Study On The Effect And Mechanism Of Baicalein On Intestinal Injury Induced By Ionizing Radiation

Aims:Intestinal injury is the main cause of death within the first week after high dose ionizing radiation(IR),which is also the main side effect of patients with pelvic and abdominal tumors after radiotherapy.Baicalein,a plant-derived flavonoid,alleviates the injury of a variety of diseases via different molecular mechanism,such as anti-oxidative stress,anti-apoptosis,anti-inflammatory and so on.In this study,we evaluated the protective effect of baicalein on intestinal injury induced by ionizing radiation and explored the mechanism of radioprotection.Methods:At the cellular level,the cell viability was detected by CCK-8 assay after pretreatment with different doses of X-ray irradiation and different concentrations of baicalein.Flow cytometry was used to detect the effects of baicalein on the levels of reactive oxygen species(ROS),y-H2AX and apoptosis in irradiated small intestinal epithelial cells(IEC-6),respectively.In the mice survival experiment,the mice were randomly divided into 4 groups:Control,Baicalein,IR and IR+Baicalein.The mice in Baicalein group and IR+Baicalein group were intraperitoneally injected with 100 mg/kg baicalein 1 h before irradiation and 1 day after irradiation,respectively.The rests including Control group and IR group were injected intraperitoneally with normal saline.The survival of mice was observed within 15 days after total body irradiation(TBI)with 10 Gy y ray.In the intestinal experiments,the mice were divided into 3 groups:Control,IR and IR+Baicalein,the dosage regimen was same as described above.The small intestinal tissue and fecal samples of mice were collected on the 3rd day after 9 Gy y ray TBI.HE staining was performed to observe the changes of intestinal structure and crypt number.The expression of Lgr 5,Axin 2,Ki67,Lysozyme and Villin in small intestine was detected by immunohistochemical staining.The changes in the number of apoptotic cells was investigated by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL).The expression of p53,8-OHdG,Caspase 3,Caspase 8,Bcl-xl and Bax proteins in intestine was detected by immunofluorescence or Western-blot.16S rRNA of feces was sequenced and the sequencing data were analyzed by α diversity analysis,β diversity analysis,abundance analysis of gut microbiota at phylum and genus levels,LefSe analysis and KEGG pathway enrichment analysis.Results:In vitro experiments,it is found that 9 Gy X-ray irradiation significantly reduced the viability of IEC-6,and 2.5 μM baicalein given 1 h before irradiation could exert the best radioprotective effects on IEC-6.Further experiments showed that baicalein could reduce the expression of ROS and γ-H2AX,and inhibit the apoptotic response among irradiated cells.In vivo experiments,it is found that baicalein could significantly improve the survival rate and prolong the survival time of irradiated mice,increase the number of small intestinal crypts and lengthen the length of small intestinal villi,markedly increase the number of Lgr 5+,Axin 2+,Ki67+,Lysozyme+ and Villin+cells.TUNEL assay showed that the number of apoptotic cells were also reduced as expected.Besides,the expression of proteins including p53,8-OHdG,Caspase 3,Caspase 8 and Bax were downregulated.Baicalein could significantly improve the structure of gut microbiota,increase the abundance of probiotics and reduce the abundance of harmful bacteria at the phylum and genus level,promote the development of gut microbiota to be beneficial to the health of mice.Conclusion:Baicalein has the effect of radioprotection for intestinal injury.The results of cellular experiments show that baicalein has a significant protective effect on the viability of irradiated IEC-6,which can significantly reduce the level of ROS and yH2AX and inhibit the apoptosis of irradiated cells.In the model of mice,it shows that baicalein can significantly increase the survival rate of irradiated mice,improve structure of small intestine and increase the number of intestinal stem cells(ISCs)and the ability of proliferation and regeneration.Further studies show that intestinal radioprotection of baicalein is closely related to rebalancing the composition of gut microbiota,inhibiting activation of p53,and suppressing p53 mediated mitochondrial apoptosis and death receptor apoptosis in intestinal cells of irradiated mice.