iNOS mRNA Expression Research Articles

Abstract PD3-01: Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer

Abstract Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in TNBC patients. Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway is one of the most common oncogenic aberrations in TNBC. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). This study investigates the role of NO in PI3K-Akt pathway activation in human TNBC cells, in conditions with extracellular NO donor exposure and increased intracellular iNOS (NOS2) expression. TNBC cell lines BT549, HCC1937, Hs578T, SUM159, MDA-MB-231, and BT-20 were exposed to the NO donor DETA NONOate at increasing concentrations (0-500 µM) for 12 hours. Significant elevation in phosphorylation of Akt (Ser473 and Thr308) in response to increase NO donor concentration was found only in cell lines with wild-type PTEN and PIK3R1, such as SUM159, MDA-MB-231, and BT20. To evaluate whether NO-induced activation of Akt was PI3K-dependent, SUM159 (wild-type PTEN) and HCC1937 (PTEN mutant) cells were also pre-treated with PI3K inhibitor alpelisib before NO exposure. PI3K inhibition was only able to eliminate basal phospho-Akt and prevent NO-induced Akt activation in the SUM159 cell line. Furthermore, compared to vehicle control treated cells, we found that NO donor-induced activation enhanced phosphorylation in 15/18 Akt signaling proteins in SUM159 cells (PTEN intact) and 3/18 Akt signaling proteins in HCC1937 cells (PTEN loss). In Hs578T cells in which NOS2 was either overexpressed or knocked down via lentiviral transduction, we found that enhanced expression of NOS2 was associated with increased p-Akt expression and knock-down of NOS2 led to reduced p-Akt. This result suggests that iNOS, as opposed to other NOS isoforms, is more associated with modulating PI3K signaling in TNBC.We also analyzed TNBC data from The Cancer Genome Atlas and found that patients with positive iNOS mRNA expression had significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.0001] expression compared to patients with no iNOS expression. When we stratified this patient cohort based on both iNOS and PTEN expression, we only found significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.01] expression in patients with positive iNOS and PTEN expression, supporting the hypothesis that NO may impair PTEN’s function as a PI3K antagonist. Ongoing studies are directed to investigate mechanisms of iNOS-PTEN physical interactions, iNOS-dependent post-translational modifications to PTEN, and whether iNOS and PTEN are dual prognostic markers in TNBC patients. Citation Format: Tejaswini P. Reddy, Liliana Guzman-Rojas, Roberto R. Rosato, Wei Qian, Hong Zhao, Jenny C. Chang. Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-01.

Antinociceptive effect of N-acetyl glucosamine in a rat model of neuropathic pain.

This study was aimed at evaluating the efficacy of glucosamine and potential mechanisms of actions in a neuropathic pain model in rats. Glucosamine (500, 1000 and 2000 mg/kg) was administered via gavage route, 1 day before the chronic constriction injury (CCI) of sciatic nerve and daily for 14 days (prophylactic regimen), or from days 5 to 14 post-injury (therapeutic regimen), as the indicators of neuropathic pain, mechanical allodynia, cold allodynia and thermal hyperalgesia were assessed on days 0, 3, 5, 7, 10 and 14 after ligation. Inducible nitric oxide synthase (iNOS) and tumour necrosis factor alpha (TNF-α) gene expressions were measured by real-time polymerase chain reaction. TNF-α protein content was measured using the enzyme-linked immunosorbent assay method. Three days after nerve injury, the threshold of pain was declined among animals subjected to neuropathic pain. Mechanical and cold allodynia, as well as thermal hyperalgesia were attenuated by glucosamine (500, 1000, 2000 mg/kg) in the prophylactic regimen. However, existing pain was not decreased by this drug. Increased mRNA expression of iNOS and TNF-α was significantly reduced in the spinal cord of CCI animals by glucosamine (500, 1000, 2000 mg/kg) in the prophylactic regimen. The overall expression of spinal TNF-α was increased by CCI, but this increase was reduced in animals receiving glucosamine prophylactic treatment. Findings suggest that glucosamine as a safe supplement may be a useful candidate in preventing neuropathic pain following nerve injury. Antioxidant and anti-inflammatory effects may be at least in part responsible for the antinociceptive effects of this drug.

