Effects of nobiletin on intestinal stem cell proliferation in vitro and in vivo

To investigate the regulatory effect of nobiletin（NOB）at typical effective doses on intestinal stem cells in vivo and in vitro.

After a 3D culture model of mouse colorectal tumor cell line MC38 was constructed, the death and survival of the obtained colonies were observed after treatment of different concentrations of NOB. Mouse small intestinal crypts were cultured in the Matrigel to generate organoids. Different concentrations of NOB were added to the culture system, and the sprouting and growth of the organoids were observed. MTT assay was used to calculate the area and absorbance values of the organoids after staining. The organoid growth was observed by removing 50 and 100 μmol/L NOB for different periods. The effects of exogenous R-spondin1 and CHIR99021（activators of Wnt pathway）on the growth of organoids were observed in the culture system with 50 and 100 μmol/L NOB treatment. C57/B6J mice were infused with different concentrations of NOB by gavage for 4 consecutive days. The ratio of intestinal crypt to villus length, cell apoptosis, and number of OLfm4 and BrdU double positive cells were calculated and observed.

Compared with the control group, 50 μmol/L NOB significantly promoted the death of MC38 cells after sphere formation and significantly reduced the colony formation rate（P<0.001）. The dose also significantly inhibited the budding of intestinal organoids, and 50~200 μmol/L NOB significantly inhibited the growth of intestinal organoids in a dose-dependent manner when compared with the control group（P<0.001）. After NOB withdrawal, the growth of intestinal organoids in the 50 μmol/L group was partially recovered, but it was difficult to recover to normal level in the 100 μmol/L NOB group. Enhanced Wnt pathway activation could partially rescue the inhibitory effect of NOB on intestinal organoids. In vivo administration of higher concentrations of NOB did not induce apoptosis of intestinal crypt cells, did not affect the crypt to villus ratio, and had no effects on the number and proliferation status of intestinal stem cells.

NOB possesses the ability to inhibit normal intestinal stem cells by regulating stem cell-related pathways such as Wnt, but this cellular inhibition cannot be phenocopied in vivo. Our study suggests the safety of NOB at regular concentration and also raises a caution to explain the inhibitory role of NOB in cancer models.