目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同...目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同浓度药物对RBL-2H3肥大细胞分泌组胺及TNF-α的影响。结果丹皮酚对RBL-2H3细胞增殖有抑制作用,IC50值为0.22 mg/mL。RBL-2H3细胞活化脱颗粒,分泌组胺的释放率在前30 m in上升较快,30 m in后上升进入平台期;TNF-α分泌的量在1h达高峰。丹皮酚抑制RBL-2H3细胞释放组胺和TNF-α作用不弱于色甘酸钠0.1,0.5 mg/mL。丹皮酚浓度的对数与其对RBL-2H3细胞释放组胺和TNF-α的抑制率呈线性相关(P=0.000<0.01)。结论丹皮酚呈剂量依赖性抑制RBL-2H3细胞分泌组胺和TNF-α。展开更多
Guided by cell-based anti-anaphylactic assay,eighteen cage-like monoterpenoid glycosides(1−18)were obtained from the bioactive fraction of P.lactiflora extract.Among these,compounds 1,5,6,11,12,15,and 17 significantly...Guided by cell-based anti-anaphylactic assay,eighteen cage-like monoterpenoid glycosides(1−18)were obtained from the bioactive fraction of P.lactiflora extract.Among these,compounds 1,5,6,11,12,15,and 17 significantly reduced the release rate ofβ-HEX and HIS without or with less cytotoxicity.Furthermore,the most potent inhibitor benzoylpaeoniflorin(5)was selected as the prioritized compound for the study of action of mechanism,and its anti-anaphylactic activity was medicated by dual-inhibiting HDC and MAPK signal pathway.Moreover,molecular docking simulation explained that benzoylpaeoniflorin(5)blocked the conversion of L-histidine to HIS by occupying the HDC active site.Finally,in vivo on PCA using BALB/c mice,benzoylpaeoniflorin(5)suppressed the IgE-mediated PCA reaction in antigen-challenged mice.These findings indicated that cage-like monoterpenoid glycosides,especially benzoylpaeoniflorin(5),mainly contribute to the anti-anaphylactic activity of P.lactiflora by dual-inhibiting HDC and MAPK signal pathway.Therefore,benzoylpaeoniflorin(5)may be considered as a novel drug candidate for the treatment of anaphylactic diseases.展开更多
Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated ...Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic展开更多
The present study aimed at developing a natural compound with anti-allergic effect and stability under latex glove manufacturing conditions and investigating whether its anti-allergic effect is maintained after its ad...The present study aimed at developing a natural compound with anti-allergic effect and stability under latex glove manufacturing conditions and investigating whether its anti-allergic effect is maintained after its addition into the latex. The effects of nine natural compounds on growth of the RBL-2H3 cells and mouse primary spleen lymphocytes were determined using MTT assay. The compounds included glycyrrhizin, osthole, tetrandrine, tea polyphenol, catechin, arctigenin, oleanolic acid, baicalin and oxymatrine. An ELISA assay was used for the in vitro anti-type I/IV allergy screening; in this process β-hexosaminidase, histamine, and IL-4 released from RBL-2H3 cell lines and IFN-γ and IL-2 released from mouse primary spleen lymphocytes were taken as screening indices. The physical stability of eight natural compounds and the dissolubility of arctigenin, selected based on the in vitro pharnacodynamaic screening and the stability evaluation, were detected by HPLC. The in vivo pharmacodynamic confirmation of arctigenin and final latex product was evaluated with a passive cutaneous anaphylaxis(PCA) model and an allergen-specific skin response model. Nine natural compounds showed minor growth inhibition on RBL-2H3 cells and mouse primary spleen lymphocytes. Baicalin and arctigenin had the best anti-type I and IV allergic effects among the natural compounds based on the in vitro pharmacodynamic screening. Arctigenin and catechin had the best physical stability under different manufacturing conditions. Arctigenin was the selected for further evaluation and proven to have anti-type I and IV allergic effects in vivo in a dose-dependent manner. The final product of the arctigenin-containing latex glove had anti-type I and IV allergic effects in vivo which were mainly attributed to arctigenin as proved from the dissolubility results. Arctigenin showed anti-type I and IV allergic effects in vitro and in vivo, with a good stability under latex glove manufacturing conditions, and a persistent anti-allergic effect after being 展开更多
Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-asso...Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-associated binder 2） siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2- mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl （DNP）-specific IgE overnight and challenged with dinitrophenyl-human serum albumin （DNP-HSA） to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of β-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcεRI-mediated mast cell release of β-hexosaminidase and histamine, reduced the production of IL-4 and TNF-α and inhibited the phosphorylation of Akt, PKC8 and p38 mitogen-activated protein kinase （MAPK）. Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the 2＋ FcεRI-mediated Ca -independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated. Cellular ＆ Molecular Immunology. 2008; 5（6）：433-438.展开更多