Ultrasound-assisted green synthesis of gold nanoparticles using citrus peel extract and their enhanced anti-inflammatory activity

Ultrasound and plant extract are two green approaches that have been used to synthesize gold nanoparticles (AuNPs); however, how the combination of ultrasound and citrus peel extract (CPE) affects the structure characteristics and the bioactivity of AuNPs remains unknown. Here we investigated the effects of ultrasound conditions on the particle size, stability, yield, phenolic encapsulation efficacy, and the anti-inflammatory activity of AuNPs. The results showed that temperature was positively correlated to the particle size and the anti-inflammatory activity of synthesized AuNPs. Increasing the power intensity significantly decreased the particle size, while increased the change of total phenolic content (ΔTPC) in the reaction mixture. The increase of ΔTPC caused the enhanced anti-inflammatory activity of AuNPs. The AuNPs synthesized with or without ultrasound treatment were characterized using UV–Vis, DLS, SEM, TEM, EDS, XRD, and FT-IR. The result verified the formation of negatively charged, spherical, stable, and monodispersed AuNPs. AuNPs synthesized with ultrasound (AuNPs-U) has smaller particle size (13.65 nm vs 16.80 nm), greater yield and anti-inflammatory activity (IC50, 82.91 vs 157.71 μg/mL) than its non-ultrasound counterpart (AuNPs-NU). HPLC analysis showed that hesperidin was the key reductant for the synthesis of AuNPs. AuNPs-U also inhibited the mRNA and protein expression of iNOS and COX-2 in the LPS-induced Raw 264.7 cells. Our research elucidates the relationship between the reaction conditions and the structure characteristics and the anti-inflammatory activity of AuNPs synthesized using CPE with the help of ultrasound, thereafter, provides a feasible and economic way to synthesize AuNPs that can be used to ameliorate inflammation.

DDAH1 SNP rs480414 that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia results in lower nitric oxide production in neonatal cord blood-derived lymphoblastoid cell lines.

Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and pulmonary hypertension (PH) is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a vasodilator and apoptotic mediator made by NO synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA), and dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Previously, in a BPD patient cohort, we identified single nucleotide polymorphism (SNP) DDAH1 rs480414 (G > A) that was protective against developing PH. This study aims to determine functional consequences of the DDAH1 SNP in lymphoblastoid cell lines (LCLs) derived from neonatal cord blood. We tested the hypothesis that DDAH1 SNP (AA) results in DDAH1 gain of function, leading to greater NO-mediated apoptosis compared to DDAH1 wild-type (GG) in LCLs. LCLs were analyzed by Western blot (DDAH1, cleaved and total caspase-3 and -8, and β-actin), and RT-PCR (DDAH1, iNOS). Cell media assayed for nitrites with chemiluminescence NO analyzer, and conversion of ADMA to L-citrulline was measured by spectrophotometry. LCLs with DDAH1 SNP had similar levels of DDAH1 protein and mRNA expression, as well as DDAH activity, compared to DDAH1 WT LCLs. There were also no changes in cleaved caspase-3 and -8 protein levels. LCLs with DDAH1 SNP had similar iNOS mRNA expression. Nitrite levels in media were lower for DDAH1 SNP LCLs compared to DDAH1 WT LCLs (p < 0.05). Contrary to our hypothesis, we found that NO production was lower in DDAH1 SNP LCLs, indicative of a loss of function phenotype.

Mechanism of protection from insulin resistance by toll-like receptor 2 deficiency in high-fat diet fed mice: involvement in macrophage polarization.

Toll-like receptor 2 (TLR2) deficiency can increase insulin sensitivity and improves glucose tolerance. However, it is not yet fully understood about its underlying mechanism. The regulation of M1/M2 macrophage polarization has been verified to involve in insulin resistance. Here, we evaluated whether the beneficial effect of TLR2 deficiency is mediated by TLR2-associated macrophage polarization in mice fed with high-fat diet (HFD). Wild-type and TLR2 knockout (TLR2-/-) mice received HFD for two months. Following intraperitoneal glucose tolerance and insulin resistance tests, peripheral monocytes were isolated, and in vitro induced for differentiation into M1 and M2 macrophages, respectively. Macrophages polarization was evaluated using flow cytometry. The expression of macrophage polarization marker genes and cytokine production in visceral adipose tissue were measured by qRT-PCR and ELISA. Compared to wild-type mice, TLR2-/- mice showed higher glucose tolerance and insulin sensitivity, along with significantly reduced the population of M1 and increased M2 count in vitro. Additionally, TLR2-/- mice demonstrated higher expression of M2 marker iNOS mRNA and interleukin-10 level in adipose tissues. Our results reveal that TLR2 knockout-mediated macrophages M2 polarization is a crucial factor for preventing against diet-induced insulin resistance in mice. These findings deepen our knowledge about the mechanism underlying HFD-induced insulin resistance.

