Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Lactate-induced spontaneous acrosomal exocytosis as a method to study acrosome function in stallion sperm.

Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 µM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo-fertility of stallions used in artificial insemination programs.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

Oral administration of Helianthus annuus leaf extract ameliorates atopic dermatitis by modulation of T cell activity in vivo.

BACKGROUND: Atopic dermatitis (AD) is multifactorial disease that is highly involved in the activity of T cells from the skin lesion. Seeds of Helianthus annuus extract have been traditionally used as anti-inflammatory reagent but few studies have been reported on leaf of H. annuus that are discarded uselessly as an immunomodulator. PURPOSE: Therefore, here, the regulatory effect of Helianthus annuus extract (HAE) on AD via suppression of T cell activity was investigated. METHODS: The efficacy of HAE was evaluated in T cells stimulated with CD3/CD28 antibody and PMA/A23187. And demonstration of the alleviating effect of HAE on AD in the ears of Balb/c female mice stimulated with mite extract and DNCB. RESULTS: Pre-treatment with HAE abrogates IL-2 production from activated T cells. It was also found that HAE suppresses the expression of surface molecules in activated T cells. Cell viability results demonstrated that HAE is not associated with cytotoxicity in resting and activated T cells. Besides, we exhibited that regulated phosphorylation of MAPK through TAK1-IKKα-NFκB by pre-treatment with HAE leads to the suppressive effect of HAE on T cell activation. Oral administration of HAE attenuates manifestations of AD including reduced thickness of dermis and epidermis, decreased IgE level in serum, and declined mRNA levels of atopic cytokines on ear tissues. The ameliorative effect of HAE on AD was found to be associated with suppressed activity of T cells from draining lymph nodes. CONCLUSION: Therefore, our results provide that HAE alleviates AD symptoms via modulation of T cell activity. In addition, these results suggest the immunomodulatory effect of HAE on T-cell mediated diseases.

Ca2+ participates in programmed cell death by modulating ROS during pollen cryopreservation.

KEY MESSAGE: After cryopreservation, the Ca2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+-chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+-chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.

Influence of Amino Acid Feeding on Production of Calcimycin and Analogs in Streptomyces chartreusis.

Streptomyces chartreusis NRRL 3882 produces the polyether ionophore calcimycin and a variety of analogs, which originate from the same biosynthetic gene cluster. The role of calcimycin and its analogs for the producer is unknown, but calcimycin has strong antibacterial activity. Feeding experiments were performed in chemically defined medium systematically supplemented with proteinogenic amino acids to analyze their individual effects on calcimycin synthesis. In the culture supernatants, in addition to known calcimycin analogs, eight so far unknown analogs were detected using LC-MS/MS. Under most conditions cezomycin was the compound produced in highest amounts. The highest production of calcimycin was detected upon feeding with glutamine. Supplementation of the medium with glutamic acid resulted in a decrease in calcimycin production, and supplementation of other amino acids such as tryptophan, lysine, and valine resulted in the decrease in the synthesis of calcimycin and of the known intermediates of the biosynthetic pathway. We demonstrated that the production of calcimycin and its analogs is strongly dependent on amino acid supply. Utilization of amino acids as precursors and as nitrogen sources seem to critically influence calcimycin synthesis. Even amino acids not serving as direct precursors resulted in a different product profile regarding the stoichiometry of calcimycin analogs. Only slight changes in cultivation conditions can lead to major changes in the metabolic output, which highlights the hidden potential of biosynthetic gene clusters. We emphasize the need to further study the extent of this potential to understand the ecological role of metabolite diversity originating from single biosynthetic gene clusters.

Oleanolic acid protects against mast cell-mediated allergic responses by suppressing Akt/NF-κB and STAT1 activation.

BACKGROUND: Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient. PURPOSE: Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders. METHODS: We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock. RESULTS: OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation. CONCLUSION: The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease.

Tripterine: A Potential Anti-Allergic Compound.

