The Mechanism Of Ginsenoside Rk1 For The Treatment Of Radiation-induced Intestinal Injury

Background and Aims.Radiation-induced intestinal injury(RIII)frequently occurs during radiotherapy;however,treatment strategies for RIII are limited.Ginsenoside Rk1(RK1)is a substance that is derived from ginseng,and it has several biological activities,such as antiapoptotic,antioxidant and anticancer activities.Therefore,the present study was designed to investigate the potential protective effect of Rk1 on RIII and the potential mechanisms.Materials and Methods.Irradiated IEC-6 cells and rats were treated with RK1.IEC-6 cell viability was determined by using CCK-8 to select the most effective concentration of RK1.After irradiation and RK1 treatment,the hepatic function and the survival status of rats were monitored.Then,intestinal samples were collected for mRNA sequencing analysis,followed by hematoxylin and eosin(H&E)staining for morphological observation.In addition,the proliferation,oxidative stress,and apoptosis and the potential mechanisms were investigated in vitro and in vivo by the way of flow cytometry,immunofluorescence,immunohistochemistry and western blotting.Moreover,in order to further explore the underlying mechanism of RK1 treatment,the overlapping differentially expressed genes from mRNA sequencing were performed by GO,KEGG functional enrichment and immune cell enrichment,as well as analyzed in protein-protein interaction networks.Results.The results showed that RK1 treatment significantly improved the survival rate of the irradiated rats and markedly ameliorated the structural injury of intestinal mucosa observed by histology.Specifically,treatment with RK1 significantly alleviated radiation-induced oxidative stress by inhibiting reactive oxygen species(ROS)generation of IEC-6 cells in vitro.In addition,RK1 treatment decreased malondialdehyde(MDA)contents from 1.49±0.55 nmol/mg prot to 0.97±0.27 nmol/mg prot and increased superoxide dismutase(SOD)levels from 15.35±4.89 U/mg prot to 24.06±4.31 U/mg prot in vivo(P<0.05).Furthermore,treatment with RK1 significantly decreased the apoptotic percentage of irradiated IEC-6 cells on day 3 and day 5(P<0.05).Immunofluorescence assay showed RK1 administration highly reduced the number of TUNEL-positive cells from 24.60±6.28 to 11.60±2.64(P<0.05).And RK1 treatment also decreased Bax protein expression and increased Bcl-2 protein expression in IEC-6 cells.Additionally,mRNA sequencing identified 388 differentially expressed genes(DEGs)and showed that the PI3K-AKT pathway may be a key signaling pathway by which RK1 exerts its therapeutic effects on RⅢ.Moreover,protein-protein interaction(PPI)network analysis shown that CD4 had a significant higher number of nodes in the entire PPI network.Also,the relative enrichment of immune cell types and shown that the activated CD4 T cell,activated CD8 T cell,effector memeory CD8 T cell,immature dendritic cell and natural killer T cell might play key roles in RK1 treatment of RⅢ.The western blotting results showed that radiation-increased expression levels of p-PI3K,p-AKT,and p-mTOR were evidently inhibited by RK1,while these effects were reversed by IGF-1.Conclusion.This study demonstrated that RK1 can alleviate RⅢ by promoting cell proliferation,inhibiting oxidative stress response and apoptosis.And the potential mechanism of the anti-apoptotic effect of RK1 may involve the suppression of the PI3K/AKT/mTOR pathway.In addition,the mechanism of RK1 treating RⅢ was probably related to the regulation of CD4 and a variety of immune cells.Altogether,this study provided a promising therapeutic agent for RⅢ.