黄芩汤调控自噬干预小鼠肠道急性移植物抗宿主病的机制

Huangqin Decoction regulates autophagy to intervene with intestinal acute graft-versus-host disease in mice

背景:如何应用中药干预肠黏膜屏障损伤后的自噬失衡状态,从而最终干预胃肠道急性移植物抗宿主病的发生是造血干细胞移植后亟需解决的问题。目的:探讨黄芩汤干预急性肠道移植物抗宿主病的具体机制。方法:CB6F1小鼠随机分成正常对照组、模型对照组、黄芩汤低剂量组、黄芩汤中剂量组、黄芩汤高剂量组,每组16只。模型对照组及黄芩汤低、中、高剂量组小鼠在^(60)Co全身照射后4 h内(照射总剂量8 Gy),经尾静脉输注Balb/c H-2d小鼠单个核细胞悬液(骨髓细胞8×10^(7)/只+脾细胞8×10^(7)/只)。造模后当天开始分别给予不同浓度黄芩汤灌胃,模型组及正常对照组灌胃等容量的生理盐水,连续应用15 d。最后一次灌胃后8 h取材,每组取6只小鼠小肠组织,采用PCR和Western blot检测LC3Ⅱ/Ⅰ、Beclin 1、P62表达水平,苏木精-伊红染色进行小肠黏膜病理分级评分,透射电镜观察小肠黏膜上皮细胞自噬泡结构;每组剩余10只(除正常对照组)小鼠进行临床急性移植物抗宿主病分级并记录生存时间。结果与结论:①应用黄芩汤后,小鼠存活时间显著延长,临床急性移植物抗宿主病评分显著降低,小肠黏膜病理分级评分显著下降,黄芩汤中、高剂量组评分较模型对照组下降最显著,且黄芩汤中、高剂量组之间无显著差异;②模型对照组小鼠小肠组织自噬相关指标LC3Ⅱ/Ⅰ、Beclin 1水平显著低于正常对照组(P<0.01),P62水平显著高于正常对照组(P<0.01),黄芩汤可以促进LC3Ⅱ/Ⅰ、Beclin1水平的恢复并下调P62水平(P<0.01);③透射电镜观察黄芩汤治疗组的自噬泡明显比模型对照组增多,且伴有如线粒体等重要细胞器形态的恢复;④结果表明,黄芩汤通过干预自噬相关蛋白,促进肠道急性移植物抗宿主病自噬稳态的恢复,保护肠黏膜屏障从而减轻了移植后肠道的排异,有望成为治疗急性移植物抗宿主病的新方法。

BACKGROUND:How to use traditional Chinese medicine to intervene the imbalance of autophagy after intestinal mucosal barrier injury,so as to ultimately intervene the occurrence of gastrointestinal acute graft-versus-host disease,is an urgent problem to be solved after hematopoietic stem cell transplantation.OBJECTIVE:To verify the precise mechanism by which Huangqin Decoction interferes with acute intestinal graft-versus-host disease.METHODS:CB6F1 mice were randomly divided into normal control group,model control group,low-dose Huangqin Decoction group,medium-does Huangqin Decoction group and high-does Huangqin Decoction group,with 16 mice per group.CB6F1 mice in the model control group,low-dose Huangqin Decoction group,medium-does Huangqin Decoction group and high-does Huangqin Decoction group were infused with mononuclear cell suspension(bone marrow cell 8×10^(7)+spleen cell 8×10^(7))obtained from Balb/c mice via caudal vein within 4 hours after ^(60)Co whole body irradiation(radiation dose was 8 Gy).Different concentrations of Huangqin Decoction were given by gavage on the same day after modeling.The rats in the model control group and the normal control group were given the same volume of normal saline by gavage for 15 days.Eight hours after the last gavage,the small intestine tissues of six mice in each group were collected.PCR and western blot assay were used to detect the expression levels of LC3II/I,Beclin1 and P62.The pathological grading of small intestinal mucosa was scored by hematoxylin-eosin staining.The autophagic vesicle structure of small intestinal mucosal epithelial cells was observed by transmission electron microscope.The remaining 10 rats in each group(except the normal control group)were used to observe the clinical grading of acute graft-versus-host disease and record the survival time.RESULTS AND CONCLUSION:(1)After the application of Huangqin Decoction,the survival time of mice was significantly prolonged;the clinical acute graftversus-host disease score was significantly decreased,and the pathological grading score of small intestinal mucosa was significantly decreased.The score of medium-does Huangqin Decoction group and high-does Huangqin Decoction group was significantly lower than that of model control group,but there was no significant difference between medium-does Huangqin Decoction group and high-does Huangqin Decoction group.(2)The LC3II/I and Beclin1 expression was significantly lower in the model control group than that in the normal control group(P<0.01),and P62 expression was significantly higher than that in the normal control group(P<0.01).Huangqin Decoction could promote the recovery of LC3II/I and Beclin1 levels and downregulate p62 levels(P<0.01).(3)Under transmission electron microscope,the number of autophagic vesicles in the treatment group was significantly higher than that in the model control group,accompanied by the recovery of important organelles such as mitochondria.(4)The results confirm that by interfering autophagy related proteins,Huangqin Decoction can promote the recovery of autophagy in acute graft-versus-host disease,protect intestinal mucosal barrier and reduce intestinal rejection after transplantation and has promise as a new treatment for acute graft-versus-host disease.