantified in serum samples (n=7050) from 2515 pregnancy during each trimester, placental inflammatory cytokine mRNA expression was assessed in 2519 placenta tissues. Geometric mean values of serum Tl concentrations were 63.57, 63.63 and 48.71ng/L for the first, second and third trimesters, respectively. After adjustment for potential confounders, serum Tl concentration was positively associated with CD68 (β: 0.30; 95% CI: 0.05, 0.56) in the first trimester and TNF-α (β: 0.12; 95% CI: 0.01, 0.23), IL-6 (β: 0.15; 95% CI: 0.05, 0.25) and CD68 (β: 0.25; 95% CI: 0.10, 0.39) in the third trimester, however was negatively associated with IL-4 (β:-0.21; 95% CI:-0.41,-0.01) and CD206 (β:-0.23; 95% CI:-0.45,-0.02) in the first trimester. Repeated measures analysis showed that TNF-α, IL-6 and CD68 increased by 0.11 (95% CI: 0.01, 0.21), 0.12 (0.15, 95% CI: 0.05, 0.25), 0.22 (95% CI: 0.10, 0.39), respectively, with each 1ln-transformed Tl increase in total samples. Gender-specific analyses revealed that these associations were largely driven by male offspring. In addition, immunohistochemistry revealed that nuclear NF-κB p65 expression increased in placenta tissue. The results of this prospective cohort study provide longitudinal evidence that prenatal Tl exposure induces a placental inflammatory response in the Chinese population.

The protective effects of resveratrol on antioxidant function and the mRNA expression of inflammatory cytokines in the ovaries of hens with fatty liver hemorrhagic syndrome

To investigate the etiopathogenesis of fatty liver hemorrhagic syndrome (FLHS) and the protective effects of resveratrol (RSV) against FLHS in laying hens, 144 healthy 90-day-old laying hens were randomly divided into 4 groups including control (Con) group, high-energy low-protein (HELP) group, RSV group, and HELP + RSV group, each of which contained 36 hens with 3 replicates. Birds in the 4 groups were fed a basal diet, HELP diet, basal diet supplemented with 400 mg/kg RSV, and HELP diet supplemented with 400 mg/kg RSV. The histopathology of the ovary lesions on day 120, egg production, antioxidative function, and mRNA expression levels of inflammatory cytokines on days 40, 80, and 120 were determined. The lipid accumulation and hemorrhaging were more severe in the HELP group than those in the HELP + RSV group. The laying rate was markedly decreased in the HELP group compared with that in the Con and HELP + RSV groups. Furthermore, the malondialdehyde concentration was significantly increased (P < 0.05), while the levels of superoxide dismutase (SOD), catalase, and glutathione were significantly decreased (P < 0.05) in the HELP group compared with those in the Con and HELP + RSV groups. The mRNA levels of antioxidant genes (Nrf2, SOD-1, and HO-1) were markedly increased (P < 0.05) in the HELP + RSV group compared with those in the HELP group. In addition, the mRNA levels of inflammation-related genes (nuclear factor kappa B, tumor necrosis factor-α, IL-1β, and IL-6) were significantly increased (P < 0.05) in the HELP group compared with those in the Con and HELP + RSV groups. Collectively, these results indicate that oxidative stress and inflammation are involved in the occurrence and development of FLHS in the ovaries of laying hens, but RSV effectively attenuates oxidative stress and inflammation in hens with FLHS. Hence, RSV can be used as an effective feed additive to protect against FLHS.

Surface-exposed loops L7 and L8 of Haemophilus (Glaesserella) parasuis OmpP2 contribute to the expression of proinflammatory cytokines in porcine alveolar macrophages

Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.

Alternative Virus-Like Particle-Associated Prefusion F Proteins as Maternal Vaccines for Respiratory Syncytial Virus.