<span style="font-family:Verdana;">Backgrou</span><span style="font-family:Verdana;">nd/Aim: Kuji amber is an interesting natural source for drug discovery</span><span>...<span style="font-family:Verdana;">Backgrou</span><span style="font-family:Verdana;">nd/Aim: Kuji amber is an interesting natural source for drug discovery</span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materia</span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">ls and </span><span style="font-family:Verdana;">Method: Biological activities of each extract were evaluated using growth-restoring </span><span style="font-family:Verdana;">activity of the mutant yeast strain involving Ca</span><sup><span style="font-family:Verdana;">2+</span></sup><span style="font-family:Verdana;">-signal transduction and inhibi</span><span style="font-family:Verdana;">tion activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents</span><span style="font-family:Verdana;"> of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers app展开更多
Objective Huangqi Injection is a preparation with an extract of Astragali Radix which has a long history of being used as a tonic to strengthen the body＇s immunity. Anaphylaxis and hemolysis are two main adverse drug...Objective Huangqi Injection is a preparation with an extract of Astragali Radix which has a long history of being used as a tonic to strengthen the body＇s immunity. Anaphylaxis and hemolysis are two main adverse drug reaction （ADR） of injections. Our study was aimed to establish an approach for the （ADR） prediagnosis of Huangqi Injection. Methods An in vitro model for anaphylactoid assay of Huangqi Injection based on the release rate of histamine and 13-hexosaminidase of RBL-2H3 cells induced by injections and a colorimetric method based on the detection of hemoglobin resulted in the erythrocyte hemolysis for prediagnostic assaying the hemolytic ADR of injections were established. Results Both histamine and ^-hexosaminidase are the anaphylactiod mediators, but 13-hexosaminidase release induced by Huangqi Injection could not be determined by spectrophotometry due to the interference of the injection itself. In addition, normal hemolysis and abnormal hemolysis were discovered during the experiment. The fingerprints and tannins in different batches of injections showed obvious differences, indicating that the content of tannins was related to abnormal hemolysis and higher histamine-secreting from RBL-2H3 cells. Conclusion The results indicate that the hemolytic assaying method is not only suitable for prediagnostic assaying of hemolytic ADR of herbal medicine injection, but also partly reflects the anaphylaxis of herbal injections, and tannins may be the major factors causing abnormal hemolysis.展开更多
文摘目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同浓度药物对RBL-2H3肥大细胞分泌组胺及TNF-α的影响。结果丹皮酚对RBL-2H3细胞增殖有抑制作用,IC50值为0.22 mg/mL。RBL-2H3细胞活化脱颗粒,分泌组胺的释放率在前30 m in上升较快,30 m in后上升进入平台期;TNF-α分泌的量在1h达高峰。丹皮酚抑制RBL-2H3细胞释放组胺和TNF-α作用不弱于色甘酸钠0.1,0.5 mg/mL。丹皮酚浓度的对数与其对RBL-2H3细胞释放组胺和TNF-α的抑制率呈线性相关(P=0.000<0.01)。结论丹皮酚呈剂量依赖性抑制RBL-2H3细胞分泌组胺和TNF-α。
基金This work was supported by the National Natural Science Foundation of China(Nos:81773996,81773589,500101135,and 81522050)Beijing Natural Science Foundation(No.JQ18026).
文摘Guided by cell-based anti-anaphylactic assay,eighteen cage-like monoterpenoid glycosides(1−18)were obtained from the bioactive fraction of P.lactiflora extract.Among these,compounds 1,5,6,11,12,15,and 17 significantly reduced the release rate ofβ-HEX and HIS without or with less cytotoxicity.Furthermore,the most potent inhibitor benzoylpaeoniflorin(5)was selected as the prioritized compound for the study of action of mechanism,and its anti-anaphylactic activity was medicated by dual-inhibiting HDC and MAPK signal pathway.Moreover,molecular docking simulation explained that benzoylpaeoniflorin(5)blocked the conversion of L-histidine to HIS by occupying the HDC active site.Finally,in vivo on PCA using BALB/c mice,benzoylpaeoniflorin(5)suppressed the IgE-mediated PCA reaction in antigen-challenged mice.These findings indicated that cage-like monoterpenoid glycosides,especially benzoylpaeoniflorin(5),mainly contribute to the anti-anaphylactic activity of P.lactiflora by dual-inhibiting HDC and MAPK signal pathway.Therefore,benzoylpaeoniflorin(5)may be considered as a novel drug candidate for the treatment of anaphylactic diseases.
