黄芩苷调控炎性因子治疗肠道急性移植物抗宿主病的研究

Study on baicalin alleviating the symptoms of intestinal acute graft versus host disease by inhibiting inflammatory factors

目的探讨黄芩苷干预肠道急性移植物抗宿主病(acute graft versus host disease,aGVHD)的作用机制。方法分离采集健康志愿者和肠道aGVHD患者的外周血单个核细胞,富集分析其测序得到的差异mRNA,取差异mRNA与黄芩苷作用靶点的交集并进行分析。制备SPF级BALB/c H-2 d雄性小鼠的单核细胞悬液(1×10^(7)骨髓细胞+1×10^(7)脾细胞),将其通过尾静脉输入4 h内照射了60Co-X射线的SPF级CB6F1雌性小鼠,造模后小鼠随意分为模型组、黄芩苷组。另取6只CB6F1雌性小鼠作为正常组。造模后黄芩苷组给予30 mg/(kg·d)黄芩苷灌胃,模型组与正常组予等容量的生理盐水灌胃,连续14 d,每日观察小鼠生存状况。干预结束,检测小鼠血清细胞因子,进行小鼠肠道aGVHD相对评分、小肠黏膜病理炎症评分,小肠组织免疫组化、免疫荧光检测肿瘤坏死因子-α(TNF-α)、白细胞介素-2(IL-2)表达。结果筛选到健康志愿者与肠道aGVHD患者差异mRNA共2131个,KEGG分析发现集中在肿瘤坏死因子等通路上。分析差异基因与黄芩苷作用靶点交集,发现TNF-α、IL-2是黄芩苷治疗肠道aGVHD的核心靶点。小鼠aGVHD模型研究发现,经过黄芩苷治疗后,与模型组比较,黄芩苷组aGVHD相对评分、小肠黏膜病理炎症相对评分降低,血清TNF-α、IL-2下降,小肠免疫组化及免疫荧光检测的TNF-α、IL-2表达均降低,均P<0.05。结论黄芩苷能够抑制血清和小肠黏膜TNF-α、IL-2的水平和浸润,降低移植后肠道aGVHD的发病率。

Objective To explore the mechanism of baicalin intervention in gastrointestinal acute graft versus host disease(aGVHD).Methods Peripheral blood mononuclear cells from patients with intestinal aGVHD and healthy volunteers were collected for high-throughput sequencing and differential mRNA enrichment analysis.The intersection of the baicalin target and differential mRNA was analyzed.CB6F1 mice were irradiated with 60Co X-rays.After irradiation,a mononuclear cell suspension(1×10^(7) bone marrow cell+1×10^(7) spleen cell)of BALB/cH-2d mice was injected into CB6F1 mice through the tail vein,and these mice were randomly devided into the model group and the baicalin group.6 SPF female mice were selected as the normal group.After modeling,the baicalin group was administered 30 mg/(kg·d)of baicalin by gavage,and the model and normal groups were gavaged with normal saline of equal volume.Serum cytokines were detected after 14 days,and aGVHD scores and small intestinal mucosa pathdogy scores,immunohistochemical,and immunofluorescence detection were performed.Results A total of 2,131 different mRNAs were identified from patients with intestinal aGVHD and healthy volunteers.Kyoto Encyclopedia of Genes and Genomes analysis indicated that these mRNAs enriched the TNF signaling pathway and other pathways.The baicalin target was intersected with the above differential genes,and TNF-αand IL-2 were the primary baicalin targets in the treatment of intestinal aGVHD.The aGVHD scores and small intestinal mucosal pathology scores after baicalin treatment were significantly better than those in the model group,serum TNF-αand IL-2 levels were significantly decreased,and the TNF-αand IL-2 protein expression levels in intestinal immunohistochemistry and immunofluorescence were also significantly recovered(P<0.05).Conclusion Baicalin can reduce the inflammatory infiltration and destruction of intestinal mucosa by reducing TNF-αand IL-2 production and reducing the incidence of intestinal aGVHD after transplantation.