Mesenchymal Stem Cells Overexpressing ACE2 Favorably Ameliorate LPS-Induced Inflammatory Injury in Mammary Epithelial Cells.

Mesenchymal stem cells (MSCs) are capable of homing injury sites to exert anti-inflammatory as well as anti-damage effects and can be used as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2) plays an important role in numerous inflammatory diseases, but fewer studies have been reported in animal mastitis. We hypothesized that MSCs overexpressing ACE2 is more effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory injury in mammary epithelial cells compared to MSCs alone. The results showed that MSC-ACE2 inhibited the LPS induction by upregulation of TNF-α, IL-Iβ, IL-6, and iNOS mRNA expression levels in EpH4-Ev cells compared with MSCs. Furthermore, results showed that both MSC and MSC-ACE2 were significantly activated IL-10/STAT3/SOCS3 signaling pathway as well as inhibited TLR4/NF-κB and MAPK signaling pathways, but MSC-ACE2 had more significant effects. Meanwhile, MSC-ACE2 promoted the expression of proliferation-associated proteins and inhibited the expression of the apoptosis-associated proteins in EpH4-Ev cells. In addition, MSC and MSC-ACE2 reversed the LPS-induced downregulation expression levels of the tight junction proteins in mammary epithelial cells, indicating that both MSC as well as MSC-ACE2 could promote blood-milk barrier repair, and MSC-ACE2 was more effective. These results suggested that MSCs overexpressing ACE2 were more anti-inflammatory as well as anti-injurious action into LPS-induced inflammatory injury in the EpH4-Ev cells. Thus, MSCs overexpressing ACE2 is expected to serve as a potential strategy for mastitis treatment.

Acetaminophen-Induced Nephrotoxicity: Suppression of Apoptosis and Endoplasmic Reticulum Stress Using Boric Acid.

Acetaminophen (APAP) is one of the popular and safe pain medications worldwide. However, due its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure. Boron is a bioactive trace element, found naturally as boric acid (BA) and borate. In this study, the effects of boric acid on the acute renal toxicity induced by APAP in rats were researched in comparison with N-acetyl cysteine (NAC). In the study, 7 groups were formed and 2g/kg dose of paracetamol per rat was prepared by suspending in 1% Carboxy Methyl Cellulose (CMC) solution of phosphate buffer saline (PBS). Boric acid dissolved in saline was administered to experimental animals by gavage at doses of 50, 100, and 200mg/kg. In this study, ER stress and apoptosis formed by paracetamol-induced nephrotoxicity were investigated. This purpose determined iNOS, PERK, ATF6, NFkB p53, caspases 3, 12, bcl-2, and bcl-xL gene mRNA expression kidney tissue. Also, the levels of kidney injury molecule-1 (KIM-1), Cysteine (Cys), and IL-18 levels, which are mentioned today as kidney damage markers were compared with BUN and creatine levels. The effect of boron on kidney damage was determined by histopathologic. Data were statistically analyzed by using SPSS-20 ANOVA and stated as means and standard deviation. According to the data obtained in our study, we believe that boric acid has a protective effect on the negative effects of paracetamol on the kidney. We believe that our study will provide useful data to the literature on the possibility of a supplement to be used as an active compound in paracetamol for the prophylaxis of boric acid and it can also be converted into a useful product.