文摘Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic
基金supported by National Science and Technology Infrastructure Program of China(No.2012BAI30B001)Technological Innovation Team of Jiangsu Higher Education+3 种基金National Natural Science Foundation of China(Nos.3087315630672524)New Century Excellent Talents in University(NCET-07-0851)Mega-projects of Science Research for the 12th Five-Year Plan of China(No.2011AX09401-007)
文摘The present study aimed at developing a natural compound with anti-allergic effect and stability under latex glove manufacturing conditions and investigating whether its anti-allergic effect is maintained after its addition into the latex. The effects of nine natural compounds on growth of the RBL-2H3 cells and mouse primary spleen lymphocytes were determined using MTT assay. The compounds included glycyrrhizin, osthole, tetrandrine, tea polyphenol, catechin, arctigenin, oleanolic acid, baicalin and oxymatrine. An ELISA assay was used for the in vitro anti-type I/IV allergy screening; in this process β-hexosaminidase, histamine, and IL-4 released from RBL-2H3 cell lines and IFN-γ and IL-2 released from mouse primary spleen lymphocytes were taken as screening indices. The physical stability of eight natural compounds and the dissolubility of arctigenin, selected based on the in vitro pharnacodynamaic screening and the stability evaluation, were detected by HPLC. The in vivo pharmacodynamic confirmation of arctigenin and final latex product was evaluated with a passive cutaneous anaphylaxis(PCA) model and an allergen-specific skin response model. Nine natural compounds showed minor growth inhibition on RBL-2H3 cells and mouse primary spleen lymphocytes. Baicalin and arctigenin had the best anti-type I and IV allergic effects among the natural compounds based on the in vitro pharmacodynamic screening. Arctigenin and catechin had the best physical stability under different manufacturing conditions. Arctigenin was the selected for further evaluation and proven to have anti-type I and IV allergic effects in vivo in a dose-dependent manner. The final product of the arctigenin-containing latex glove had anti-type I and IV allergic effects in vivo which were mainly attributed to arctigenin as proved from the dissolubility results. Arctigenin showed anti-type I and IV allergic effects in vitro and in vivo, with a good stability under latex glove manufacturing conditions, and a persistent anti-allergic effect after being
基金supported by Youth Foundation of Jinan University (2007, No. 51208022)
文摘Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-associated binder 2） siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2- mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl （DNP）-specific IgE overnight and challenged with dinitrophenyl-human serum albumin （DNP-HSA） to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of β-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcεRI-mediated mast cell release of β-hexosaminidase and histamine, reduced the production of IL-4 and TNF-α and inhibited the phosphorylation of Akt, PKC8 and p38 mitogen-activated protein kinase （MAPK）. Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the 2＋ FcεRI-mediated Ca -independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated. Cellular ＆ Molecular Immunology. 2008; 5（6）：433-438.
文摘<span style="font-family:Verdana;">Backgrou</span><span style="font-family:Verdana;">nd/Aim: Kuji amber is an interesting natural source for drug discovery</span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract</span></span></span><span><span><span style="font-family:;" "=""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materia</span></span></span><span><span><span style="font-family:;" "=""><span style="font-family:Verdana;">ls and </span><span style="font-family:Verdana;">Method: Biological activities of each extract were evaluated using growth-restoring </span><span style="font-family:Verdana;">activity of the mutant yeast strain involving Ca</span><sup><span style="font-family:Verdana;">2+</span></sup><span style="font-family:Verdana;">-signal transduction and inhibi</span><span style="font-family:Verdana;">tion activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents</span><span style="font-family:Verdana;"> of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers app
基金National Natural Science Foundation of China(No.81373939)Innovation Team Project of Colleges and Universities in Liaoning Province at 2013(LT2013020)
文摘Objective Huangqi Injection is a preparation with an extract of Astragali Radix which has a long history of being used as a tonic to strengthen the body＇s immunity. Anaphylaxis and hemolysis are two main adverse drug reaction （ADR） of injections. Our study was aimed to establish an approach for the （ADR） prediagnosis of Huangqi Injection. Methods An in vitro model for anaphylactoid assay of Huangqi Injection based on the release rate of histamine and 13-hexosaminidase of RBL-2H3 cells induced by injections and a colorimetric method based on the detection of hemoglobin resulted in the erythrocyte hemolysis for prediagnostic assaying the hemolytic ADR of injections were established. Results Both histamine and ^-hexosaminidase are the anaphylactiod mediators, but 13-hexosaminidase release induced by Huangqi Injection could not be determined by spectrophotometry due to the interference of the injection itself. In addition, normal hemolysis and abnormal hemolysis were discovered during the experiment. The fingerprints and tannins in different batches of injections showed obvious differences, indicating that the content of tannins was related to abnormal hemolysis and higher histamine-secreting from RBL-2H3 cells. Conclusion The results indicate that the hemolytic assaying method is not only suitable for prediagnostic assaying of hemolytic ADR of herbal medicine injection, but also partly reflects the anaphylaxis of herbal injections, and tannins may be the major factors causing abnormal hemolysis.
