肠安Ⅰ号方含药血清对致敏肥大细胞活化脱颗粒的影响

Effect of Medicated Serum Prepared with Chang’an Ⅰ Prescription on Sensitized Mast Cell Degranulation

目的:探讨肠安Ⅰ号方含药血清对免疫球蛋白E(IgE)介导的大鼠嗜碱性粒细胞(RBL-2H3)活化脱颗粒的影响及对非受体型酪氨酸蛋白激酶/脾酪氨酸蛋白激酶/丝裂原活化蛋白激酶(Lyn/Syk/MAPK)信号通路的调节作用。方法:制备肠安Ⅰ号方含药血清,将SD雄性大鼠随机分为肠安Ⅰ号方高、中、低剂量组及空白组,每组10只,给药剂量:空白组给予10 mL·kg-1蒸馏水灌胃,肠安Ⅰ号方低、中、高剂量组分别予1.15,2.30,4.60 g·kg-1药液灌胃,给药体积10 mL·kg-1,1次/d,连续灌胃7 d。细胞分组:空白组,予正常大鼠血清;模型组,予正常大鼠血清;酮替芬组,予正常大鼠血清+30μmol·L-1酮替芬。肠安Ⅰ号方低、中、高剂量组分别为予肠安Ⅰ号方低、中、高剂量含药血清。建立IgE介导的RBL-2H3细胞活化、脱颗粒模型,采用甲苯胺蓝染色进行肥大细胞计数;采用比色法检测细胞脱颗粒β-氨基己糖释放率;采用酶联免疫吸附测定(ELISA)检测细胞上清中肥大细胞类胰蛋白酶(MCT),肿瘤坏死因子-α(TNF-α),单核细胞趋化蛋白-1(MCP-1)及组胺含量;采用蛋白免疫印迹法(Western blot)检测Lyn/Syk/MAPK通路蛋白表达。结果:对于细胞活化脱颗粒,与空白组比较,模型组细胞β-氨基己糖释放率显著升高(P<0.01),脱颗粒率明显升高(P<0.05);与模型组比较,肠安Ⅰ号方含药血清各剂量组细胞β-氨基己糖释放率显著下降(P<0.01),细胞脱颗粒率明显下降(P<0.05)。对于活性介质释放,与空白组比较,模型组细胞上清组胺,MCT,TNF-α及MCP-1含量均显著增高(P<0.01);与模型组比较,肠安Ⅰ号方各剂量组细胞上清组胺,MCT,TNF-α及MCP-1含量均显著减低(P<0.01)。与正常组比较,模型组细胞Lyn和Syk及细胞外调节蛋白激酶1/2(ERK1/2),c-Jun氨基末端激酶(JNK),p38 MAPK磷酸化水平明显升高(P<0.05);与模型组比较,肠安Ⅰ号方各剂量Lyn,Syk及ERK1/2,JNK及p38蛋白磷酸化水平明显降低(P<0.05)。结论:肠安Ⅰ号方含药血清通过下调RBL-2H3细胞活化上游信号通路关键蛋白Lyn,Syk和下游ERK1/2,JNK及p38蛋白的磷酸化水平,抑制肥大细胞活化脱颗粒,减少组胺,MCT,TNF-α及MCP-1等活性介质释放,这可能是其抑制肥大细胞活化,治疗腹泻型肠易激综合征(IBS-D)内脏高敏感的机制之一。

Objective:To observe the influence of Chang’an Ⅰ prescription drug-containing serum on IgE-mediated RBL-2 H3 cell degranulation model,and explore the mechanism of Chang’an Ⅰ prescription in inhibiting RBL-2 H3 activation degranulation and releasing inflammatory mediators with v-yes-1 Yanaguchi sarcoma viral related oncogene homolog(Lyn)/spleen tyrosine protein kinase(Syk)/mitogen-activated protein kinase(MAPK)signal pathway. Method: Preparation for Chang’an Ⅰ prescription serum. Animal group,SD male rats were randomly divided into Chang’an Ⅰ prescription serum high,medium,low dose,and blank control groups with 10 rats in each group. Dosage:10 mL·kg-1 distilled water was given to blank control group,while Chang’an Ⅰ prescription serum high,medium and low dose groups were respectively given to the Chang’an Ⅰ prescription concentrated crude drug with concentration of 1.15,2.30,4.60 g·kg-1,respectively once a day for 7 days continuously and then blood was taken from aorta ventralis and centrifuged. Ketotifen as the positive control drug. Mast cells are counted with toluidine blue staining. Cellular release of β-aminohexose was detected by colorimetric method. Contents of MCT,TNF-α,MCP-1 and histamine were measured by enzymelinked immunosorbent assay(ELISA)kits,Lyn/Syk/MAPK protein levels were detected by immunoblotting.Result: For cell activation and degranulation,compared with the blank control group,the model group had more cell degranulation(P<0.05),compared with model group,the cell degranulation rate of each dose group of Chang’an Ⅰ prescription decreased(P<0.05). The release rate of β-hexosamine in each dose group of Chang’an Ⅰ prescription decreased significantly(P<0.01). For the release of active mediators,compared with the blank control group,the contents of histamine,MCT,TNF-α and MCP-1 all increased in the model group(P<0.01),compared with the model group,the contents in each dose group of Chang’an Ⅰ prescription all decreased significantly(P<0.01). Compared with the normal group,the phosphorylation levels of Lyn and Syk,extracellular regulatory protein kinase 1/2(ERK1/2),c-Jun N-terminal kinase(JNK),and mitogen-activated protein kinase p38 increased in the model group(P<0.05). Compared with the model group,the Lyn,Syk and ERK1/2,JNK and p38 protein phosphorylation levels reduced in Chang’an Ⅰ prescription group(P<0.05).Conclusion: Chang’an Ⅰ prescription drug-containing serum down-regulates the phosphorylation levels of proteins Lyn,Syk,and ERK1/2,JNK,and p38,inhibits RBL-2 H3 cell activation and degranulation,reduces the release of cytokines and chemokines,such as histamine,MCT,TNF-α and MCP-1,it may be one of its mechanisms for treating IBS-D visceral hypersensitivity.