Inhibitory Effect And Mechanism Of Rhizoma Coptidis Extract On Hyphal Invasion Into Oral Of OPC Mice

OBJECTIVE: To explore the intervention effect and mechanism of Coptidis Rhizoma extract on OPC(Oropharyngeal candidiasis)mice invaded by Candida albicans hyphae,in order to provide experimental basis for treatment of OPC.Methods: 1.Extract of Coptidis Rhizoma(ECR)from Coptidis Rhizoma was extracted by reflux alcohol,and the experiment was carried out in vitro and in vivo.(1)In vivo experiment: To establish murine OPC model,using crystal violet staining,HE staining,PAS staining and the detection of symptoms in mice to verify the success of the OPC model.The group was divided into normal control group,model group,ECR high concentration group(240 mg/mL),ECR medium concentration group(120 mg/mL),ECR low concentration group(60 mg/mL)and nystatin positive drug group(135 mg/mL).The mice in the high,medium and low concentration ECR and nystatin were orally smeared three times a day,while the mice in the normal control group and model group were smeared with normal saline.After 10 days of continuous intervention,the efficacy of ECR was evaluated by symptoms and signs of mice,fungal microscopy observation,fungal load measurement and histopathological examination,and the effects of ECR on oral mucosa invasion by Candida albicans hyphae were observed by scanning electron microscopy and fluorescent microscopy.(2)In vitro experiments: the effect of ECR on the metabolic activity of Candida albicans hyphae was evaluated by XTT reduction method;the effect of ECR on the marginal growth of Candida albicans colonies was observed on solid medium;the effect of ECR on the hyphal growth of Candida albicans on semi-solid medium;the effect of ECR on the hyphal growth of Candida albicans was observed on liquid medium;the effect of ECR on the hyphal morphology of Candida albicans was observed under scanning electron microscope;The effects of ECR on the morphology and viability of Candida albicans hyphae were observed under light microscope,and the effects of ECR on the expression of invasin ALS3 and SSA1 genes in Candida albicans hyphae were detected by qRT-PCR.RESULTS: In vivo experiments showed that crystal violet staining,HE staining,PAS staining and the detection of symptoms and signs of mice could validate the successful construction of OPC model;ECR in medium concentration group had certain curative effect on the tongue mucosal injury of OPC mice,ECR in high concentration group could completely restore the tongue mucosal injury of OPC mice;XTT method showed that with the increase of ECR concentration(64,128,256 mg/mL),the metabolic activity of Candida albicans hyphal gradually decreased;128,256 mg/mL ECR was tested on solid,semi-solid and liquid medium to inhibit the growth of colonial margin,hyphae formation and invasive ability of Candida albicans;128,256 mg/mL ECR was also found by scanning electron microscopy and fluorescence microscopy to significantly affect hyphal formation;qRT-PCR showed that 64,128,256 mg/mL ECR could significantly inhibit hyphal formation and down-regulate the expression of invasin ALS3 and SSA1 gene.CONCLUSION: In vivo and in vitro experiments demonstrated that the extract of Coptidis Rhizoma(ECR)could reduce the invasiveness of oral mucosa by inhibiting the hyphal formation of Candida albicans,inhibiting the expression of ALS3 and SSA1 genes,thus exerting its therapeutic effect on OPC.