Glaucocalyxin A attenuates allergic responses by inhibiting mast cell degranulation through HMGB1/TLR4/NF-κB signaling pathways / 药学学报

The study is to investigate the effect of glaucocalyxin A (GLA) on mast cell-mediated anaphylaxis. The animal welfare and experimental process of this experiment followed the regulations of the Animal Ethics Committee of Yanbian University. BALB/c mice were used in the animal experiment and randomly divided into five groups, control group, model group, and GLA low, medium, and high dose groups (10, 20, and 40 mg·kg-1). Mice were sensitized by intradermal injection of anti-dinitrophenyl-immunoglobulin E (DNP-IgE) into the ears and challenged with a mixture of DNP-human serum albumin (HSA) and 4% evans blue into the tail veins to prepare an animal skin passive cutaneous anaphylaxis (PCA) model, which was collected from both ears for measurement of dye staining and histology. Rat peritoneal mast cells (RPMCs) were used in the cell experiment and divided into control, IgE + antigen (Ag), and IgE + Ag + GLA groups to determine histamine release as well as calcium influx levels. High-affinity IgE receptor (FcεRI)-mediated signaling pathway proteins and HMGB1/TLR4/NF-κB (high mobility group box 1/toll like receptor 4/nuclear transcription factor kappa B) signaling proteins were detected by Western blot. The results of animal experiments suggest that GLA inhibits PCA, reduces evans blue dye exudation, and reduces ear inflammation and ear thickness in mice. The results of cellular experiments suggested that GLA could reduce histamine release and calcium influx, and inhibit tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-13, and IL-1β production; Western blot results showed that GLA inhibited FcεRI-mediated phosphorylation levels of spleen tyrosine kinase (Syk), Lck/Yes novel tyrosine kinase (Lyn), tyrosine kinase Fyn (Fyn), growth-factor receptor-bound protein 2 (Gab2), and phospholipase C (PLC) γ1, while GLA inhibited HMGB1/TLR4 signaling pathway to limit NF-κB p65 nuclear metastasis. The results indicate that GLA inhibits mast cell degranulation and attenuates allergic inflammation through the HMGB1/TLR4/NF-κB signaling pathway.

Effect of electroacupuncture on expressions of Lyn and Syk in mast cells of subcutaneous loose connective tissue in rats with urticarial / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria.@*METHODS@#A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg•d) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells.@*RESULTS@#In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (<0.01).@*CONCLUSION@#Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.

Mechanism of Yupingfeng Granule on CU Rats Based on IL-23/IL-17 Inflammation Axis / 中国实验方剂学杂志

Objective::To study the effect of Yupingfeng granule on the degranulation of skin mast cells in chronic urticaria (CU) rats and the intervention mechanism of interleukin-23(IL-23), interleukin-17(IL-17) inflammation axis. Method::Totally 60 SPF SD rats were selected and randomly divided into normal group (normal saline), model group (normal saline), and loratadine group (0.9 mg·kg-1·d-1), high-dose Yupingfeng granules group (4.05 g·kg-1·d-1), middle-dose group (2.7 g·kg-1·d-1), low-dose group (1.35 g·kg-1·d-1). The CU rat model was reproduced through intraperitoneal injection of ovalbumin with aluminum hydroxide suspension and DTP vaccine. Histopathological changes of rat skin were observed by hematoxylin-eosin (HE) staining. Degranulation of mast cells in rat skin was determined by toluidine blue staining. IL-23 and IL-17 protein expressions in skin tissue were determined by immunohistochemistry (IHC). IL-23 and IL-17 mRNA transcription levels in skin tissue were determined by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result::Yupingfeng granules can significantly alleviate the pathological manifestations of dermal edema, collagen beam distance, inflammatory cell infiltration of CU rats, and reduce the degranulation reaction of skin tissue mast cells in CU rats. The IL-23, IL-17 mRNA and protein expressions of the skin of model group were significantly increased compared with the normal group (P<0.05, P<0.01). Compared with the model group, Yupingfeng granules can significantly down-regulate IL-23 mRNA and protein expressions of CU rats (P<0.05, P<0.01). Yupingfeng granules had no significant regulatory effect on IL-17. Conclusion::Yupingfeng granule can significantly reduce the degranulation of mast cells in skin tissue of CU rats, and improve the pathological manifestations, such as dermal edema, serous exudation and inflammatory cell infiltration. The mechanism may be related to inhibiting the secretion of IL-23 pro-inflammatory cytokines and improving CU lesions.

