加味丹玄口康调控口腔黏膜下纤维化大鼠Wnt/β-catenin信号通路机制研究

Mechanism of Jiawei Danxuan Koukang regulating expression of Wnt/β-catenin signaling pathway in oral submucosal fibrosis rats

目的:探讨加味丹玄口康调控口腔黏膜下纤维化大鼠Wnt/β-catenin信号通路表达作用机制。方法:从35只SPF级成年雄鼠中随机挑选5只作为正常组,剩余30只大鼠采用槟榔提取物建模,成功建立25只口腔黏膜下纤维化(OSF)大鼠模型。将25只OSF模型大鼠随机分为OSF模型组、加味丹玄口康低剂量组、加味丹玄口康中剂量组、加味丹玄口康高剂量组和Wnt抑制剂组,每组各5只。加味丹玄口康低剂量组、加味丹玄口康中剂量组、加味丹玄口康高剂量组大鼠分别给予4、8、12 ml/kg加味丹玄口康灌胃,Wnt抑制剂组大鼠给予25 mg/kg的KYA1797K腹腔注射,OSF模型组和正常组大鼠给予4 ml/kg的0.9%氯化钠溶液灌胃。1次/d,持续给药4周。分别于治疗前、治疗2周、治疗4周比较各组大鼠张口度和颊黏膜评分。给药4周后处死大鼠,采用PCR和Western blot分别检测Wnt-1、β-catenin、Cylin D1和Axin基因及蛋白表达情况。结果:治疗2周及治疗4周后,Wnt抑制剂组、加味丹玄口康中剂量组、加味丹玄口康高剂量组大鼠的张口度增大,颊黏膜评分减小,加味丹玄口康高剂量组大鼠的张口度最大、颊黏膜评分最低,差异有统计学意义(均P<0.05);且以上各组大鼠张口度和颊黏膜评分与正常组比较,差异无统计学意义(均P>0.05)。加味丹玄口康高剂量组与OSF模型组、加味丹玄口康低剂量组、加味丹玄口康中剂量组、Wnt抑制剂组比较,Wnt-1、β-catenin、Cylin D1 mRNA和蛋白表达水平降低,Axin mRNA和蛋白表达水平升高,差异有统计学意义(均P<0.05)。加味丹玄口康高剂量组Wnt-1、β-catenin、Cylin D1和Axin基因及蛋白表达与正常组比较,差异无统计学意义(均P>0.05)。结论:加味丹玄口康可有效改善OSF模型大鼠临床症状,通过抑制Wnt/β-catenin信号通路相关基因及蛋白表达,抑制大鼠口腔黏膜纤维化。

Objective:To explore mechanism of Jiawei Danxuan Koukang regulating expression of Wnt/β-catenin signaling pathway in oral submucosal fibrosis rats.Methods:5 rats were randomly selected from 35 SPF adult male rats as the normal group,the remaining 30 rats were modeled with ANE,and 25 OSF rat models were successfully established.Rats were randomly divided into OSF model group,Jiawei Danxuan Koukang low dose group,Jiawei Danxuan Koukang medium dose group,Jiawei Danxuan Koukang high dose group,and Wnt inhibitor group,with 5 rats in each group.4,8,12 ml/kg of Jiawei Danxuan Koukang were given by gavage,the rats in the Wnt inhibitor group were given 25 mg/kg KYA1797K intraperitoneal injection,and the rats in the OSF model group and the normal group were given 4 ml/kg of 0.9%sodium chloride solution by gavage.The dose was administered once a day for 4 weeks.Mouth opening and buccal mucosa score of rats in each group were compared before treatment,2 and 4 weeks after treatment.4 weeks after administration,PCR and Western blot were used to detect gene and protein expressions of Wnt-1,β-catenin,Cylin D1 and Axin.Results:After 2 and 4 weeks of treatment,mouth opening increased and buccal mucosa score decreased in the Wnt inhibitor group,Jiawei Danxuan Koukang medium dose group and Jiawei Danxuan Koukang high dose group,while mouth opening was the highest and buccal mucosa score the lowest in the Jiawei Danxuan Koukang high dose group,differences statistically significant(all P<0.05).There were no significant differences in mouth opening degree and buccal mucosa score between the above groups and normal group(all P>0.05).Compared with the OSF model group,the Jiawei Danxuan Koukang low dose group,Jiawei Danxuan Koukang medium dose group and the Wnt inhibitor group,the mRNA and protein expression levels of Wnt-1,β-catenin and Cylin D1 in the Jiawei Danxuan Koukang high dose group were decreased,and the expression levels of Axin mRNA and protein were increased,differences statistically significant(all P<0.05).There were no significant differences in gene and protein expressions of Wnt-1,β-catenin,Cylin D1 and Axin in the Jiawei Danxuan Koukang high dose group compared with the normal group(all P>0.05).Conclusion:Jiawei Danxuan Koukang can effectively improve clinical symptoms of OSF model rats,and inhibit the fibrosis of oral mucosa in rats by inhibiting the expression of genes and proteins related to Wnt/β-catenin signaling pathway.