Objective To investigate the mechanism of Dangshen (Codonopsis Radix) containing serum (CRCS) on inflammatory factors release and epithelial cell excessive apoptosis in vitro model of ulcerative colitis (UC). Methods Bioinformatics analysis tools were used to predict and regulate the upstream miRNAs of phosphatase and tensin homolog (PTEN). qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of PTEN and the expressions of miR-21, miR-30a-3p, miR-198, miR-152-3p, miR-301a-3p, miR-320 and miR-3691-5p in colon tissues of UC rats model induced by enema 5% 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)/ethanol composite solution. Lipopolysaccharide (LPS, 10 μg/mL) was used to induce human normal colon epithelial NCM460 cells to construct an in vitro model of UC, 1%, 2%, 4%, 6% and 8% CRCS intervention was administered for 24 h. The cell viability was detected by CCK-8 method. Subsequently, miR-21 mimics and miR-21 inhibitor were transfected into model cells, respectively, which were cultured in 6 % CRCS complete medium for 24 h. The levels of inflammatory factors interleukin-6 (IL-6), IL-17, IL-1β, IL-8, nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) in each group were detected by ELISA. The expression of miR-21 in each group was detected by qRT-PCR. Western blotting was used to detect the expressions of PTEN, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved cystein-asparate protease-3 (cleaved Caspase-3) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway related proteins. The binding sites of miR-21 and PTEN were predicted by bioinformatics, and the targeting effect of miR-21 on PTEN was verified by dual luciferase reporter gene detection system. Results A total of seven miRNAs that may target PTEN were screened by bioinformatics analysis. Among them, the expression of miR-21 was decreased significantly after CRCS intervention (P < 0.01), while the expressions of miR-30a-3p, miR-198, miR-152-3p, miR-301a-3p, miR-320 and miR-3691-5p had no statistical significance. The results of CCK-8 showed that 2%, 4%, 6% and 8% CRCS could significantly improve cell viability (P < 0.01). Compared with model group, CRCS and miR-21 inhibitor increased the expression levels of PTEN and Bcl-2 proteins in cells (P < 0.01), and decreased the expression levels of miR-21, Bax, cleaved Caspase-3, p-PI3K/PI3K and p-Akt/Akt (P < 0.01). Compared with CRCS group, miR-21 mimics significantly attenuated the up-regulation of PTEN and Bcl-2 expressions by CRCS (P < 0.01) and the down-regulation of miR-21, Bax, cleaved Caspase-3, p-PI3K/PI3K, p-Akt/Akt expressions (P < 0.05, 0.01). The results of dual luciferase reporter assay showed that in the transfected wt-Luc-PTEN plasmocytes, CRCS intervention could significantly increase luciferase activity (P < 0.01), and miR-21 mimics could significantly reduce luciferase activity (P < 0.01). In the combined treatment, miR-21 mimics could partially weaken the effect of CRCS on luciferase activity (P < 0.01). In transfected mut-Luc-PTEN plasmocytes, CRCS intervention and miR-21 mimics did not cause significant changes in luciferase activity. Conclusion CRCS could alleviate UC by inhibiting pro-inflammatory factors release and excessive apoptosis of epithelial cells. The mechanism is at least partially dependent on miR-21/PTEN/PI3K/Akt axis.

Codonopsis Radix  /  ulcerative colitis  /  epithelial cell excessive apoptosis  /  miR-21/PTEN axis  /  PI3K/Akt pathway  /  lobetyolin