The Proliferative And Regenerative Activities As Well As Mechanism Of Intestinal Crypt Cells Upon Radiation Exposure Mediated By MPLA

Soldiers on nuclear-related military missions unavoidably suffer from the damage induced by ionizing radiation(IR).Acute radiation syndrome(ARS),which is also called acute radiation sickness,often occurs after whole body exposure to a high dose of radiation in a short period of time.Gastrointestinal(GI)tract represents one of the most susceptible organs to IR injury,in which the intestinal cells will be killed massively upon IR exposure,thereafter the regeneration of villi is severely impaired and the epithelial integrity along the entire GI tract is compromised.Nevertheless,there is currently no effective medical strategy to cure severe hematopoietic syndrome or GI syndrome caused by IR exposure due to the serious damage on GI.There is a great need to develop agents diminishing intestinal injury induced by IR exposure and clarify the underlying mechanism.As the source of intestinal regeneration,the crypts serve as the niche for intestinal stem cells.Upon IR injury,a mass of intestinal stem cells(ISCs)are irreparably killed,which will then impede the regeneration of intestine.The mechanism involved in proliferation and regeneration of intestinal crypt cells upon IR damage may shed new light on GI syndrome treatment.Toll-like receptors(TLRs)family agonists represent a series of effective agents in facilitating the nullification of the IR-induced injuries.Since the activation of TLR5 was demonstrated to counteract radiation-induced damage on mice and monkey,the stimulation of TLRs family,including TLR2/6,TLR4 and TLR9,have been reported to be an effective strategy to relieve radiation-induced damage.In previous study,our group have demonstrated that TLR4 agonists lipopolysaccharide(LPS)and monophosphoryl lipid A(MPLA)could significantly protect mice against IR injury,in which the survival rate was improved and the GI toxicity was alleviated.However,the mechanism involved in the radioprotective effects of LPS or MPLA on intestine was unclear.In the present study,the proliferative effect and the associated mechanism of LPS or MPLA on intestinal crypt cells upon IR exposure were investigated.With immunofluorescence assay,the TLR4 receptor was found to be restricted on intestinal crypts,indicating the modulated effect of TLR4 on intestinal stem cells.The proliferative effect of LPS or MPLA on intestinal crypt cells mediated by TLR4 was confirmed by employing mouse intestinal organoids.Besides,with immunohistochemistry and intestinal organoids cultivation,we found that LPS or MPLA treatment also preserved the structure of intestinal organoids after IR exposure,and mitigated radiation-induced injury on intestine in vivo.To explore the underlying mechanism associated with the proliferative effect of LPS or MPLA on intestinal crypt cells upon IR exposure,intestinal crypt cells of mice receiving abdominal ionizing radiation(AIR)or receiving MPLA treatment followed by AIR were collected.The related pathways were investigated by RNA sequencing,in which the biomarkers of active intestinal stem cells(Lgr5,Olfm4)were significantly downregulated while the biomarkers of quiescent intestinal stem cells(Clu,Bmi1)upregulated upon AIR exposure,but Lgr5 was remarkably upregulated with MPLA treatment after AIR damage.Besides,pathways(including Wnt singaling,Notch signaling,Hippo signaling,Hedgehog signaling)and genes(including SOX9,c-Myc,MSI)which were reported to facilitate intestinal stem cells regeneration were inhibited upon AIR injury,but MPLA treatment stimulated these pathways and genes again.Interestingly,pathways involved in cell apoptosis,necrosis,ferroptosis,RNA or amino acids degradation were activated by AIR exposure,and MPLA treatment inhibited theses pathways.The above study indicated that the mitigated effect of MPLA against AIR exposure might be due to the modulation of intestinal stem cells as well as the inhibition of apoptosis or necrosis.Proteins of intestinal organoids and crypt cells were extracted after LPS or MPLA treatment,and the corresponding intestine issues were collected.With western-blot assay and immunofluorescence assay,we found that Wnt signaling,Notch signaling as well as cMyc,MSI2,SOX9,YAP1 were stimulated by LPS or MPLA treatment.When Wnt signaling,Notch signaling and c-Myc proteins were inhibited by selected inhibitors,the proliferative effect of LPS or MPLA on intestinal organoids were diminished.With adeno-associated virus(AAV)which can selectively knock-down MSI2,SOX9 or YAP1 on intestine,we found that the proliferative effect of LPS or MPLA on intestinal organoids and crypts without or with IR exposure were all diminished.Besides,the lentiviruses were constructed to knockdown MSI2,SOX9 or YAP1 on intestinal organoids,combined with western-blot and immunofluorescence assay on mice treated by AAV,we found that LPS and MPLA stimulated c-Myc-MSI2-SOX9-YAP1 signal axis on intestinal organoids and crypts.To investigate the interaction of related molecules upon TLR4 agonist stimulation,human intestinal organoids cultivation and co-immunoprecipitation were employed,in which the recruitment of c-Myc,MSI2,SOX9,YAP1 by My D88 or Trif were increased by MPLA stimulation.Further experiment showed that,there is remarkable difference of proteins recruitment pattern between LPS and MPLA treatment,in which MSI2,SOX9,YAP1 are more important for the recruitment of associated proteins upon MPLA treatment.Besides,MSI2,SOX9,YAP1 are essential for Trif to recruit c-Myc and ?-catenin.The interaction between My D88 and ?-catenin,SOX9,YAP1 or c-Myc were eliminated without MSI2.Finally,we found that LPS or MPLA treatment could enhance the interaction among MSI2,SOX9 and YAP1.With AAV which can selectively knock-down MSI2,SOX9 or YAP1 on intestine,we demonstrated that the formation of MSI2-SOX9-YAP1 complex was critical for the recruitment of ?-catenin or c-Myc by MSI2,SOX9 or YAP1.Besides,MSI2 could not recruit SOX9 or YAP1 until the formation of SOX9-YAP1 complex.To sum up,LPS and MPLA were confirmed to stimulate the proliferation of intestinal crypt cells with or without IR exposure,in which the proliferative effects were mediated by Wnt signaling,Notch signaling,c-Myc,MSI2,SOX9 and YAP1.A novel signal axis(c-MycMSI2-SOX9-YAP1)was discovered in intestinal organoids and crypt cells upon LPS or MPLA stimulation,and the interaction pattern between the related proteins in intestinal crypt cells upon LPS or MPLA treatment,as well as the different patterns between My D88 and Trif recruiting related protein were also clarified.