Maternal vaccination may be the most effective and safest approach to the protection of infants from respiratory syncytial virus (RSV) infection, a severe acute lower respiratory tract disease in infants and young children worldwide. We previously compared five different virus-like particle (VLP)-associated, mutation-stabilized prefusion F (pre-F) proteins, including the prototype DS-Cav1 F VLPs. We showed that alternative versions of prefusion F proteins have different conformations and induce different populations of anti-F protein antibodies. Two of these alternative pre-F VLPs, the UC-2 F and UC-3 F VLPs, stimulated in mice higher titers of neutralizing antibodies than DS-Cav1 F VLPs (M. L. Cullen, R. M. Schmidt, M. G. Torres, A. A. Capoferri, et al., Vaccines 7:21-41, 2019, https://doi.org/10.3390/vaccines7010021). Here we describe a comparison of these two pre-F VLPs with DS-Cav1 F VLPs as maternal vaccines in cotton rats and report that UC-3 F VLPs significantly increased the neutralizing antibody (NAb) titers in pregnant dams compared to DS-Cav1 F VLPs. The neutralizing antibody titers in the sera of the offspring of the dams immunized with UC-3 F VLPs were significantly higher than those in the sera of the offspring of dams immunized with DS-Cav1 VLPs. This increase in serum NAb titers translated to a 6- to 40-fold lower virus titer in the lungs of the RSV-challenged offspring of dams immunized with UC-3 F VLPs than in the lungs of the RSV-challenged offspring of dams immunized with DS-Cav1 F VLPs. Importantly, the offspring of UC-3 F VLP-immunized dams showed significant protection from lung pathology and from induction of inflammatory lung cytokine mRNA expression after RSV challenge. Immunization with UC-3 F VLPs also induced durable levels of high-titer neutralizing antibodies in dams.IMPORTANCE Respiratory syncytial virus (RSV) is a significant human pathogen severely impacting neonates and young children, but no vaccine exists to protect this vulnerable population. Furthermore, direct vaccination of neonates is likely ineffective due to the immaturity of their immune system, and neonate immunization is potentially unsafe. Maternal vaccination may be the best and safest approach to the protection of neonates through the passive transfer of maternal neutralizing antibodies in utero to the fetus after maternal immunization. Here we report that immunization of pregnant cotton rats, a surrogate model for human maternal immunization, with novel RSV virus-like particle (VLP) vaccine candidates containing stabilized prefusion RSV F proteins provides significant levels of protection of the offspring of immunized dams from RSV challenge. We also found that antibodies induced by VLPs containing different versions of the prefusion F protein varied by 40-fold in the extent of protection provided to the offspring of vaccinated dams upon RSV challenge.

Morin alleviates LPS-induced mastitis by inhibiting the PI3K/AKT, MAPK, NF-κB and NLRP3 signaling pathway and protecting the integrity of blood-milk barrier

Mastitis is a common veterinary clinical disease that restricts the development of dairy farming around the world. Morin, extracted from Mulberry Tree and other herbs, has been reported to possess the function of anti-bacteria, anti-oxidant, and anti-inflammatory. However, whether morin could protect lipopolysaccharide (LPS)-induced mouse mastitis in vivo has not well known. This study firstly aims to evaluate the effects of morin on LPS-induced mouse mastitis in vivo, and then try to illustrate the mechanism involved in the process. Before injected with LPS, mice were intraperitoneally pre-injected with different concentrations of morin, and mice of the control and LPS group were injected with the same amount of saline. Pathologic changes of mammary gland were determined by histopathological examination. Myeloperoxidase (MPO) activities of mammary gland were determined by the MPO kits. The mRNA expressions of inflammatory cytokines including TNF-α, IL-1β and IL-6, and those of chemokine factors CCL2 and CXCL2, and those of tight junctions occludin claudin-3 were examined by qRT-PCR analysis. The activities of IκB, p65, ERK, P38, AKT, PI3K, NLPR3, claudin-1, claudin-3 and occludin were determined by western blotting. The results showed that morin alleviated LPS-induced edema, destructed structures and infiltrated inflammatory cells of mammary gland. Morin administration significantly decreased LPS-induced TNF-α, IL-1β, IL-6, CCL2 and CXCL2 mRNA expressions. Furthermore, western blot analysis also showed that morin significantly reduced LPS-induced phosphorylation of p65, IκB, p38 and ERK, and enhanced LPS-induced phosphorylation of AKT and PI3K. It was also found that LPS-decreased claudin-3 and occludin expressions were also inhibited by morin treatment. In summary, above results suggest that morin indeed protect LPS-induced mouse mastitis in vivo, and the mechanism was through inhibiting the PI3K/AKT, MAPK, NF-κB and NLRP3 signaling pathways and protecting the integrity of blood-milk barrier by regulating the tight junction proteins expressions.

ANGIOTENSIN (1-7) EXPRESSING LACTOBACILLUS DOSE-DEPENDENTLY BENEFITS THE GUT-BRAIN AXIS IN AGED RATS

Aging is associated with gut dysbiosis – a condition linked with altered central nervous system function (i.e the “gut-brain axis”). Age-related health benefits have been ascribed to the renin-angiotensin system (RAS), mediated partially via the angiotensin (1-7) or Ang(1-7) axis. This pre-clinical study explored dosing of a genetically modified probiotic expressing Ang(1-7) – which we previously showed to induce dose-dependent increases in circulating Ang(1-7) – in modulating the gut-brain axis. Twenty-nine male F344BN rats were randomized at 24 months of age to receive oral gavage of Ang(1-7) Lactobacillus paracasei (LP) zero (control), one, three, or seven times/week over 28 days. At day 29, samples of feces, serum and pre-frontal cortex (PFC) were collected. Microbiome taxonomic analysis of fecal samples was performed via 16S-based PCR. Serum samples were analyzed for tryptophan and downstream metabolites via LC-MS. PFC was evaluated for mRNA expression of select inflammatory cytokines. PCoA revealed that groups differed in the overall fecal microbiota community structure as determined by Unweighted UniFrac. Indices of alpha-diversity, including richness and phylogenetic diversity, displayed significant group differences – with the most dramatic effects observed in the 3-times/week group. Compared to control, serum serotonin and 2-Picolinic Acid were significantly increased in the 3-times/week group. The 3-times/week regimen also significantly reduced COX2, IL1β, and TNFα mRNA expression, and 7-times/week reduced COX2 and IL1β expression in PFC. Therefore, we conclude that short-term treatment with Ang(1-7) LP dose-dependently benefits the gut-brain axis in aged rats, with 3-times/week appearing to be the optimal dosing regimen.