Ameliorative efficacy of oxypaeoniflorin, a traditional chinese medicine monomer against adjuvant-induced arthritic inflammation and pain

Background: Rheumatoid arthritis is a chronic, common autoimmune disorder identified by progressive dysfunction of joints and cartilage damage. Oxypaeoniflorin (OPA), a Traditional Chinese Medicine monomer, has been reported for potential against inflammation and oxidative stress. Objectives: The objective of the study is to evaluate the potential of OPA against Freund complete adjuvant (FCA)-induced inflammatory pain in experimental arthritic rats. Materials and Methods: FCA was administered in female rats (Wistar strain) to induce polyarthritis, followed by 28 days of oral treatment with either vehicle, leflunomide (10 mg/Kg), or OPA (10, 20, and 40 mg/kg). Various parameters were assessed to determine the effect of OPA on pain and inflammatory pathway. Results: Adjuvant-induced arthritis (AIA)-induced elevated paw withdrawal latency and threshold suggested induction of arthritic pain. However, OPA (20 and 40 mg/kg) treatment diminished arthritic pain reflected by amelioration of hyperalgesia and allodynia. In addition, OPA showed an effective inhibition (P < 0.05) in FCA-induced alterations in alkaline phosphatase, alanine transaminase, aspartate aminotransferase, serum albumin, and C-reactive protein levels. The AIA-induced elevated oxido-nitrosative stress, protein levels of tumor necrosis factor-α, and interleukins in synovial tissue were effectively reduced (P < 0.05) by OPA. Moreover, OPA effectively downregulated (P < 0.05) enhanced nuclear factor-kappa beta (NF-κB), Ikβα, iNOs, and COX-2 mRNA expressions in synovial tissue. OPA also reduced histopathology alteration induced in the tibiotarsal joint by FCA. Conclusion: OPA exerts its antiarthritic property through inhibition of NF-κB/IκBα pathway to downregulate the activation of pro-inflammatory cytokines and inflammatory mediator thus, ameliorated arthritic inflammation and pain.

Selected lactobacilli strains inhibit inflammation in LPS-induced RAW264.7 macrophages by suppressing the TLR4-mediated NF-κB and MAPKs activation

Probiotics are known to provide the host with immune-modulatory effects, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers.Lactobacillus acidophilus KLDS 1.0901, Lactobacillus helveticus KLDS 1.8701, and Lactobacillus plantarum KLDS 1.0318 were isolated from Chinese fermented dairy food with antibacterial activity, antioxidant activity, and immunomodulatory activity, respectively. This study evaluated the anti-inflammatory potential of the tested strains in LPS (Lipopolysaccharide)-induced RAW264.7 cells. The results showed that all tested strains could inhibit the mRNA expression of iNOS and COX-2 and reduce the concentration of NO and PGE2 production. Furthermore, all tested strains markedly reduced proinflammatory cytokines' production (TNF-α, IL-1β, and IL-6). Moreover, these results may be associated with inhibiting TLR4-mediated NF-κB and MAPKs signaling pathway activation. These results indicated that L. acidophilus KLDS 1.0901, L. helveticus KLDS 1.8701, and L. plantarum KLDS 1.0318 possesses an anti-inflammatory potential and provide a molecular basis regarding the development of functional probiotic products.

Effects of Vitamin A and K3 on Immune Function and Intestinal Antioxidant Capacity of Aged Laying Hens

This study was conducted to investigate effects of vitamin A (VA) and vitamin K3 (VK3) on immune function and intestinal antioxidant capacity of aged laying hens. In a 3 × 3 factorial arrangement, the diets of 1080 Roman Pink laying hens (87 weeks old) was formulated with deficient, adequate and excess VA and VK3, including 0, 7000 and 14000 IU/kg VA and 0, 2.0 and 4.0 mg/kg VK3 for 8 weeks. Interactive effects between VA and VK3 were observed that VA and VK3 decreased the splenetic mRNA expression of inducible nitric oxide synthase (iNOS) and tumour necrosis factor α (TNF-α), but increased the plasma immunoglobulin G (IgG) content and jejunal mRNA expression of nuclear factor-like 2 (Nrf2). Hens fed adequate or excess VA had higher spleen index, mRNA expression of interleukin-10 (IL-10) in spleen, sIgA content, catalase (CAT), glutathione peroxidase and total dismutase (T-SOD) activity, and mRNA expression of polymeric immunoglobulin receptor (pIgR) in jejunum and lower mRNA expression of IL-1β in jejunum and iNOS, TNF-α in spleen. Furthermore, adequate or excess VK3 significantly increased plasma IgG content, the CAT, T-SOD and total antioxidant capacity activities, up-regulated the mRNA expression of pIgR, Nrf2, SOD1 and CAT in jejunum and down-regulated the mRNA expression of iNOS and TNF-α in spleen. In conclusion, dietary addition of adequate VA (7000 IU/kg) and VK3 (2.0 mg/kg) improved the immune function and intestine antioxidant capacity of aged laying hens and excess levels did not exhibit superior effects.

