Nocardia Rubra Cell-Wall Skeleton Mitigates Radiation-Induced Intestinal Injury In Mice Via Regulating Macrophage Function

Objective:Radiation-induced intestinal injury is commonly associated with structural and functional damage to the intestine tissue caused by nuclear accident exposure and abdominal or pelvic tumor radiotherapy.At present,there is a lack of effective clinical treatment strategy.Nocardia rubra cell wall skeleton(Nr-CWS)is an immunomodulator and antitumor agent with potential radioprotective effects.The purpose of this study was to explore the role and possible mechanism of NR-CWS in the treatment of radiation-induced intestinal damage,and to provide a new method for the treatment of radiation-induced intestinal injury.Methods:1.The effect of Nr-CWS in alleviating radiation-induced intestinal injury in vivoA mouse model of acute radiation-induced intestinal injury was established by irradiating the whole abdomen with 12 Gy X-ray,and Nr-CWS(20μg/kg)was administered intraperitoneally until the third day post-radiation.The survival curve,body weight curve,diarrhea and gross morphology of the mice were observed to evaluate the effect of Nr-CWS on prevention and treatment of radioactive intestinal damage.To further evaluate the effect of Nr-CWS in radiation-induced intestinal damage,the mice were stained by HE staining of intestine tissue sections at different time points(6 h and 3.5 d),immunofluorescence staining of ZO-1,an indicator of mucosal barrier evaluation,AB-PAS staining,immunofluorescence staining ofγH2AX,a marker of DNA damage,and immunofluorescence staining of apoptosis-related markers(TUNEL and Cleaved caspase 3),Lgr5immunohistochemical staining,Lysozyme immunofluorescence staining,PCNA and Ki-67 staining,and 16S r DNA high-throughput gene sequencing were used to analyze the changes of intestinal flora after irradiation.The therapeutic effect of Nr-CWS on radiation-induced intestinal damage was comprehensively evaluated.2.The mechanism of Nr-CWS on alleviating radiation-induced intestinal injuryThe m RNA levels of inflammatory factors such as IL-1β,IL-6 and TNF-αin intestine tissues were determined by q-PCR and protein levels of IL-1α,TIMP-1,KC,Fasl and MCP-1 were detected by protein microarray to clarify the regulatory effect of Nr-CWS on intestinal inflammation.F4/80 staining used to determine whether Nr-CWS recruited macrophages to intestine tissue or not.To determine whether Nr-CWS-induced radioprotection was macrophage-dependent,macrophages in mice were depted with chlorophosphate liposomes and then evaluated by gross observation and HE staining of intestinal tissue.Furtherly,to investihged the effect of Nr-CWS peritoneal macrophages were used to explore the mechanism.The m RNA levels of secretory factors(TNF-α,IL-1β,IL-6 and CD16)and chemokines(CXCL1,CXCL3 and CXCL12)were detected by q-PCR.The phagocytosis of fluorescent microbeads by macrophages was detected by immunofluorescence and flow cytometry.The effect of Nr-CWS on macrophage migration was detected by Transwell assay and immunofluorescence and WB were used to detect whether Nr-CWS activated NF-κB signaling pathway.Results:1.Nr-CWS attenuates radiation-induced intestinal injuryIn the model of radiation-induced intestinal injury,Nr-CWS reduced the mortality,rate of weight loss and diarrhea in irradiated mice.Gross anatomy showed that irradiation caused a decrease in intestinal contents and obvious bloody or watery diarrhea,Nr-CWS treatment made intestinal contents close to normal.HE staining of intestine showed that Nr-CWS reduced the damage of morphological structure of intestine.ZO-1 immunofluorescence staining showed that Nr-CWS treatment alleviated radiation-induced intestinal epithelial cell injury and promoted the recovery of intestinal epithelial mucosal barrier.AB-PAS staining showed that Nr-CWS reduced the loss of goblet cells.