BACKGROUND: Tripterine (TRI), an active monomer in Tripterygium wilfordii, has significant pharmacological activities, such as anti-inflammatory, immunosuppressive and anti-tumor activities. TRI may be used to treat allergic diseases because of its characteristics of immunosuppression. OBJECTIVE: This study aims to explore the anti-allergic effect of TRI. METHODS: It was tested in vivo and in vitro in this study. RESULTS: The results showed that TRI could significantly inhibit histamine release from rat peritoneal mast cells; the inhibitory effect of TRI on histamine release was stronger than that of other known histamine inhibitors such as disodium cromoglyceride. TRI also significantly inhibited systemic anaphylactic shock induced by compound 48/80 and skin allergy induced by IgE, and inhibited the expression of inflammatory factors secreted by Human Mast Cells (HMC-1) induced by Phorbol 12-Myristate 13- Acetate (PMA) and calcium carrier A23187. In the animal model of allergic rhinitis induced by Ovalbumin (OA), the scores of friction, histamine, IgE, inflammatory factors and inflammatory cells decreased after TRI was administered orally or nasally. CONCLUSION: TRI, as an active immunoregulatory factor, has great potential in the treatment of mast cell-mediated allergic diseases.

In vivo and in vitro anti­allergic and anti­inflammatory effects of Dryopteris crassirhizoma through the modulation of the NF­Ä¸B signaling pathway in an ovalbumin­induced allergic asthma mouse model.

Dryopteris crassirhizoma (DC) has a wide range of pharmacological effects, including antibacterial, anti­influenza virus, anti­tumor, anti­reverse transcriptase and antioxidant effects. However, the inhibitory effect of DC on allergic inflammatory response remains unclear; therefore, the current study used an experimental ovalbumin (OVA)­induced allergic asthma mouse model and phorbol myristate acetate (PMA)­ and A23187­stimulated HMC­1 cells to reveal the effects of DC in regulating airway inflammation and its possible mechanism. Allergic asthma was initiated in BALB/c mice via exposure to OVA emulsified in aluminum, on days 1 and 14. Thereafter, the mice were treated with DC or dexamethasone (Dex) orally, before being challenged, from days 15 to 26. Subsequently, the mice were challenged with OVA on days 27, 28 and 29. The results of histological analysis indicated that the administration of DC decreased the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and suppressed eosinophilic infiltration, mucus production and collagen deposition in the lung tissue. DC treatment increased the level of T helper type 1 (Th1) cytokines (IL­10 and interferon (IFN)­Î³) and decreased the levels Th2 cytokines (IL­4, IL­5 and IL­13) and proinflammatory cytokines (IL­6 and TNF­α). Furthermore, DC treatment inhibited the activation of NF­κB signaling (NF­κB, p­NF­κB, IκB and p­IκB), both in BALF and lung homogenates. Serum levels of total IgE and OVA­specific IgE and IgG1 were significantly lower after DC treatment compared with after OVA treatment. However, the anti­inflammatory effect of OVA­specific IgG2a was higher after DC treatment. In addition, DC treatment attenuated the production of proinflammatory cytokines, including IL­6 and TNF­α, and the activation of NF­κB signaling (NF­κB and p­NF­κB), in PMA and calcium ionophore A23187­stimulated HMC­1 cells. In summary, the current study demonstrated that DC acts a potent anti­allergic and anti­inflammatory drug by modulating the Th1 and Th2 response and reducing the allergic inflammatory reaction in PMA and A23187­stimulated HMC­1 cells via NF­κB signaling in an OVA­induced allergic asthma model.

Clinacanthus nutans Leaves Extract Reverts Endothelial Dysfunction in Type 2 Diabetes Rats by Improving Protein Expression of eNOS.

Diabetes mellitus is associated with endothelial dysfunction; it causes progressive vascular damage resulting from an impaired endothelium-dependent vasorelaxation. In the diabetes state, presence of hyperglycemia and insulin resistance predisposes to endothelial dysfunction. Clinacanthus nutans, widely used as a traditional medicine for diabetes is reported to have hypoglycemic, hypolipidemic, antioxidant, and anti-inflammatory properties. However, the possibility of C. nutans affecting the vascular endothelial function in diabetes remains unclear. This study was aimed at evaluating the effects of C. nutans methanolic leaves extract (CNME) on endothelial function in a type 2 diabetes (T2DM) rat model. Sixty male Sprague-Dawley rats were divided into five groups (n = 12 per group): nondiabetic control, nondiabetic treated with four weeks of CNME (500 mg/kg/daily), untreated diabetic rats, diabetic treated with metformin (300 mg/kg/daily), and diabetic treated with CNME (500 mg/kg/daily). T2DM was induced by a single intraperitoneal injection of low-dose streptozotocin (STZ) to rats fed with high-fat diet (HFD). Endothelial-dependent and endothelial-independent relaxations and contractions of the thoracic aorta were determined using the organ bath. Aortic endothelial nitric oxide synthase (eNOS) expression was determined using Western blotting. Endothelial-dependent relaxation was reduced in diabetic rats. Both diabetic groups treated with CNME or metformin significantly improved the impairment in endothelium-dependent vasorelaxation; this was associated with increased expression of aortic eNOS protein. CNME- and metformin-treated groups also reduced aortic endothelium-dependent and aortic endothelium-independent contractions in diabetics. Both of these diabetic-treated groups also reduced blood glucose levels and increased body weight compared to the untreated diabetic group. In conclusion, C. nutans improves endothelial-dependent vasodilatation and reduces endothelial-dependent contraction, thus ameliorating endothelial dysfunction in diabetic rats. This may occur due to its effect on increasing eNOS protein expression.

