摘要
目的探讨加味丹玄口康对大鼠口腔黏膜下纤维化(OSF)及其自噬水平的影响。方法选取SPF级SD雄性大鼠24只(180~200 g,4~8周龄),采用随机数字表法将其分为空白组、模型组和加味丹玄口康组,每组8只。模型组和加味丹玄口康组在大鼠双侧颊黏膜组织注射槟榔碱0.2 ml(10 mg/ml),1次/2 d,同时每天使用小牙刷沾取适量槟榔碱均匀施力刷大鼠双侧颊黏膜,持续8周,观察到大鼠颊黏膜出现白色病变确认造模成功,空白组不做干预。造模成功后,加味丹玄口康组以8 ml/(kg·d)加味丹玄口康灌胃,空白组和模型组灌胃等量0.9%氯化钠溶液,三组均灌胃4周。实验后取三组双侧颊黏膜进行苏木精-伊红染色和Masson染色观察病理变化和胶原纤维沉积情况,透射电镜观察三组自噬小体数量。采用免疫组织化学染色检测三组上皮钙黏素和微管相关蛋白轻链3(LC3)阳性细胞表达情况;Western blot检测上皮钙黏素、LC3-Ⅱ和选择性自噬接头蛋白p62的表达;RT-PCR检测LC3-Ⅱ和p62 mRNA表达。结果模型组口腔黏膜上皮明显萎缩,胶原纤维显著变粗、增多,上皮层变薄,黏膜下层胶原纤维大量沉积,加味丹玄口康组口腔黏膜上皮层较厚,胶原纤维减少,上皮层萎缩程度减轻。模型组自噬小体数量低于空白组,上皮钙黏素、LC3阳性细胞数低于空白组,上皮钙黏素、LC3-Ⅱ蛋白表达低于空白组,LC3-ⅡmRNA表达低于空白组,p62蛋白及其mRNA表达高于空白组(P<0.05)。加味丹玄口康组自噬小体数量高于模型组,上皮钙黏素、LC3-Ⅱ阳性细胞数高于模型组,上皮钙黏素、LC3-Ⅱ蛋白表达高于模型组,LC3-ⅡmRNA表达高于模型组,p62蛋白及其mRNA表达低于模型组(P<0.05)。结论加味丹玄口康可改善大鼠OSF,可能与其自噬水平一定程度升高有关。
Objective To investigate the influence of Jiawei Danxuan Koukang on oral submucous fibrosis(OSF)and the level of autophagy in rats.Methods Twenty-four SPF male SD rats(180-200 g,4-8 weeks old)were randomly divided into blank group,model group,and Jiawei Danxuan Koukang group,with eight rats in each group.In the model group and Jiawei Danxuan Koukang group,arecoline 0.2 ml(10 mg/ml)was injected into the bilateral buccal mucosa of the rats,once every two days,and a small toothbrush was used to brush the bilateral buccal mucosa of the rats every day for eight weeks.White lesions were observed in the buccal mucosa of the rats to determine the success of the model.After the model was successfully established,the rats in the Jiawei Danxuan Koukang group were treated with 8 ml/(kg·d)Jiawei Danxuan Koukang by gavage,and the blank group and the model group were treated with the same amount of 0.9%sodium chloride solution by gavage for four weeks.After the experiment,the bilateral buccal mucosa of the three groups were collected for hema-toxylin-eosin staining and Masson staining to observe the pathological changes and collagen fiber deposition,and the number of autophagosomes in the three groups was observed by transmission electron microscope.Immunohistochemical staining was used to detect the expression of E-cadherin and microtubule-associated protein light chain 3(LC3)positive cells in the three groups.Western blot was used to detect the expression of E-cadherin,LC3-Ⅱ,and selective autophagy adaptor protein p62.The mRNA expression of LC3-Ⅱand p62 was detected by RT-PCR.Results In the model group,the oral mucosal epithelium was significantly atrophed,the collagen fibers were significantly thickened and increased,the epithelial layer was thinner,and a large amount of collagen fibers were deposited in the submucosal layer.In the Jiawei Danxuan Koukang group,the oral mucosal epithelium was thicker,the collagen fibers were reduced,and the atrophy of the epithelial layer was alleviated.In model group,the number of autophagosomes was lower than that in blank group,the number of E-cadherin and LC3 positive cells were lower than those in blank group,the expression of E-cadherin and LC3-Ⅱprotein were lower than those in blank group,the expression of LC3-ⅡmRNA was lower than that in blank group,and the expression of p62 protein and its mRNA were higher than those in blank group(P<0.05).In Jiawei Danxuan Koukang group,the number of autophagosomes was higher than that in model group,the expression of E-cadherin and LC3-Ⅱprotein were higher than those in model group,the expression of LC3-ⅡmRNA was higher than that in model group,and the expression of p62 protein and its mRNA were lower than those in model group(P<0.05).Conclusion Jiawei Danxuan Koukang can improve OSF in rats,which may be related to the increased level of autophagy.
作者
贺珺
张婷
胡亮
潘世杰
邹红
连科荃
谭劲
胡兆勇
HE Jun;ZHANG Ting;HU Liang;PAN Shijie;ZOU Hong;LIAN Kequan;TAN Jin;HU Zhaoyong(School of Medicine,Hunan University of Chinese Medicine,Hunan Province,Changsha 410208,China;Department of Stomatology,the First Affiliated Hospital of Hunan University of Chinese Medicine,Hunan Province,Changsha 410007,China)
出处
《中国医药导报》
CAS
2023年第24期9-15,共7页
China Medical Herald
基金
国家自然科学基金资助项目(81874496)
湖南省教育厅科学研究一般项目(20C1389)
湖南省级大学生创新创业训练计划项目(S202110541057)。