γ-H2AX immunofluorescence staining showed that Nr-CWS reduced the level of radiation-induced DNA damage in intestinal epithelial cells.TUNEL and Cleaved caspase 3 immunofluorescence staining showed that NR-CWS reduced radiation-induced apoptosis cells in intestinal crypt cells.Lgr5~+immunohistochemical staining showed that Nr-CWS maintained the survival of the intestinal crypt stem cells post-radiation.Lysozyme immunofluorescence staining showed that Nr-CWS reversed the reduction of Paneth cells in intestinal crypts induced by radiation.The results of PCNA and Ki-67 staining showed that Nr-CWS alleviated the proliferation inhibition of intestinal tissue cells induced by radiation,protected intestinal crypt cells and promoted intestinal epithelial regeneration.16S r DNA gene sequencing analysis of intestinal flora changes post-irradiation showed that irradiated mice shared 21 OTUs with unirradiated mice,and irradiated mice shared 227 OTUs with unirradiated mice after Nr-CWS treatment and the total number of OTUs after Nr-CWS treatment was close to that of non-irradiated mice.Irradiation significantly reduced theαdiversity of intestinal flora,while Nr-CWS treatment restored theαdiversity of intestinal flora.The relative abundance histogram showed that Nr-CWS increased the abundance of Lachnospiraceae,Akkermansiaceae,and Prevotellaceae at the family level.At the phylum level,Nr-CWS reduced the relative abundance of Proteobacteria and increased the relative abundance of Bacteroidetes;NMDS analysis showed that the structure of intestinal flora changed after IR exposure and Nr-CWS treatment.At the genus level,heat map analysis showed that Nr-CWS increased the relative abundance of Alistipes,Akkermansia and Faecalibaculum;IDA effect analyzed the differences in intestinal flora species after IR exposure and Nr-CWS treatment.The results showed that IR enriched Candidatus-Arthromitus,while Nr-CWS enriched Verrucomicrobiae,Akkermansiaceae,and Lachnospiraceae.In summary,the results showed that Nr-CWS treatment improved the survival rate of irradiated mice,alleviated weight loss,reduced the damage of intestinal morphology and function,reduced intestinal epithelial cell DNA damage and intestinal crypt cell apoptosis,and promoted the proliferation and differentiation of intestinal crypt Lgr5~+intestinal stem cells.In addition,Nr-CWS treatment improved the composition and relative abundance of intestinal flora.2.Nr-CWS alleviates radiation-induced intestinal injury by activating macrophage functionThe results of q-PCR showed that radiation increased the m RNA levels of inflammatory factors such as IL-1β,IL-6 and TNF-αin the intestine,and Nr-CWS treatment reduced the expression of these inflammatory factors.Protein microarray results showed that radiation caused elevated protein levels of KC,IL-1αand TIMP-1in intestinal tissues,and Nr-CWS treatment down-regulated these proteins and upregulated MCP-1 and Fas L protein levels.F4/80 immunofluorescence staining showed that Nr-CWS treatment promoted macrophage recruitment in the intestinal mucosa.After depleting macrophages with chlorophosphate liposomes in vivo,it was found that the therapeutic effect of Nr-CWS was reversed,and severe bloody diarrhea also occurred in the Nr-CWS treatment group.HE staining of intestinal tissue sections showed that mice in the Nr-CWS-treated group given chlorophosphate liposomes did not have improved intestinal epithelial integrity in terms of intestinal morphological structure,length of villi and number of surviving crypt foci compared to irradiated mice receiving chlorophosphate liposomes alone,suggesting that Nr-CWS promotes the repair of radioactive small intestinal injury dependent on macrophages.At the cytological level,q-PCR results showed that Nr-CWS increased the m RNA expression levels of inflammatory factors and chemokines such as TNF-α,IL-1β,IL-6,CD14,CXCL1 and CXCL3,immunofluorescence and flow cytometry results showed that NR-CWS treatment enhanced the phagocytosis of macrophages.Transwell results showed that Nr-CWS increased the migration ability of macrophages.Immunofluorescence and WB results showed that Nr-CWS promoted nuclear translocation and phosphorylation of NF-κB,indicating that NF-κB signaling was activated.The above studies suggest that the preventive and curative effects of Nr-CWS on radioactive small intestinal injury may promote macrophage phagocytosis and migration through activation of NF-κB signaling pathway,and reduce the inflammatory response of injured tissues,but the specific mechanism needs further in-depth study.Conclusions:1.Nr-CWS increases the survival rate of irradiated mice and ameliorates WAI-induced disruption of small intestinal structure and barrier function.2.Nr-CWS maintaines the survival of intestinal stem cells and promotes the regeneration of intestinal epithelium.3.Nr-CWS maintains intestinal flora homeostasis and improves the composition of intestinal flora components.4.Nr-CWS-induced radiation protection is macrophage-dependent,activating the NF-κB signaling pathway and promoting macrophage phagocytosis and migration capacity.