Two Japanese pepper (Zanthoxylum piperitum) fruit-derived compounds attenuate IgE-mediated allergic response in vitro and in vivo via inhibition of mast cell degranulation.

Zanthoxylum piperitum (ZP, 'Japanese pepper') is a traditional medicine and pepper used in Asian countries such as Japan. Hydroxy-α-sanshool, a pungent-tasting substance contained within ZP, has been reported to slightly suppress immunoglobulin E (IgE)-mediated mast cell degranulation. The current study aims to newly identify anti-allergic compounds derived from ZP. We examine the inhibitory mechanisms behind IgE-mediated mast cell degranulation. By inhibitory effect-guided isolation, we identified degranulation inhibitory compounds derived from ZP fruit: 1-acetoxy-7-hydroxy-3, 7-dimethylocta-2E, 5E-diene (ZP1) and 8-hydroxygeranyl acetate (ZP2). ZP1 and ZP2 inhibited IgE-mediated degranulation and A23187-mediated degranulation in RBL-2H3 mast cells. Our findings suggest the inhibition of degranulation by ZP1 and ZP2 was by inhibition of Lyn phosphorylation, followed by inhibition of intracellular Ca2+ mobilization, protein kinase C alpha phosphorylation, membrane ruffling, and granule-to-plasma membrane fusion. Oral administration of ZP1 or ZP2 attenuated an IgE-mediated passive cutaneous anaphylactic reaction in mice. Histological observation suggests that this effect occurred via inhibition of mast cell degranulation. These findings indicate that ZP1 and ZP2 attenuate allergic reaction via inhibition of IgE-mediated mast cell degranulation.

Long-term nitric oxide synthase inhibition prevents 17ß-estradiol-induced suppression of cyclooxygenase-dependent contractions and enhancement of endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries of ovariectomized rats.

Endothelial dysfunction is associated with a reduced bioavailability of nitric oxide (NO). In this study, the effects of 17ß-estradiol supplement on endothelial function were examined in ovariectomized (OVX) rats following long-term inhibition of NO synthases with L-NAME. Female Sprague Dawley rats were ovariectomized at 12 weeks old. They were supplemented with 17ß-estradiol (25 µg/kg/day, intramuscularly) or its vehicle (olive oil) until they were killed. At 18 weeks old, they were administered daily with NO synthase inhibitor L-NAME (60 mg/kg, by gavage) or its vehicle (distilled water) for 6 weeks. Rats were then anesthetized for blood pressure measurement and for isolation of mesenteric arteries and aortae for isometric tension measurement. Long-term L-NAME-treatment, without or with 17ß-estradiol supplement, resulted in reduced plasma nitrite/nitrate level without causing an increase in blood pressure in OVX rats. Acute inhibition of cyclooxygenase (COX) with indomethacin improved relaxations of mesenteric arteries to the calcium ionophore A23187 in OVX rats, and in those with long-term L-NAME-treatment without or with 17ß-estradiol supplement, but not in those with female hormone supplement only. 17ß-estradiol supplement or long-term L-NAME-treatment resulted in a greater endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries. In the quiescent aorta, 17ß-estradiol supplement or long-term L-NAME-treatment unmasked the COX-dependent components of A23187-induced contractions, but prevented that of the smooth muscle contractions to U46619 in OVX rats. In summary, long-term 17ß-estradiol-supplement results in differential effects in different blood vessel types, and its beneficial vascular effects are masked under the conditions with NO synthase inhibition.

Usnic acid as calcium ionophore and mast cells stimulator.

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

Xanthone suppresses allergic contact dermatitis in vitro and in vivo.