Electroacupuncture improves cutaneous allergic reaction by inhibiting degranulation of intrape-ritoneal mast cells, MAPK signaling and inflammatory factor levels in urticaria rats / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on degranulation of intraperitoneal mast cells (MCs) and expression of mitogen-activated protein kinase (MAPK) signaling related proteins, tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) in urticaria rats, so as to reveal its mechanisms underlying improvement of urticaria. METHODS: Thirty-two SD rats were randomly divided into control，model，EA and medication groups (n＝8 in each group). The urticaria model was established by using passive cutaneous anaphylaxis (PCA) reaction method. EA (2 Hz /15 Hz, 1 mA) was applied to bilateral "Zusanli"(ST36), "Quchi "(LI11) and "Xuehai"(SP10) for 20 min，once daily for 7 consecutive days before antigen attack. Rats of the medication group were treated by gavage of Loratadine(1 mg•kg－1•d－1)for 7 days. The diameter of cutaneous Evan's blue spots was measured to evaluate the severity of PCA. Intraperitoneal fluid smears were prepared to observe the degranulation state of MCs. The contents of TNF-α and IL-6 in the intraperitoneal fluid were detected by ELISA, and the expression of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, c-Jun N-terminal kinase (JNK), p-JNK, P38MAPK and p-P38MAPK of the acquired intraperitoneal MCs was detected by Western blot. RESULTS: The diameter of cutaneous Evan's blue spot was significantly increased in the model group than that in the control group (P<0.01), and considerably decreased in both EA and medication groups compared with the model group(P<0.01). After modeling，the percentage of degranulated MCs, contents of TNF-α and IL-6, and expression levels of ERK, p-ERK, JNK, p-JNK, P38MAPK and p-P38MAPK were remarkably increased in the mo-del group than those in the control group (P<0.01, P<0.05). After the treatment, the percentage of degranulated MCs, contents of TNF-α and IL-6, and expression levels of p-ERK, JNK, p-JNK and p-P38MAPK were obviously decreased in both EA and medication groups relevant to the model group (P<0.01, P<0.05), while no significant changes were found in the expression of ERK in both EA and medication groups, and P38MAPK in the EA group. Compared with the model and EA groups, expression levels of P38MAPK were down-regulated in the medication group (P<0.05). CONCLUSION: EA can reduce skin allergic reaction in rats with urticaria, which may be related to its effects in inhibiting the degranulation of intraperitoneal MCs, down-regulating the expression of MAPK signaling-related proteins and the level of pro-inflammatory factors TNF-α and IL-6 in intraperitoneal MCs.

Pathogenic effect of bacterial flagellin in TNBS-induced colitis in mice / 中国病理生理杂志