조리방법에 따른 가지(Solanum melongena L.)의 항염증 활성 평가

Purpose: This study was conducted to investigate the anti-inflammatory effects of Solanum melongena L. (SM) using 3 kinds of cooking methods. Methods: To estimate the anti-inflammatory effect of SM, the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and cytokines, and nuclear factor kappa B (NF-κ B) protein were measured. Results: Compared to raw SM, there was a decrease in the production of NO and pro-inflammatory cytokine mRNA in the cooked SM. All samples (10, 20, 50 μg/mL) dose-dependently decreased the production of NO and PGE2. Roasted SM (RSM) and steamed SM (SSM) treatment down-regulated iNOS, COX-2 and cytokines mRNA expression, and p50 and IκBα phosphorylation compared to the vehicle. Specifically, SSM showed the greatest anti-inflammatory effect. Conclusion: Thus, the cooking method may have a bearing on the anti-inflammatory effect of SM. This is presumed to be due to the change in the functional components during heating. However, further studies to identify the active compounds are required.

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.

Indole derivative XCR-5a alleviates LPS-induced inflammation in vitro and in vivo

Context Few studies on anti-inflammatory drugs with indole groups have been published. This is the first study that demonstrates the anti-inflammatory effects of indole derivative XCR-5a in vitro and in vivo. Objective This study aimed to discover more anti-inflammatory drugs with indole groups and investigate their anti-inflammatory mechanisms. Materials and methods First, a series of indole derivatives was synthesized, then screened for XCR-5a, a compound with anti-inflammatory effects. Second, the in vitro production of IL-1β, IL-6, TNF-α, inducible nitric oxide synthase (iNOS), and cyclo-oxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced primary cells of mice pretreated with XCR-5a was determined using qPCR and ELISA. Finally, the effect of XCR-5a on LPS-induced NF-κB signaling activation was determined by Western blotting. An in vivo mouse sepsis model was established. In mouse lung tissue, the production of IL-1β, IL-6, and TNF-α was determined and H&E staining was performed. Results Our findings showed that XCR-5a could suppress the production of LPS-induced IL-1β, IL-6, and TNF-α, as well as mRNA expression of iNOS and COX-2. Pretreatment with XCR-5a inhibited the LPS-induced inflammatory response in septic mice in vivo by decreasing pro-inflammatory cytokines production in serum and reducing immune cell infiltration. Mechanistically, XCR-5a suppressed LPS-induced activation of the NF-κB signaling pathway. Conclusions XCR-5a has anti-inflammatory effects in vitro and in vivo. Therefore, XCR-5a could be a potential drug candidate for the treatment of inflammatory diseases.

A Novel Polysaccharide Isolated From Fresh Longan (Dimocarpus longan Lour.) Activates Macrophage via TLR2/4-Mediated PI3/AKT and MyD88/TRAF6 Pathways.

A novel immunomodulatory polysaccharide (LP4) with a molecular weight 6.31 × 104 g/mol was purified from fresh longan pulp. It was composed of mannose, glucose, glucuronic acid, galactose, xylose, arabinose, galacturonic acid, fucose, and rhamnose in a molar percentage of 36:31:10:7:4:4:3:2:2, and mainly linked by (1→6)-β-Man, (1→4)-β-Glc and (1→6)-α-Glc. LP4 can obviously enhance the phagocytosis of macrophages and promote the proliferation of lymphocytes. After treating macrophages with LP4 (12.5–50 μg/ml), the production of IL-1β and TNF-α was significantly increased. These increases of cytokines were suppressed when the TLR2/TLR4 receptors were inhibited by anti-TLR2 and/or anti-TLR4 antibodies. Moreover, the mRNA expression of INOS, AKT, PI3K, TRAF6 and MyD88 was significantly suppressed by TLR2/TLR4 antibodies. These results indicated that LP4 induced macrophage activation mainly via the TLR2 and TLR4-induced PI3K/AKT and MyD88/TRAF6 pathways.

