中药复方对复发性口腔溃疡大鼠模型的治疗效果及作用机制研究

Study on the therapeutic effects and mechanisms of traditional Chinese medicine compound on rats with recurrent oral ulcers

摘要

[目的]本文旨在研究中药复方对复发性口腔溃疡(recurrent oral ulcers,ROU)模型大鼠的药效及作用机制。[方法]首先选择用金银花、知母、麦冬和淡竹叶4味中药组成中药复方,采用液质联用(LC-MS)法对中药复方中化学成分进行定性分析,接着将35只SD大鼠随机均分为阳性药对照组(20mg·kg^-1左旋咪唑)、中药复方低(15mg·kg^-1中药)剂量组、中药复方高(45mg·kg^-1中药)剂量组、模型组、空白对照组。除空白组外,其他4组用免疫化学联合诱导法建立大鼠ROU模型,然后,各组分别连续给药7d。给药期间每日拍照记录大鼠溃疡恢复情况及大鼠体重和采食量,给药结束后立即处死大鼠,制备大鼠口腔溃疡组织切片,观察大鼠口腔溃疡愈合情况;酶联免疫吸附法检测大鼠血清中炎性相关因子含量;免疫组化法检测大鼠溃疡组织VEGF蛋白的表达水平;水溶性四唑蓝法、硫代巴比妥酸法和比色法检测溃疡组织中氧化应激相关因子的含量;高通量测序法检测大鼠盲肠粪便微生物种群的丰度。[结果]ROU模型成功构建后,与空白组相比,建模的4组大鼠体重均显著降低(P<0.05),建模组之间无明显差异;给药结束后,与空白组相比,模型组大鼠体重及采食量显著降低,溃疡明显;溃疡组织中VEGF表达量显著降低;血清中IL-6、TNF-α水平显著升高(P<0.05);溃疡组织中SOD、GSH-Px活性下降,MDA含量显著升高(P<0.05);大鼠肠道微生物菌群失调。与模型组比较,中药复方组大鼠体重及采食量显著升高(P<0.05),溃疡组织愈合明显;溃疡组织中VEGF表达量明显提高;血清中炎性因子水平显著下调(P<0.05),溃疡组织中SOD、GSH-Px活性显著升高(P<0.05),MDA含量显著降低(P<0.05);大鼠粪便微生物菌群平衡恢复,在门水平上,厚壁菌门平均相对丰度下降,趋近于空白对照组。[结论]经大鼠复发性口腔溃疡模型验证,中药复方具有抗炎、抗氧化、促进溃疡组织血管生长,调节肠道微生物菌群进而促进ROU大鼠溃疡恢复的作用。

Abstract

[Objectives]The paper aimed to investigate the pharmacological effects and mechanisms of action of a traditional Chinese medicine compound on a rat model of recurrent oral ulcers (ROU). [Methods]First, a compound herbal formula consisting of four Chinese medicinal herbs-honeysuckle, anemarrhena, ophiopogon, and bamboo leaf-was selected, and liquid chromatography-mass spectrometry (LC-MS) was employed to perform qualitative analysis of the chemical constituents in the formula. And thirty-five SD rats were randomly and evenly divided into five groups: a positive control group (20 mg·kg^-1 levamisole), a low-dose group of traditional Chinese medicine compound (15 mg·kg^-1 of traditional Chinese medicine), a high-dose group of traditional Chinese medicine compound (45 mg·kg^-1 of traditional Chinese medicine), a model group, and a blank control group. Except for the blank control group, the other four groups were induced to establish a rat ROU model using an immunochemical combined induction method, and each group was administered medication continuously for 7 days. During the medication period, photographs were taken daily to record the recovery of the rat's ulcers as well as the rats' body weight and food intake. After the medication was completed, the rats were immediately euthanized, and oral ulcer tissue sections were prepared to observe the healing of the rat's oral ulcers. The content of inflammatory-related factors in the rat serum was detected using the enzyme-linked immunosorbent assay (ELISA) method; the expression of VEGF protein content in the rat ulcer tissue was detected using the immunohistochemical method; the content of oxidative stress-related factors in the ulcer tissue was detected using the water-soluble tetrazolium blue method, thiobarbituric acid method, and colorimetric method; and the richness of microbial species in the rat cecal feces was detected using high-throughput sequencing methods. [Results]After the model was established, the body weights of the four modeling groups of rats significantly decreased compared with the blank group (P < 0.05), with no significant differences among the modeling groups. After drug administration, compared with the blank group, the body weight and food intake of the model group rats significantly decreased, and ulcers were evident. The expression of VEGF in the ulcer tissue was significantly reduced. The levels of IL-6 and TNF-α in the serum significantly increased (P<0.05). The activities of SOD and GSH-Px in the ulcer tissue decreased, while the content of MDA significantly increased (P<0.05). The fecal microbiota of the rats was also imbalanced. Compared with the model group, the body weight and food intake of rats in the traditional Chinese medicine compounds significantly increased (P<0.05); the healing of ulcer tissue was markedly evident; the expression of VEGF in the ulcer tissue was significantly enhanced; the levels of inflammatory factors in the serum were significantly downregulated (P<0.05), and the activities of SOD and GSH-Px in the ulcer tissue were significantly elevated (P<0.05); the content of MDA was significantly reduced (P<0.05); the balance of microbial flora in the rat feces was restored, and at the phylum level, the average relative abundance of Firmicutes decreased, approaching that of the blank control group. [Conclusions]The immunochemical combined induction method successfully established a rat model of complex oral ulcers. The traditional Chinese medicine compound demonstrated anti-inflammatory, antioxidant, and angiogenic properties in ulcer tissues, and regulated gut microbiota, thereby promoting ulcer healing in ROU rats.

关键词

中药复方 / 复发性口腔溃疡 / 炎性因子 / 氧化应激 / 肠道菌群

Key words

traditional Chinese medicine compound / recurrent oral ulcers / inflammatory factors / oxidative stress / gut microbiota

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基金资助

国家重点研发计划项目(2022YFD1801103)

企业横向项目(HMQY23052)