Xanthone is a phenolic compound found in a few higher plant families; it has a variety of biological activities, including antioxidant, anti-inflammatory, and anticancer properties. However, the molecular and cellular mechanisms underlying the activity of xanthone in allergic contact dermatitis (ACD) remain to be explored. Therefore, this study aimed to investigate the regulatory effects of xanthone in ACD in human keratinocytes (HaCaT cell), and human mast cell line (HMC-1 cell) in vitro and in an experimental murine model. The results demonstrated that treatment with xanthone reduced the production of pro-inflammatory cytokines and chemokines including interleukin (IL)-1ß, IL-6, IL-8, and expression of chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells. Xanthone also suppressed the production of pro-inflammatory cytokines, chemokines, and allergic mediators in phorbol myristate acetate/A23187 calcium ionophore (PMACI)-stimulated HMC-1 cells. Xanthone significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) and activation of caspase-1 signaling pathway in vitro model. Additionally, xanthone administration alleviated 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis like-skin lesion by reducing the serum levels of immunoglobulin E (IgE), histamine, and pro-inflammatory cytokines and suppressing MAPKs phosphorylation. Xanthone administration also inhibited mortality due to compound 48/80-induced anaphylactic shock and suppressed the passive cutaneous anaphylaxis (PCA) reaction mediated by IgE. Collectively, these results suggest that xanthone has a potential for use in the treatment of allergic inflammatory diseases.

The Role of Rhodomyrtus tomentosa (Aiton) Hassk. Fruits in Downregulation of Mast Cells-Mediated Allergic Responses.

Rhodomyrtus tomentosa, a flowering plant of Myrtaceae family from southern and southeastern Asia, was known to possess a rich source of structurally diverse and various biological activities. In this study, the inhibitory effect of R. tomentosa fruit extract (RFE) on allergic responses in calcium ionophore A23187-activated RBL-2H3 mast cells was investigated. The result showed that RFE was able to inhibit mast cell degranulation via decreasing ß-hexosaminidase release and intracellular Ca2+ elevation at the concentration of 400 µg/ml. Moreover, the suppressive effects of RFE on the production of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were evidenced. In addition, RFE effectively scavenged DPPH radical and suppressed the reactive oxygen species generation in a dose-dependent manner. Notably, the pretreatment of RFE caused the downregulation of tyrosine kinase Fyn phospholipid enzyme phospholipase Cγ (PLCγ), extracellular-signal-regulated kinase (ERK), and nuclear factor kappa B (NF-κB) phosphorylation. These results indicated that RFE could be a promising inhibitor of allergic responses and may be developed as bioactive ingredient for prevention or treatment of allergic diseases.

Microparticle Release from Cell Lines and Its Anti-Influenza Activity.

Microparticles (MPs) are vesicles that are released by budding from plasma membrane of living cells. Recently, the role of MPs in antiviral activity has been proposed. We investigated quantity and anti-influenza activity of MPs from human alveolar epithelial cells A549, human bronchial epithelial cells BEAS-2B, human colon adenocarcinoma cells HT-29, and the human lung fibroblast cells MRC-5. MPs were found from all four cell lines. However, anti-influenza activity against an H1N1 influenza virus was found only from MPs of A549 and BEAS-2B. BEAS-2B cell differentiation did not increase MP release. Methyl-ß-cyclodextrin (MßCD) increased MP release and anti-influenza activity in HT-29 and A549. MP release increased after calcium ionophore A23187 treatment in three cell lines but only in HT-29 after forskolin treatment. These findings provide in vitro data supporting the role of MPs as an innate defense against influenza virus and as an approach to enhance the defense.

Effects of calcium Ionophore A23187 on the apoptosis of hepatic stellate cells stimulated by transforming growth factor-ß1.

BACKGROUND: Our previous study showed that during in vitro experiments changes in calcium concentration were associated with apoptosis. We presumed that the calcium ion might play a role as intermediate messenger for apoptosis-related genes. No such evidence has been reported in the literature. Here, we investigate the effect of calcium ionophore A23187 on the apoptosis of rat hepatic stellate cells (HSCs) stimulated by transforming growth factor-ß1 (TGF-ß1) to explore the mechanism of apoptosis through the endoplasmic reticulum stress pathway. METHODS: The apoptotic rate was determined using flow cytometry. The changes in Ca2+ level in HSCs were examined with laser confocal microscopy. The expressions of caspase-12 GRP78 and caspase-9 were assayed via western blot. RESULTS: The respective apoptosis rates for the blank group, the TGF-ß1 group and the TGF-ß1 + low, medium and high dose calcium ionophore A23187 groups were 3.40 ± 0.10%, 1.76 ± 0.12%, 5.86 ± 0.31%, 11.20 ± 0.48% and 15.08 ± 0.75%, with significant differences between the groups (p < 0.05). The concentration of Ca2+and the expression of the GRP78, caspase-9 and caspase-12 proteins significantly increased with increasing calcium ionophore A23187 doses (p < 0.05). CONCLUSION: Calcium ionophore A23187 increased intracellular Ca2+ and activated endoplasmic reticulum stress, which promoted HSC apoptosis.