Comparison of the anti-inflammatory activities of furanocoumarins from the roots of Angelica dahurica

Background: The roots of Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier (Apiaceae) have traditionally been used for inflammatory skin diseases. A. dahurica roots (Byakushi) contain furanocoumarins, such as imperatorin and byakangelicin. To elucidate which constituents are responsible for the anti-inflammatory effects, we evaluated the potency of crude A. dahurica root extract fractions by monitoring the production of the inflammatory mediator nitric oxide (NO) in hepatocytes.Methods: The dried roots of A. dahurica were collected in South Korea and extracted with methanol. The resulting extract was fractionated into ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β and each fraction for 8 h, and then the NO production and lactate dehydrogenase activity in the medium were measured. The expression of inducible nitric oxide synthase (iNOS) was detected by Western blotting, and its mRNA expression level was measured by quantitative reverse transcription-polymerase chain reaction.Results: Among the three fractions, the EtOAc-soluble fraction markedly suppressed NO production without showing cytotoxicity and decreased iNOS expression in hepatocytes. From this hydrophobic fraction, we isolated five furanocoumarins: isoimperatorin, imperatorin, phellopterin, oxypeucedanin, and oxypeucedanin methanolate. Phellopterin and oxypeucedanin methanolate significantly suppressed NO production and reduced the mRNA expression of iNOS and tumor necrosis factor α. In contrast, the other three constituents did not affect NO production. Comparison of their chemical structures suggests that a methoxy group at carbon 5 and a side chain at carbon 8 in the furanocoumarin skeleton may be essential for NO production suppression.Conclusion: These data imply that phellopterin and oxypeucedanin methanolate, which are hydrophobic furanocoumarins, may contribute to the anti-inflammatory effects of A. dahurica roots by suppressing iNOS gene expression.Keywords: Inflammation, nitric oxide, hepatocyte, coumarin, Kampo medicine

Antioxidant and Anti-inflammatory Activities of Different Solvent Extracts from Ipomoea pes-caprae (L.) R. Br. in Lipopolysaccharide Stimulated RAW 264.7 Macrophages

The oxidative stress and inflammatory environment trigger an unhealthy circle, which can lead to various inflammatory diseases. Ipomoea pes-caprae, a traditional medicine mangrove plant, posed many pharmacological activities, including antioxidant, anti-inflammatory and anticancer effects. However, the possible mechanisms involved in Ipomoea pes-caprae are still unclear. This study aimed to investigate the antioxidant and anti-inflammatory effects of different solvent extracts from Ipomoea pes-caprae on lipopolysaccharide (LPS) stimulated macrophages. Three different solvent gradients were prepared orderly from non-polar to polar: hexane (Hex), supercritical fluid extraction using carbon dioxide plus EtOH as co-solvent (SCO2) and ethanol (EtOH). All 3 extracts were screened for the cytotoxicity on RAW264.7 cells by MTT assay. The non-toxic doses were investigated for reactive oxygen species (ROS) scavenging by DPPH and DCFH-DA assays and evaluated their anti-inflammatory activities via inhibition against LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression. All 3 extracts (25 - 50 μg/mL) exhibited DPPH scavenging and reduced intracellular ROS level in the order of SCO2 &gt; EtOH &gt; Hex. Further, these extracts suppressed NO and PGE2 production by regulating iNOS and COX-2 mRNA expression in the order of SCO2 &gt; Hex &gt; EtOH. Additionally, their inhibitory effects were in a similar pattern as the standard drugs L-NAME and celecoxib. These findings support the traditional use of Ipomoea pes-caprae in treating inflammatory diseases due to its attenuation of inflammation in activated macrophage. Also, a wide range of secondary metabolites in unique ecology may be useful as one of the alternative therapies for inflammatory diseases.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Ipomoea pes-caprae employ a sort of secondary metabolites under stressful high salt conditions with benefit for new drug discoveries&#x0D; SCO2 extract exhibited the strongest activities in the LPS-induced ROS and PGE2 production&#x0D; These bioactive compounds contained in Ipomoea pes-caprae confirm the traditional use against jellyfish stings and may targeting inflammatory pathways&#x0D; &#x0D; GRAPHICAL ABSTRACT

Heparin-binding protein (HBP) worsens the severity of pancreatic necrosis via up-regulated M1 macrophages activation in acute pancreatitis mouse models

