The CBS/H2S axis regulates intestinal stem cell homeostasis and radiation-induced intestinal damage

Background: The cycling intestinal stem cells (ISCs) exhibit radiosensitivity, and their death or impaired regenerative capacity following irradiation may result in intestinal barrier dysfunction. The cystathionine-β-synthase (CBS)/H2S axis plays a critical role in regulating cell proliferation, Reactive Oxygen Species scavenging, and the DNA damage response. However, it remains unclear whether the CBS/H2S axis modulates ISC homeostasis and tissue radiosensitivity.

Methods: Intestinal epithelium specific conditional CBS knockout mice were generated by crossing CBS^fl/+ mice with Villin-CreERT2 mice. CAGGCre-ER™ mice were crossed with CBS^fl/fl mice to achieve CBS knockout in multiple tissues and cell types. The Lgr5-Tdtaomato-Flag mice were generated by CRISPR/Cas9 system. The CBS inhibitor AOAA or the H2S donor GYY4137 was used to treat mice or intestinal crypt organoids. Hematoxylin and eosin, immunohistochemistry, immunofluorescence, Western blot, qRT-PCR, et al. were employed to investigate the role of the CBS/H2S axis in ISCs homeostasis and radiation-induced intestinal damage.

Results: Lgr5 + ISCs and progenitor cells expressed higher levels of CBS than differentiated cells. The cecum and colon expressed significant higher CBS levels than the small intestine. Treatment with the H2S donor GYY4137 enhanced the proliferation of intestinal organoids in vitro, while inhibition of CBS by AOAA reduced this effect. Genetic knockout of CBS in the intestinal epithelium or global downregulation of CBS driven by CAGG-CreER™ in vivo did not affect ISC proliferation or differentiation under physiological conditions. Pharmacological regulation of the CBS/H2S axis in vitro failed to protect organoids from radiation-induced damage. Interestingly, administration of AOAA in vivo reduced radiation-induced atrophy of the intestinal mucosa. Furthermore, global downregulation of CBS significantly promoted ISC recovery after irradiation exposure. However, intestinal epithelium-specific CBS knockout did not confer radioprotective effects.

Conclusions: Our findings suggest that the CBS/H2S axis contributes to the regulation of ISC homeostasis and represents a potential target for radiation protection, mediated through the intervention of non-epithelial cells.

CBS; H2S; Intestinal stem cells; Radiation; Radiation enteritis.