The Promotive Effect of the Active Ingredients of Atractylodes macrocephala on Intestinal Epithelial Repair Through Activating Ca2+ Pathway

Atractylodes macrocephala ( AM) is a famous traditional Chinese medicine for intestinal epithelial restitution through activating Ca2+ channels. However, the roles of specific AM compositions in intestinal epithelial restitution are sparse. Therefore, this study aimed to compare the concrete effects of the 4 active ingredients (atractylon, β-eudesmol, atractylenolide II, atractylenolide III) of AM and their combination on intestinal epithelial repair and the Ca2+ pathway in intestinal epithelial cell (IEC-6) cells. First, the best combination of the 4 ingredients with an optimal mixing ratio of atractylon: β-eudesmol: atractylenolide II: atractylenolide III = 1:2:2:2 was demonstrated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide orthogonal experiment. Subsequently, enzyme-linked immunosorbent assay was used to measure anti-inflammatory cytokine levels, the migratory ability was evaluated by cell scratch experiments, cell cycle analysis and [Ca2+]cyt concentration in cells were detected by flow cytometry, and the expression of the Ca2+ pathway-related genes was detected by immunofluorescence staining, quantitative polymerase chain reaction and whole blood assays. Our result showed that atractylon, β-Eudesmol, atractylenolide II, and atractylenolide III showed different abilities to promote the IEC-6 cells proliferation, migration, and the expression of anti-inflammatory cytokines interleukin (IL)-2, IL-10, and ornithine decarboxylase, as well as the intracellular [Ca2+]cyt concentration through stromal interaction molecule 1 transposition to activate Ca2+ pathway. Thereinto, atractylenolide III was the main active ingredient of AM for pro-proliferation and anti-inflammation, and the combination of 4 AM ingredients performed better beneficial effects on IEC-6 cells. Therefore, our study suggested that atractylenolide III was the active ingredient of AM for intestinal epithelial repair through activating the Ca2+ pathway, and the 4 ingredients of AM have a synergy in intestinal epithelial repair.

RNA-binding protein HuR regulates translation of vitamin D receptor modulating rapid epithelial restitution after wounding

Homeostasis of the intestinal epithelium is tightly regulated by numerous extracellular and intracellular factors including vitamin D and the vitamin D receptor (VDR). VDR is highly expressed in the intestinal epithelium and is implicated in many aspects of gut mucosal pathophysiology, but the exact mechanism that controls VDR expression remains largely unknown. The RNA-binding protein human antigen R (HuR) regulates the stability and translation of target mRNAs and thus modulates various cellular processes and functions. Here we report a novel role of HuR in the posttranscriptional control of VDR expression in the intestinal epithelium. The levels of VDR in the intestinal mucosa decreased significantly in mice with ablated HuR, compared with control mice. HuR silencing in cultured intestinal epithelial cells (IECs) also reduced VDR levels, whereas HuR overexpression increased VDR abundance; neither intervention changed cellular Vdr mRNA content. Mechanistically, HuR bound to Vdr mRNA via its 3′-untranslated region (UTR) and enhanced VDR translation in IECs. Moreover, VDR silencing not only inhibited IEC migration over the wounded area in control cells but also prevented the increased migration in cells overexpressing HuR, although it did not alter IEC proliferation in vitro and growth of intestinal organoids ex vivo. The human intestinal mucosa from patients with inflammatory bowel diseases exhibited decreased levels of both HuR and VDR. These results indicate that HuR enhances VDR translation by directly interacting with its mRNA via 3′-UTR and that induced VDR by HuR is crucial for rapid intestinal epithelial restitution after wounding.