Human Colonic Epithelial Cells

Co-Culture of Epithelial Cells with Clostridium scindens

A dynamic interaction between the human gastrointestinal epithelium and intestinal microbiota is key to understanding and treating intestinal diseases. Human colonic epithelial cells from a healthy donor were cultured with Clostridium scindens (C. scindens), Staphylococcus aureus (S. aureus), or both. A special platform with controlled oxygen levels created separate anaerobic and aerobic environments. Epithelial cells and C. scindens remained viable in co-culture for 48 hours.

C. scindens was tested with epithelial cells to see if they can coexist without an immune system. Epithelial cell viability and barrier function were evaluated: no significant cell death was observed (control: 9.8% vs. C. scindens: 10.8%, p = 0.64, Figure 1A). No significant change in cell death indicators (control: 9.5% vs. C. scindens: 6.1%, p = 0.24, Figure 1B). TEER values decreased over time but were not significantly different (control: 168 ± 18 vs. 150 ± 37 Ω·cm² with C. scindens, p = 0.45). ZO-1 tight junctions were similar with and without C. scindens (Figure 1C). Cell measurements showed no significant differences (area, circularity, perimeter). These results indicate C. scindens proliferation did not harm cells, except at extremely high numbers (~5×10⁶ cells), which increased cell death.

Fig. 1. Co-culture of primary human colonic epithelial cells with C. scindens (Wang H, Kim R, et al., 2024).

Circ_0085323 Depletion Ameliorated TNF-a–Induced Inflammation and Apoptosis of NCM460 Cells

Previous studies have demonstrated that circRNA is involved in the occurrence of ulcerative colitis (UC). However, the mechanism of circ_0085323 in the occurrence of UC has not been reported. In the present study, AN's team explored whether the circ_0085323/miR-495-3p/TRAF3 axis was involved in the occurrence of UC in vitro.

First, the effects of si-hsa_circ_0085323 on circ_0085323 expression in Normal human colonic epithelial cells (NCM460) were examined. The results showed that si-hsa_circ_0085323 could significantly downregulate the expression of circ_0085323 in NCM460 cells (Fig. 2A). Then, the effects of si-hsa_circ_0085323 on the proliferation, viability, IL-1β, IL-6, and IL-8 secretion, LDH activity, and apoptosis of TNF-α-stimulated NCM460 cells were detected. The results showed that TNF-α induced the decrease of cell proliferation and viability, but the effects were reversed after the silencing of circ_0085323 (Fig. 2, B and C). TNF-α promoted the secretion of IL-1β, IL-6, and IL-8 and the activity of LDH, and the effects were reversed after the silencing of circ_0085323 (Fig. 2, D and E). TNF-α induced the increase of apoptosis in NCM460 cells, and the effects were reversed after the silencing of circ_0085323 (Fig. 2F). TNF-α induced the increase of the expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP, and the effects were reversed after the silencing of circ_0085323 (Fig. 2G). These results showed that the silencing of circ_0085323 could reverse the TNF-α-induced injury of NCM460 cells.

Fig. 2. Circ_0085323 knockdown attenuated TNF-α–caused NCM460 cell injury (An Q, Yang SS, et al., 2023).