Since the intestinal epithelium is one of the fastest self-renewing tissue in mammals and therefore sensitive to IR damage, IR-induced GI toxicity is the limited factor in abdominal pelvic radiotherapy and a great threat to public health in the potential nuclear accident . Nonetheless, there are no effective strategies approved to alleviate the pain of victims with GI syndrome, and the currently reported agents have unfavorable side effect profiles [8, 9]. The development of novel agents protecting against IR-induced intestinal damage is of great significance. Currently, the radio-protective effects of HKST on small intestine was investigated. As is shown in our results, the radiation-induced damage on intestine was dramatically mitigated by pretreating with HSKT in vitro and in vivo, in which the structure of intestinal organoids and small intestine of the HKST-treated group was more integrated than of the radiation damage group, and HKST pretreatment remarkably promoted the proliferation of intestinal cells post IR exposure (Figs. 1 and 2). HKST is the potent co-agonist of TLRs family which has been reported to alleviate the radiation-induced injury on bone marrow, spleen and testis in our previous study . It has been demonstrated that the attenuated Salmonwlla typhimurium could be employed to stimulate immune system or developed into vaccine thereby protecting against malaria or tumor [17, 18]. Besides, the therapeutic use of bacteria in preventing IR-induced GI toxicity has begun to receive more attention [27–29]. Our study demonstrates that HKST is of great potential alleviating injury on intestine induced by radiation and facilitating the relief of victims with GI syndrome in clinical practice.
Generally, the critical target of IR is considered to be DNA . Upon IR exposure, the irreparable DNA damage is induced and the unrepaired or error-repaired DNA damage subsequently leads to cellular damage or death . DNA double-strand breaks (DSBs) is regarded as the most deleterious form of DNA damage, which can rise chromosomal aberrations, loss of genetic materials, the cell death, or other detrimental consequences . Homologous recombination (HR) and non-homologous end joining (NHEJ) and are the two main modes to repair DSBs in mammalian cells, in which ATR and ATM are confirmed to be the key components of DNA damage response (DDR) pathway [22, 23]. As DDR is critical for the maintenance and modulation of genome integrity in intestinal cells after radiation exposure [33, 34], we speculated that the radio-protective effects of HKST might be involved in DDR. As our results show that, after radiation exposure, levels of P-ATM, P-ATR, P-CHK1 and p21 were significantly increased in cells pretreated with HKST (Fig. 3). The dysfunction of genome integrity caused by radiation damage requires DDR to prevent the transmission of incompletely replicated or damaged chromosomes. DNA damage repair reactions in mammalian cells require the cell cycle arrest by the activation of a DNA damage checkpoint . ATM and ATR are the main regulators of two major checkpoint pathways, in which ATR-CHK1 checkpoint signaling is essential for HR repair pathway while the conversion of ATM to monomers by autophosphorylation and then phosphorylates CHK2 appear to be essential for the checkpoint response [36, 37]. In some condition, the cell-cycle arrest is initiated by ATR-CHK1 signaling but maintained by ATM-CHK2 signaling . Cell cycle arrest will activate the tumor suppressor p53 which then targets its transcriptional protein named cyclindependent kinase inhibitor (p21) . As the elevated levels of P-ATR, P-CHK1 and p21 after radiation exposure in HKST-treated cells were observed, and there were minor changes on P-ATM and P-CHK2, between HKST treatment and IR group, the ATR pathway might be more crucial in facilitating the protective effects of HKST against IR.
The maintenance and self-renew of intestinal cells, especially the stem cells, in adult mammalian requires Wnt signaling pathway, whereas the inactivation of the essential components of Wnt signaling leads to the deficiency in intestine development and regeneration [40, 41]. Previous study have demonstrated that DDR activation was amplified by Wnt signaling in mammalian cells, whereas the inhibition of Wnt signaling fail to resolve DSBs after radiation [42–44]. As the protective activity of HKST against radiation injury was confirmed to be related to DDR, and the pretreatment of HKST effectively preserved intestinal stem cells when exposed to IR (Fig. 4), the radio-protective effect of HKST might be mediated by Wnt signaling pathway. As expected, the stimulation of ATR and ATM signaling upon IR in HKST-treated cells and the radio-protective effects of HKST on intestinal stem cells in mice were diminished by the selective inhibitor of Wnt signaling pathway (Figs. 3 and 4). Previous study have shown that the kinases ATM/ATR and CHK1/2 were involved in the inflammatory responses induced by TLRs signaling , and TLRs signaling was reported to modulate the DNA repair reaction in cells . As HKST is a co-agonist of TLRs, the role of TLRs signaling in DDR mediated by Wnt signaling pathway upon IR damage remains further investigation. As HKST is the co-agonist of TLRs family, and TLRs family are expressed in different organs. We presumed that HKST might activates other cells and the protective effects of HKST against IR is a whole body protective effects. As the results showed that, besides small intestine, HKST pretreatment significantly activates NF-kB signaling pathway in liver and spleen.