ABSTRACT Acute pancreatitis (AP) is one of the most widespread clinical emergencies. Macrophages are the most common immune cells in AP pancreatic tissue and are closely associated with pancreatic necrosis and recovery. The level of heparin-binding protein (HBP) is closely linked to inflammation. In this study, we assessed the effect of HBP on AP tissue necrosis severity and whether HBP is associated with M1 macrophages in pancreatic necrosis. We observed the dynamic changes of HBP levels in the pancreas during acute inflammation in the caerulein-induced AP mice model. We used hematoxylin–eosin staining to evaluate pancreatic edema and necrosis, and to detect infiltration of macrophages by immunohistochemistry. Moreover, expressions of the maker and cytokines of macrophages, including inducible nitric oxide synthase (iNOS), and arginase 1 (Arg-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNA, were detected by real-time polymerase-chain reaction (RT-PCR). High levels of HBP in the pancreas were detected at 48 h, and heparin inhibited HBP expression in AP pancreatic tissue. Inhibiting HBP expression by injecting heparin before AP can alleviate pancreatic necrosis and inhibit F4/80 labeled M1 macrophage infiltration and IL-6, TNF-α, and iNOS mRNA expression. Clodronate liposome (CLDL) intraperitoneally treated mice showed no change in pancreatic HBP levels, but pancreatic macrophage-specific antigen F4/80 and TNF-α, IL-1β, and IL-6 mRNA levels decreased after CLDL treatment. HBP is critical for pancreatic necrosis response in acute pancreatitis by increasing the infiltration of M1 macrophages and promoting the secretion of inflammatory factors, such as TNF-α, IL-6, IL-1β, which can be reduced by heparin.

Salmonella chitosan nanoparticle vaccine administration is protective against Salmonella Enteritidis in broiler birds.

Salmonella control strategies include vaccines that help reduce the spread of Salmonella in poultry flocks. In this study we evaluated the efficacy of administering a live Salmonella vaccine followed by a killed Salmonella chitosan nanoparticle (CNP) vaccine booster on the cellular and humoral immunity of broilers. The CNP vaccine was synthesized with Salmonella Enteritidis (S. Enteritidis) outer-membrane-proteins (OMPs) and flagellin-proteins. At d1-of-age, one-hundred-sixty-eight chicks were allocated into treatments: 1) No vaccine, 2) Live vaccine (Poulvac®ST), 3) CNP vaccine, 4) Live+CNP vaccine. At d1-of-age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7-of-age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14-of-age, birds were challenged with 1×109 CFU/bird S. Enteritidis. There were no significant differences in body-weight-gain (BWG) or feed-conversion-ratio (FCR). At 8h-post-challenge, CNP and Live+CNP-vaccinated birds had 17% and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d28-of-age, CNP, Live, and Live+CNP-vaccinated birds had 33%, 18%, and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d14-of-age, Live+CNP-vaccinated birds had 46% greater levels (P<0.05) of anti-Salmonella OMPs IgY in serum, compared to control. At d21-of-age, splenocytes from CNP and Live-vaccinated birds had increased (P<0.05) T-lymphocyte proliferation at 0.02 mg/mL OMPs stimulation compared to the control. At d28-of-age, CNP and Live+CNP-vaccinated birds had 0.9 Log10 CFU/g and 1 Log10 CFU/g decreased S. Enteritidis cecal loads (P<0.05), respectively, compared to control. The CNP vaccine does not have adverse effects on bird’s BWG and FCR or IL-1β, IL-10, IFN-γ, or iNOS mRNA expression levels. It can be concluded that the CNP vaccine, as a first dose or as a booster vaccination, is an alternative vaccine candidate against S. Enteritidis in broilers.