Interleukin‑37 is increased in peripheral blood mononuclear cells of coronary heart disease patients and inhibits the inflammatory reaction.

It has been universally acknowledged that interleukin-37 (IL-37) has an immunosuppressive effect on various inflammatory disorders. However, whether IL-37 participates in the acute inflammation associated with coronary heart disease (CHD) has not yet been clarified. In the present study, the association between the serum levels of IL-37 and the clinical indexes of CHD were analysed. In addition, the anti-inflammatory effects of IL-37 on peripheral blood mononuclear cells (PBMCs) were studied in CHD patients. PBMCs from 46 healthy controls (HCs) and 92 CHD patients were cultured in vitro and stimulated using the recombinant IL-37 protein. The protein levels, as well as the mRNA expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-17) were analysed by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR). Spearman's correlation test was performed to examine the association between the serum level of IL-37 and the levels of pro-inflammatory cytokines, certain clinical indexes, and disease activity during CHD. Compared to the HCs, the CHD patients, especially those with acute myocardial infarction, exhibited higher levels of IL-37 in their PBMCs and sera. Serum levels of IL-37 were associated with the levels of IL-17, IL-6, and TNF-α, and clinical indexes such as the left ventricular ejection fraction (LVEF), amino-N-terminal pro-plasma brain natriuretic peptide (NT-proBNP) levels, and cardiac troponin T (cTnT) levels in CHD patients. Compared to the HC group, the production of inflammatory cytokines such as IL-17, IL-6, TNF-α, and IL-1β increased in the PBMCs of CHD patients and significantly decreased after the stimulation of the cells with recombinant IL-37. The IL-37 levels in CHD patients were high, and were correlated with the levels of CHD-related pro-inflammatory cytokines and disease activity. Notably, the expression of CHD-related pro-inflammatory cytokines in the PBMCs of CHD patients decreased following the stimulation of the cells with recombinant IL-37, indicating that IL-37 exerts anti-inflammatory effects during CHD.

Effects of vitamin B6 on growth, diarrhea rate, intestinal morphology, function, and inflammatory factors expression in a high-protein diet fed to weaned piglets1.

Vitamin B6 (VB6) is an important coenzyme factor which participates in many metabolic reactions, especially amino acid metabolism. There are few reports on how VB6 mediates weaned piglet intestinal health. This study purposed to investigate dietary VB6 effects on growth, diarrhea rates, and intestinal morphology and function in weaned piglets fed a high-crude protein (22% CP) diet. Eighteen 21-d-old weaned [(Yorkshire × Landrace) × Duroc] piglets with body weights of 7.03 ± 0.15 (means ± SEM) kg were randomly assigned into 3 VB6-containing dietary treatments. Vitamin B6 content was: 0, 4, and 7 mg/kg, respectively. The feeding period lasted 14 d. The results showed that no significant difference existed for the growth performance. The 7 mg/kg VB6 group had a tendency to decrease diarrhea rate (P = 0.065). Blood biochemical parameters analysis demonstrated that total protein, cholesterol, and high-density lipoprotein significantly increased in the 7 mg/kg VB6 group (P < 0.05). In the jejunum, no significant differences were detected for villus height, villus width, crypt depth, villus height and crypt depth ratios, and positive Ki67 counts and the mRNA expression of inflammatory cytokines. Vitamin B6 significantly increased the mRNA expression of SLC6A19 and SLC6A20 (P < 0.05) and decreased the mRNA expression of SLC36A1 (P < 0.05). In the ileum, VB6 significantly increased villus height and villus width (P < 0.05) while decreased positive Ki67 cell counts for 7 mg/kg VB6 group (P < 0.05). Vitamin B6 had significantly increased the mRNA expression of interleukin-1β, tumor necrosis factor-α,cyclo-oxygen-ase-2, and transforming growth factor-β (P < 0.05). Vitamin B6 also had significantly increased mRNA expression of SLC6A19, SLC7A6, SLC7A7, and SLC36A1 (P < 0.05). These findings suggest that dietary supplementation with VB6 may affect the intestinal morphology and absorption and metabolism of protein in weaned piglets fed a high-protein diet by altering the expression of intestinal inflammatory cytokines and amino acid transporters.