Résumé
ObjectiveThis study aims to investigate the mechanism in which Celastrus orbiculatus extract （COE） affects the proliferation and differentiation of gastric organoids and the expression of Lgr5 and thus reverses the precancerous lesions of gastric cancer （PLGC） by regulating the leucine-rich repeat containing G protein-coupled receptor 5 （Lgr5）/Wingless （Wnt）/β-catenin signaling pathway based on a gastric organoid injury model. MethodGastric organoids were established based on stem cells of the mouse gastric gland. Gastric organoid injury models were constructed by treating gastric organoids with N-methyl-N'-nitro-N-nitrosoguanidine （MNNG， 0.02 mg·L-1）. Gastric organoid injury models were randomly divided into normal group， model group （0.02 mg·L-1 MNNG）， low， medium， and high dose （5， 10， 20 mg·L-1） groups of COE， and Wnt inhibitor Dickkopf-related protein 1 （DKK1） （0.5 mg·L-1） group， and they were treated with respective agents for 24 h. The number and volume of gastric organoids under different drug concentrations were observed under a microscope. The viability of the gastric organoid injury models was detected by Methyl thiazolyl tetrazolium （MTT） assay. The morphology and pathology of gastric organoids were observed using Hematoxylin and Eosin （HE） staining. The expression levels of Lgr5， Mucin2 （MUC2）， Mucin5AC （MUC5AC）， Mucin6 （MUC6）， Wnt， and β-catenin in gastric organoids under different drug concentrations were detected by Western blot （WB）. ResultCompared with the normal group， the number， volume， and activity of gastric organoids in the model group were decreased （P<0.01）， while the expressions of Lgr5， MUC2， Wnt， and β-catenin were significantly increased （P<0.01）. The expressions of MUC5AC and MUC6 were significantly decreased （P<0.01）. Compared with the model group， the number and volume of gastric organoids in the low， medium， and high dose groups of COE were all improved （P<0.01）， and the vitality of gastric organoids was significantly enhanced （P<0.01）. The effect was the most significant at a COE concentration of 20 mg·L-1 （P<0.01）. The expressions of Lgr5 and MUC2 in the medium and high dose groups of COE were significantly reduced （P<0.01）， while the expression of MUC5AC and MUC6 were significantly increased in the low， medium， and high dose groups of COE （P<0.05， P<0.01）. Compared with the model group， Wnt inhibitors could promote the expression of MUC5AC and MUC6 in gastric organoids （P<0.05， P<0.01） and reduce the expression of MUC2， Wnt， and β-catenin. In addition， the combined use of COE at high concentrations and Wnt inhibitors could further promote this trend （P<0.01）. ConclusionCOE inhibits the Wnt/β-catenin pathway by inhibiting the expression of Lgr5， MUC2， Wnt， and β-catenin and promoting the expression of MUC5AC and MUC6， thus promoting the proliferation and differentiation of gastric organoids and reversing the PLGC process.
Résumé
Objective@#To investigate the effect of Celastrus orbiculatus extract ( COE) on the growth of gastric organoids and the expression of E-cadherin in gastric epithelial cells of mice．@*Methods@#The gastric pylorus of 8-week-old C57 mice was isolated and cultured into gastric organoids．The dynamic changes of gastric organoid formation were observed under light microscope ; the intercellular structure of gastric epithelium was observed by HE staining ; the expression of epithelial-cadherin E-cadherin in gastric epithelial cells was observed by immunofluorescence staining．After passage to the third-generation ，the organoids were treated with different concentrations of COE (0，5 ，10，20 μg/ ml) ，the organoids were collected ，their numbers were counted ，their diameters were measured，their cellular activities were measured by methyl thiazolyl tetrazolium( MTT) colorimetry，and Western blot was used to detect the expression of E-cadherin in organoids after COE treatment. @*Results @#At 24 to 48 h， cystlike structures were formed and three-dimensional cell clusters with cystic gland-like central structure appeared， and gradually budding and forming gastric organoids after 72 h，suggesting that the organoids were successfully constructed．The epithelial cell marker E-cadherin was expressed in the organoid，which further confirmed the formation of organoid．Compared with the control group，the number and diameter of gastric organoids in the COE group significantly increased，cell activity was significantly enhanced (P＜0. 05) ，and the expression of E-cadherin increased with the increase of COE dose (P＜0. 01) ．@*Conclution @#Low dose COE can promote the expression of E-cadherin and the growth and formation of organoids，which may affect the repair of gastric mucosa injury.
Résumé
Programmed cell death 4 (PDCD4) is a newly discovered tumor suppressor gene, whose expression is regulated by methylation of PDCD4 gene and many kinds of microRNAs.Protein of PDCD4 enco-ding can improve the sensitivity of tumor cells to anticancer agents, inhibit tumor development and metastasis process, and play an important role in a variety of tumors.It is expected to be a clinical indicator to determine the prognosis of tumor.
Résumé
This study was aimed to investigate whether the extracts of Celastrus orbiculatus enhanced the invasion function of maspin tumor inhibitor gene through the construction of maspin overexpression human gastric carcinoma MGC803 cell line. Maspin was cloned into plasmid GV208-EGFP eukaryotic expression vector. And then, the recombinant plasmid GV208-maspin-EGFP was transfected into human gastric carcinoma MGC803 cells. After the maspin overexpression MGC803 cell were treated with Celastrus orbiculatus extracts in different concentrations (10, 20, 40 μg·mL-1), the invasion effects were detected by Transwell chamber assay. The results showed that after the successful construction of maspin overexpression cell line, the number of cells invading through Matrigel was obviously decreased in the Transwell chamber assay. It also showed drug concentration dependency. It was concluded that maspin gene can inhibit invasion of gastric carcinoma MGC803 cells. Simultaneously, the extracts of Celastrus orbiculatus can enhance the function of maspin gene.