AIM:To detect the expression of CBir1 in the serum and colon tissue and mast cell degranulation in the tissue of 2,4,6-trinito-benzene-sulfonic acid ( TNBS)-induced colitis in mice with different interventions. ME-THODS:SPF male BALB/c mice were randomized into 6 groups (12 mice in each group):normal control group, normal saline group, 50% alcohol group, 50% alcohol+TNBS group, 50% alcohol+TNBS+lipopolysaccharide (LPS) +ovalbu-min (OVA) group and 50% alcohol+TNBS+ketotifen group. Corresponding treatment was given to each group, and the disease activity index (DAI) of the mice was evaluated. The mice were sacrificed on day 22 after treatment. The colon tis-sues were evaluated by histological index (HI) scoring. Serum concentrations of anti-CBir1, mast cell tryptase (MCT) and histamine were measured by ELISA. The expression of CBir1, toll-like receptor 5 (TLR5) and MCT in the colon tissues was detected by immunohistochemistry. RESULTS:Compared with the normal control group, the DAI score, HI score and CBir1, anti-CBir1, MCT, TLR5, histamine concentrations in colon tissues and serum were all significantly higher in 50% alcohol+TNBS group, 50% alcohol+TNBS+ketotifen group and 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, CBir1, MCT, histamine levels in 50% alcohol+TNBS group were lower than those in 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, TLR5, hista-mine, CBir1 Levels in 50% alcohol+TNBS group were higher than those in 50% alcohol+TNBS+ketotifen group ( P<0.05). Normal saline group and 50% alcohol group had no statistically significant difference in comparison with normal control group. In TNBS model group, serum concentration of anti-CBir1 was positively correlated with MCT concentration (r=0.648, P<0.01) and histamine concentration (r=0.751, P<0.01). CONCLUSION:The heavier degree of in-flammation in TNBS-induced colitis, the higher levels of the CBir1 and the degranulation of mast cells. There is a positive correlation between the expression of CBir1 and the degranulation of mast cells in TNBS-induced colitic mice.

Ameliorative effects of type-A procyanidins polyphenols from cinnamon bark in compound 48/80-induced mast cell degranulation / 대한해부학회지

Allergic diseases are a significant health concern in developing countries. Type-A procyanidin polyphenols from cinnamon (Cinnamomum zeylanicum Blume) bark (TAPP-CZ) possesses antiasthmatic and antiallergic potential. The present study was aimed at the possible anti-allergic mechanism of TAPP-CZ against the compound 48/80 (C48/80)–induced mast cell degranulation in isolated rat peritoneal mast cells (RPMCs). TAPP-CZ (1, 3, 10, and 30 µg/ml) was incubated for 3 hours with isolated, purified RPMCs. The C48/80 (1 µg/ml) was used to induce mast cell degranulation. The mast cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay whereas histamine, β-hexosaminidase (β-HEX), and interleukin-4 (IL-4) levels were determined in RPMCs. TAPP-CZ (3, 10, and 30 µg/ml) showed significant and dose-dependent decrease in a number of degranulated cells and levels of markers (histamine, β-HEX, and IL-4) as compared with C48/80 control. In conclusion, TAPP-CZ stabilizes mast cell and cause inhibition of the allergic markers such as histamine, IL-4, and β-HEX in IgE-mediated manner. The present study supports mast cell stabilization as a possible mechanism of action of TAPP-CZ against immune respiratory disorders such as asthma and allergic rhinitis.

Effects of artemisinin on early - phase response of allergic conjunctivitis in a murine model induced by pollen / 国际眼科杂志(Guoji Yanke Zazhi)

? AIM: To investigate the immunosuppression of artemisinin in the early-phase response( EPR) of allergic conjunctivitis in a murine model induced by pollen.?METHODS:Twenty-five Balb/c mice were divided into five groups at random: A: model group, B: artemisinin topical treatment group, C: oral artemisinin group, D:dexamethasone topical treatment group, E: negative control group. Balb/c mice were first sensitized with mixture of ragweed crude extract and complete Freund’s adjuvant by left footpad and root of tails injection at days 0. At days 7 and 14, mice were strengthen sensitized by intraperitoneal injection. Negative control group replacd with a mixture of equal amounts of Freund’s adjuvant and PBS mixture. Treatment time was between 21-27d:group B received 1% topical artemisinin eye drops 4 times per day; group C received artemisinin at doses of 600mg/kg orally once per day; group D received topical dexamethasone eye drops 4 times per day. The model and naive groups replaced with solvent for control. After treatment, mice were excited by ragweed pollen crude infusion drops. Within 1h after the excitation, the eyes were taken for histopathology testing, the serum for detection of specific IgE for ragweed pollen.?RESULTS:After excitation, allergy symptoms of model group were more significantly compared with negative control group and the treatment groups; allergy symptom score, mast cell degranulation ratios and the ragweed pollen specific IgE in serum in the topical treatment groups and oral group were higher than that in the negative control group, but significantly lower than that in model group.?CONCLUSION: Artemisinin topical treatment and oral treatment for mice could inhibit the symptoms of pollen allergic conjunctivitis, early-phase response of mast cell degranulation ratio and specific IgE generation for ragweed pollen, suggesting that artemisinin has some therapeutic effect for pollen allergic conjunctivitis.

Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 microg/ml BG, 100 microg/ml peptidoglycan (PGN), or 10 microM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation.

Detection of mast cell degranulation by fluorescent molecular diffusion dynamic measurement / 中国病理生理杂志

AIM:To establish a rapid in vitro method for mast cell degranulation tracing by raster image corre-lation spectroscopy (RICS).METHODS:RBL-2H3, a basophilic granulocyte mast cell line transfected with CD 63-GFP plasmid, was used for evaluating the methods , including β-hexosaminidase ( HEX) colorimetric assay, scanning electron microscopy (SEM) and RICS in the detection of mast cell degranulation .The sensibilities of these methods were com-pared.RESULTS:The sensitivities of β-HEX colorimetric assay and SEM were 5 mg/L and 3.9 ×10 -2 mg/L, respec-tively.RICS detection showed obvious decrease in the diffusion coefficient at dose of 3.9 ×10 -2 mg/L.CONCLUSION:Fluorescent molecular diffusion dynamic measurement can be used for rapid tracing of allergic substances in vitro.Accord-ing to the results, RICS can achieve nearly the same extent of sensitivity as the SEM does and is far more sensitive than β-HEX colorimetric assay.Compared with SEM, RICS has several advantages: it is faster, simpler and cheaper; it can be used in living cells;it is more suitable for rapid in vitro allergenic compounds tracing .Therefore, RICS is applicable in clinic allergic antigen screening and may also be used in pharmaceutical quality control .

Evaluation of anti-allergic and anti-anaphylactic activity of ethanolic extract of Zizyphus jujuba fruits in rodents

The present study reports the anti-allergic activity of ethanolic extract of Zizyphus jujuba Mill., Rhamnaceae, and its possible mode of action. The effect of extract of Z. jujuba at different doses (250, 500 and 1000 mg/kg, orally) was simulated on studied animal models of asthma and allergy: a) milk induced eosinophilia and leukocytosis; b) compound 48/80 induced mast cell degranulation; and, c) active and passive cutaneous anaphylaxis. In addition, extract of Z. jujuba's effect on sensitized guinea pig ileum (ex vivo) and tracheal chain preparations (in vitro) were investigated.Treatment with extract of Z. jujuba at all doses significantly: prevented the milk-induced eosinophilia and compound 48/80 induced degranulation of mesenteric mast cells; decreased passive cutaneous and active anaphylactic reactions. In addition, extract of Z. jujuba inhibited acetylcholine as well as histamine induced tracheal chain contraction, and also antigen induced contraction of sensitized guinea pig ileum (Shultz-Dale inhibition test). Furthermore, it exhibited also free radicals scavenging activity (in vitro). The observed anti-allergic and anti-anaphylactic activity of extract of Z. jujuba may be largely through the stabilization of mast cells by the membrane presence of phytoconstituents (steroidal saponins and flavonoids).

Anti-allergic, anti-inflammatory and anti-lipidperoxidant effects of Cassia occidentalis Linn.

Cassia occidentalis Linn. mast cell degranulation at a dose of 250 mg/kg, showed dose dependent stabilizing activity towards human RBC, with is widely used in traditional medicine of India to treat a number of clinical conditions including allergy and inflammatory manifestations. In the present study anti-allergic, anti-inflammatory and anti-oxidant properties of C. occidentalis whole plant ethanolic extract (CO) was investigated. Effects of CO on rat mast cell degranulation inhibition and human red blood cell (HRBC) membrane stabilization were studied in vitro following standard methods. The anti lipidperoxidant effects of CO were also studied in vitro. Effect of CO on carrageenan-induced mouse paw oedema inhibition was also assessed. CO significantly decreased maximum protection of 80.8% at 15 μg/ml. The extract also caused significant reduction in malondialdehyde (MDA) levels of murine hepatic microsomes at 100 μg/ml (56%) and significantly reduced carrageenan induced inflammation in mice at a dose of 250 mg/kg. Results of the present study indicated that CO inhibited mast cell degranulation, stabilized HRBC membrane thereby alleviating immediate hypersensitivity besides showing anti oxidant activity.

