加味四君子汤通过影响5-羟色胺及其受体对斑马鱼肠道出血模型的止血效应及其机制探讨  

The hemostatic effect and its mechanism of supplemented Sijunzi Decoction on the zebrafish intestinal bleeding model through the influence of 5-hydroxytryptamine and its receptors

作　　者：王珺[1] 郎海燕 周佳丽 张雪莹 亢炎红 张蕴[1] 陈信义 马薇 WANG Jun;LANG Haiyan;ZHOU Jiali;ZHANG Xueying;KANG Yanhong;ZHANG Yun;CHEN Xinyi;MA Wei(The First Affiliated Hospital of Zhejiang Chinese Medical University,Zhejiang Provincial Hospital of Chinese Medicine,Hangzhou 310000,China;Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;Hunter Biotechnology,Inc,Hangzhou 310051,China)

机构地区：[1]浙江中医药大学附属第一医院(浙江省中医院),杭州310000 [2]北京中医药大学东直门医院 [3]杭州环特生物科技股份有限公司

出　　处：《北京中医药大学学报》2024年第4期516-522,共7页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.81803904);浙江省自然科学基金项目(No.LQ23H270001);浙江省中医药管理局项目(No.2024ZL407)。

摘　　要：目的探讨加味四君子汤(党参、茯苓、白术、炙甘草、黄芪、阿胶、茜草)通过影响5-羟色胺(5-HT)及其受体(5-HT2aR、5-HT2bR)对斑马鱼肠道出血模型的止血作用及其机制。方法以受精后4 d的斑马鱼为研究对象,6μmol/L辛伐他汀诱导24 h建立肠道出血模型。将斑马鱼利用随机数字表法分为正常组,模型组,加味四君子汤低、中、高质量浓度组(55.6、167、500 mg/L),造模后,给予相应质量浓度加味四君子汤,24 h后收样。观察血小板计数(PLT)及出血情况,比色法检测5-HT含量及Caspase 3/7活性,实时PCR法检测5-HT2aR、5-HT2bR、5-HT转运蛋白(SERT)基因表达,蛋白质印迹法检测蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、细胞外调解蛋白激酶(ERK)、磷酸化ERK(p-ERK)蛋白表达。结果模型组肠道出血发生率为70.0%,加味四君子汤低、中、高质量浓度组出血发生率为36.7%、40.0%、80.0%,肠道止血作用分别为54%、52%、7%。与模型组比较,加味四君子汤低、中质量浓度组PLT减少;各质量浓度组5-HT含量升高、5-HT2aR及5-HT2bR基因表达上调,低、中质量浓度组SERT基因表达上调;中、高质量浓度组Caspase 3/7活性降低;低、中质量浓度组p-AKT、p-ERK蛋白表达上调(均P<0.05)。结论加味四君子汤可减少斑马鱼肠道出血模型的出血发生率及出血程度,其机制可能是通过5-HT及其受体激活AKT和ERK信号通路实现的。Objective We aimed to investigate the hemostatic effect and mechanism of supplemented Sijunzi Decoction(MSJZT),consisted of tangshen,India bread,largehead atractylodes rhizome,liquorice root,milkvetch root,ass hide glue,and India madder root,on the zebrafish intestinal bleeding model through the influence of 5-hydroxytryptamine(5-HT)and its receptors(5-HT2aR,5-HT2bR).Methods Zebrafish at 4 days post fertilization were used as the research object.An intestinal bleeding model was established by induction with 6μmol/L simvastatin for 24 hours.The zebrafish were divided into normal group,model group,MSJZT low-,mid-and high-concentration groups(55.6,167,500 mg/L),and after modeling,the corresponding concentration of MSJZT was administration.The samples were collected after 24 hours.Platelet count(PLT)and bleeding status were observed.The content of 5-HT and caspase 3/7 activity were detected by colorimetry.The gene expressions of 5-HT2aR,5-HT2bR and serotonin transporter(SERT)were detected by PCR.The protein expressions of protein kinase B(AKT),phosphorylated AKT(p-AKT),extracellular signal-regulated protein kinase(ERK),and phosphorylated ERK(p-ERK)were detected by Western blotting.Results The intestinal bleeding rate in the model group was 70.0%,and that in the MSJZT low-,mid-and high-concentration groups was 36.7%,40.0%,and 80.0%,respectively;The intestinal hemostatic effect was 54%,52%,and 7%,respectively.Compared with the model group,the PLT in the MSJZT low-and mid-concentration groups decreased;the content of 5-HT in all MSJZT groups increased,and the gene expressions of 5-HT2aR and 5-HT2bR were up-regulated.The gene expression of SERT was up-regulated in the low-and mid-concentration groups;the caspase3/7 activity in the mid-and high-concentration groups was reduced;the expressions of p-AKT and p-ERK proteins were up-regulated in the low-and mid-concentration groups(all P<0.05).Conclusion MSJZT can reduce the incidence and severity of intestinal bleeding in zebrafish intestinal bleeding models.The mechanism ma

关 键 词：免疫性血小板减少症 加味四君子汤 5-羟色胺 5-羟色胺受体 蛋白激酶B 细胞外调解蛋白激酶 斑马鱼 

分 类 号：R285.5[医药卫生—中药学]