Anti-Oxidative and Anti-Inflammatory Activities of Astragalus membranaceus Fermented by Lactiplantibacillus plantarum on LPS-Induced RAW 264.7 Cells

Astragalus membranaceus (AM) has been used for anti-oxidative, anti-inflammatory, anti-cancer, and immunomodulatory activities. In this study, we confirmed that the anti-oxidative and anti-inflammatory effects of AM were enhanced after it was fermented by Lactiplantibacillus plantarum. The anti-oxidative effect was measured by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical levels, total phenolic contents (TPC), reducing power, and H2O2 levels. AM-LP MG5145 and MG5276 showed higher free radical scavenging activity than AM-NF (51.22%). In addition, AM-LP MG5145 and MG5276 showed higher TPC (49.11 mg GAE/mL), reducing power (OD700 = 0.37), and H2O2 (1.71 µM) than AM-NF. The calycosin contents determined to AM-NF (17.24 ng/mL), AM-LP MG5145 (139.94 ng/mL), and MG5276 (351.01 ng/mL) using UPLC-ESI-MS/MS. Anti-inflammatory effects were analyzed by investigating the inhibitory effects of fermented AM on cytotoxicity, NO production, and mRNA expression of COX-2, iNOS, NF-κB, and TNF-α in LPS-induced RAW 264.7 cells. AM-LP MG5145 and MG5276 showed no cytotoxicity. AM-LP MG5145 (50.86%) and MG5276 (51.66%) inhibited NO production in LPS-induced RAW 264.7 cells. Moreover, AM-LP MG5145 and MG5276 downregulated macrophage iNOS, COX2, TNF-α, and NF-κB expression. In conclusion, A. membranaceus fermented by L. plantarum MG5145 and MG5276 can be used in cosmetics and health foods as natural antioxidant compounds.

Anti-proliferation and anti-inflammation effects of corilagin in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways

Ethnopharmacological relevanceThe dried aboveground part of Geranium Wilfordii Maxim. (G. Wilfordii) is a traditional Chinese herbal medicine named lao-guan-cao. It has long been used for dispelling wind-dampness, unblocking meridians, and stopping diarrhea and dysentery. Previous investigations have revealed that 50% ethanolic extract of G. Wilfordii has anti-inflammatory and anti-proliferation activities on TNF-α induced murine fibrosarcoma L929 cells. Corilagin (COR) is a main compound in G. Wilfordii with the content up to 1.69 mg/g. Pharmacology study showed that COR has anti-inflammatory, anti-tumor, anti-microorganism, anti-oxidant, and hepatoprotective effects. However, there is no any investigation on its anti-proliferation and anti-inflammation effects in rheumatoid arthritis (RA). Aim of the studyThe present study aimed to evaluate the potential pharmacological mechanisms of anti-proliferation and anti-inflammation effects of COR in RA. Materials and methodsIn vitro, MH7A cells model induced by IL-1β was used. The anti-proliferation activity of COR was assessed by Cell Counting Kit-8 (CCK-8) assay, and the anti-migration and anti-invasion activity of COR was determined by wound healing assay and transwell assay, respectively. Furthermore, apoptosis assay by flow cytometer was used to measure the pro-apoptotic effect of COR. The mRNA expressions of Bax, Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS were measured by qRT-PCR, and related protein were further verified by ELISA kits or Western blot. Moreover, protein levels associated with NF-κB and MAPK signaling pathways of p65, P-p65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK1/2/3, p38, and P-p38 were determined by Western blot. The nuclear translocation of NF-κB-p65 was detected by immunofluorescent staining. In vivo, adjuvant-induced arthritis (AIA) rat model was used, and the body weight, paw swelling, and arthritis score during the entire period were measured. Histopathological analysis of joints of synovial tissues was also determined. The expression of pro-inflammatory cytokines in serum including IL-6, TNF-α, IL-1β, and IL-17 were measured. ResultsThe in vitro results showed that COR could dose-dependently inhibit the proliferation, migration, and invasion of IL-1β-induced MH7A cells, as well as promote its apoptosis. Moreover, it also suppressed the over-expression of Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS while up-regulated the level of Bax. Besides, the ratios of P-p65/p65, P-IκBα/IκBα, P-ERK/ERK, P-JNK/JNK, and P-p38/p38 were decreased, and the nuclear translocation of p65 induced by IL-1β was blocked by COR. In vivo results indicated that COR significantly reduced the paw swelling and arthritis score in AIA rats, and inhibited synovial tissue hyperplasia and erosion, as well as inflammatory cells infiltration. It also decreased the serum pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, and IL-17) production. ConclusionThese results revealed that COR exerted anti-rheumatoid arthritis effect, and its underlying mechanisms may be related to inhibiting the proliferation, migration, and invasion of synovial fibroblasts, enhancing cell apoptosis, and suppressing inflammatory responses via downregulating NF-κB and MAPK signaling pathways.