Molecular mechanisms of anti-inflammatory effect of chrysophanol, an active component of AST2017-01 on atopic dermatitis in vitro models.

AST2017-01 mainly consists of Rumex crispus and -Cordyceps militaris and has been widely consumed as an herbal medicine or functional food in Korea. Here we investigated the influences of AST2017-01 and its active component, chrysophanol on human mast cell (HMC-1 cell) and human keratinocyte (HaCaT cell)-mediated inflammatory reactions. Pretreatment with AST2017-01 or chrysophanol suppressed intracellular calcium levels and histamine release in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-treated HMC-1 cells. Levels of phosphorylated-mitogen-activated protein kinase increased by PMACI stimulation were reduced by AST2017-01 or chrysophanol pretreatment. Protein levels of IκB kinaseß and receptor-interacting protein 2 in PMACI-treated HMC-1 cells were decreased by AST2017-01 or chrysophanol pretreatment. Pretreatment with AST2017-01 or chrysophanol significantly blocked PMACI-induced activation of caspase-1 and nuclear factor-κB. In addition, pretreatment with AST2017-01 or chrysophanol significantly decreased the PMACI-induced levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and thymic stromal lymphopoietin (TSLP) on HMC-1 cells. In activated HaCaT cells, pretreatment with AST2017-01 or chrysophanol significantly reduced production of TSLP and activation of caspase-1. In conclusion, these findings indicate that chrysophanol is an active component of AST2017-01 and AST2017-01 acts as a novel potent anti-inflammatory herbal medicine or functional food.

Golgi-independent routes support protein disulfide isomerase externalization in vascular smooth muscle cells.

Extracellular pools of intracellular molecular chaperones are increasingly evident. The peri/epicellular(pec) pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1(PDI) is involved in thrombosis and vascular remodeling, while PDI externalization routes remain elusive. In endothelial cells, vesicular-type PDI secretion involves classical and unconventional pathways, while in platelets PDI exocytosis involves actin cytoskeleton. However, little is known about pecPDI in vascular smooth muscle cells(VSMC). Here, we showed that VSMC display a robust cell-surface(cs) PDI pool, which binds to cs independently of electrostatic forces. However, contrarily to other cells, soluble secreted PDI pool was undetectable in VSMC. Calcium ionophore A23187 and TNFα enhanced VSMC csPDI. Furthermore, VSMC PDI externalization occurred via Golgi-bypass unconventional route, which was independent of cytoskeleton or lysosomes. Secreted PDI was absent in ex vivo wild-type mice aortas but markedly enhanced in PDI-overexpressing mice. Such characterization of VSMC pecPDI reinforces cell-type and context specific routes of PDI externalization.

Cynanchum atratum inhibits the development of atopic dermatitis in 2,4-dinitrochlorobenzene-induced mice.

Cynanchum atratum Bunge (Apocynaceae) is a folk medicine to treat skin inflammatory diseases. However, the effects of C. atratum on atopic dermatitis have not been elucidated. In this study, we evaluate the effects of aqueous extract of C. atratum (CA) and its molecular mechanism on atopic dermatitis (AD). 1 and 100mg/mL CA were topically applied to 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions for 11 days. The number of scratching behavior was evaluated for 20min. AD-like symptoms including elevated serum IgE, skin hyperplasia and mast cell infiltration were investigated. The expressions of pro-inflammatory cytokines and mediators were analyzed in AD-like skin legions. In addition, pro-inflammatory cytokine production was confirmed in human mast cells (HMC)-1 stimulated with PMA plus A23187 (PMACI). Topical application of CA attenuated total serum IgE level and scratching behavior. Skin hyperplasia including epidermis and dermis was ameliorated in CA-treated skin legions. The number of infiltrated mast cells was significantly decreased by CA treatment. In addition, CA reduced pro-inflammatory cytokines, such as IL-6, IL-1ß and TNF-α and Th2 cytokine, IL-4, in both of AD-like skin lesions and PMACI-sensitized HMC-1 cells. Furthermore, CA decreased the expressions of NF-κB, phospho-IκBα and MAP kinase. These results suggest the inhibitory effects of CA on the development of AD by regulating pro-inflammatory cytokines and mediators. CA could be an effective substance for the treatment of AD.