Inhibitory effect of Cortex mori on the substance P-induced mast cell degranulation, granular leukocyte infiltraion, and vascular permeability in vivo / 대한해부학회지

Cortex mori (Morus alba L.), the root bark of mulberry tree, has been used as an antiphlogistic, diuretic, and expectorant in herbal medicine. It has been reported tha hot water extract of Cortex mori has an inhibitory effect on substance P- and compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells. Substance P (SP), a decapeptide which is present in sensory neurons, is believed to be a major mediator of neurogenic inflammations. N-terminal peptide of SP causes mast cell degranulation and its C-terminal peptide directly activates vascular endothelial cells to increase the vascular permeability. It has recently been shown that SP induces leukocyte infiltration in the skin, which is mediated through mast cell degranulation. The purpose of this study was to investigate whether Cortex mori could inhibit SP-induced leukocyte infiltration in mouse skin. Subcutaneous administration of SP (10(-8) to 10(-5) M) induced leukocytes infiltration in the skin of BALB/c mice 8h after the injection. Leukocyte infiltration of mouse skin was associated with mast cell degranulation which was induced by SP1-9 (10(-7) to 10(-5) M), but not by SP6-11 (10(-7) to 10(-5) M) which was found to increase the vascular permeability of endothelial cells in mouse skin. However, SP6-11 (10(-7) to 10(-5) M) enhanced SP1-9-induced leukocyte infiltration in the skin without any significant increase in mast cell degranulation. Cortex mori (10 mg/ml) inhibited SP- and SP1-9-induced mast cell degranulation, but did not inhibite SP6-11-induced increase in vascular perameability. Taken together, the data indicate that the hot water extract of Cortex mori contains some substances with an activity to inhibit SP-induced leukocyte infiltration in to the mous skin. These activity of Cortex mori is likely due to the inhibition of release of chemical mediators from the mast cells which participate in neurogenic inflammations.

Effect of Periplogenin on Mast Cell Degranulation and Histamine Release / 中国药房

OBJECTIVE:To investigate the effects of Periplogenin, a compound extracted and purified from traditional Chinese medicine Cortex Periplocae, on mast cell degranulation and histamine release in mice and rats. METHODS: Sensitization was induced by injecting pertussis vaccine injected in rats' abdominal cavity and ovalbumin injected in mice's hind leg to detect mast cell degranulation reaction and prepare anti-serum; rats were injected intraperitoneally with the diluted serum sample taken form sensitized rats to detect mast cell degranulation reaction; and the concentration of histamine was determined by fluorescence spectrophotometry. RESULTS: Periplogenin showed significant inhibitory effect on histamine release of mast cell cultured in vitro, and at experimental dose it could decrease histamine release by(69.4?8.6)% in an obvious dose-depe-ndent manner. Periplogenin also showed obvious inhibitory effect on histamine release of mast cells in antigen-sensitized rats,and at a concentration of 20 ?g?mL-1,it could decrease histamine release by 73.55%, and at an oral dose of 50 mg?kg-1, it could dose-dependently reduce histamine release by above 80% in sensitized rats. CONCLUSION: Periplogenin showed significant inhibitory effects on histamine release of mast cells either cultured in vitro or in antigen-sensitized rats. Oral administration of Periplogenin can result in the significant reduction of histamine release of mast cells in rats. In view of the role that the mast cell histamine release and degranulation play in inflammation reaction, periplogenin can be regarded as one of the active anti-inflammation components of traditional Chinese medicine Cortex Periplocae.