CRX-527 induced differentiation of HSCs protecting the intestinal epithelium from radiation damage.

Abstract:

Background: Recently, Toll-like receptors (TLRs) have been extensively studied in radiation damage. However, the inherent defects of high toxicity and low efficacy of most TLR ligands limit their further clinical transformation. CRX-527, as a TLR4 ligand, has rarely been reported to protect against radiation.

Methods: In vivo, C57BL/6 mice and TLR4 knockout mice were used to observe the radiation protection of CRX-527 on the hematopoietic system and intestinal system of mice. The proportion and number changes of LSK, MPP, MDSC and macrophages were detected by flow cytometry. The changes of intestinal protein expression were observed by immunohistochemistry, immunofluorescence and Western blot. In vitro, RAW264.7-MODE-K and THP-1-HIEC co-culture systems were performed to observe the proliferation of cells under different irradiation doses in clone formation experiments, as well as the expression of proteins in the co-culture system under the same irradiation doses.

Results: CRX-527 improved the survival rate of total body irradiation (TBI) to 100% in wild-type mice but not in TLR4^-/- mice. After TBI, hematopoietic system damage was significantly alleviated, and the recovery period was accelerated in CRX-527-treated mice. Moreover, the stimulation of CRX-527 significantly increased the proportion and number of LSK cells and promoted their differentiation into macrophages, activating immune defense. Furthermore, we proposed an immune defense role for hematopoietic differentiation in the protection against intestinal radiation damage, and confirmed that macrophages invaded the intestines through peripheral blood to protect them from radiation damage. Meanwhile, CRX-527 maintained intestinal function and homeostasis, promoted the regeneration of intestinal stem cells, and protected intestinal injury from lethal dose irradiation. Furthermore, CRX-527 protects the hematopoietic and intestinal systems from irradiation through the activation of TLR4-related pathways.

Conclusion: CRX-527 induced differentiation of HSCs to maintain the haematopoiesis. The activated macrophage protected the intestinal epithelium from radiation damage through TLR4-related pathways. We demonstrated that CRX-527 was safe and effective in protecting radiation damage in vivo and in vitro.