Polydatin Acted As A Radioprotector Of The Intestine And A Radiosensitizer Of Cancer In Mouse Colorectal Carcinoma Model

Radiation therapy is one of the most important therapies for the treatment of human malignancies,but its acute and late toxic effects on normal tissues limit the effect on oncotherapy~1.The small intestine is the most sensitive gastrointestinal organ to ionizing radiation~2.Intestinal damage is a major clinical problem in patients receiving abdominal or pelvic irradiation,which limits the radiotherapy of pelvic,abdominal and retroperitoneal malignant tumors.Therefore,to search effective small intestine radioprotective drugs become a problem to be solved as soon as possible,and they not only should have radioprotective effect on normal tissue,but also must not promote tumor growth.Polydatin(PD)is the fourth kind of monomer extracted from the dried root and rhizome of Polygonumviviparum,as well as a natural precursor of resveratrol.PD has been extensively studied because it has great pharmacological activities,including inhibition of the production of inflammatory mediators~3,induction of antioxidant production~4,regulation of immune function~5,prevention of mutations~6,induction of tumor cell apoptosis~7,protection of the liver~8 and prevention of cardiovascular disease~9.Therefore,we speculate that PD can be used as a candidate for radioprotective drugs.This study mainly investigates the radioprotective effect of PD on the small intestine of colorectal cancer in-situ mice model with 10Gy abdominal irradiation,and explores the suppressive effect of PD combined with radiotherapy on the growth of colorectal cancer.In addition,we investigate theeffect of PD on Colorectal cancer cell lines and Lgr5~+cancer stem cells(CSCs)in vitro.Part One:The radioprotective effect of PD on small intestineObjective:To study the radioprotective effect of PD on small intestine in mice and human intestinal epithelial cells.Methods:The azoxymethane(AOM)/dextran sodium sulfate(DSS)induced in-situ colorectal cancer model was established in C57BL/6 mice.The model mice were divided into 5 groups:control group(control),PD mono-treated group(PD),10Gy irradiation group(IR),PD combined irradiation treatments group(PD+IR),amifostine administration combined irradiation group(AMI+IR).Abdominal was exposed to 10Gy X-ray(200 cGy/min)once a week for a total of 4 times.PD was given by intraperitoneal injection 30min before radiotherapy at the concentration of 25mg/kg.Five mice of each group were randomly selected to record the survival rate,and the other five were sacrificed at the fourth week,and the small intestines were collected to make paraffin section.Then HE staining was used to observe the change of intestinal crypt and villus,and the expressions of Ki67 and CD31 in the intestine were studied by immunohistochemical staining.The survival time of the mice was observed as well.In addition,FHs74Int intestinal epithelial cells were cultured in vitro and the effects of PD on the toxicity and proliferation of FHs74Int cells were observed by CCK8 assay.The effects of different concentrations of PD on the survival time of C57BL/6 mice were observed at 30 min before and after 20Gy abdominal irradiationResults:Compared with IR group,PD+IR group had longer villi of small intestine in mice and increased the number of small intestine crypts in vivo.PD+IR group could promote the proliferation of small intestinal epithelial cells and increase intestinal microvascular density,reduce the microvascular apoptosis,and prolong the survival time of mice.PD had little cytotoxic effect on FHs74Int cell and can promote the proliferation of cells after irradiation.Moreover PD was able to prolong the survival time of C57BL/6 mice after 20 Gy irradiation.Conclusion:PD can protect the small intestine from damage of irradiation.Part 2:Study on PD combined with radiotherapy in colorectal cancerObjective:To study the role of PD in colorectal cancer radiotherapy and to explore its mechanism.Methods:The colorectal cancer model and treatments were same as before.The mouse were sacrificed at the fourth week to observe the number and volume of tumors.Meanwhile,expressions of Ki67 and cleaved caspase-3 in tumors were quantified.The effect of PD on the proliferation and apoptosis of colon cancer cells was also observed in vitro.Furthermore,Lgr5~+Cancer stem cells(CSCs)were sorted from CT26 colon cancer cells and treated with PD to observe their proliferation and apoptosis.The mechanism of PD was explored preliminary.Results:In vivo experiments,PD+IR group can significantly reduce the tumor volume compared with control group,PD group,IR group,AMI+IR group.PD+IR group can inhibit the proliferation of mouse colorectal tumor cells and promote its apoptosis compared with IR group.In vitro experiments,PD+IR group compared with IR group can inhibit the proliferation of colon cancer cell lines&Lgr5~+CSCs and promote their apoptosis.Compared with control group,PD could promote the apoptosis of HCT116 cells and Lgr5~+CSCs.100?M of PD could significantly inhibit the proliferation of Lgr5~+CSCs compared with control group.6.4 nM of type I BMP receptor inhibitor K02288 given before PD+IR combined treatments greatly promoted the proliferation of Lgr5~+CSCs and significantly reversing the cytotoxicity of PD combined irradiation on Lgr5~+CSCs.Conclusion:In vivo and in vitro experiments,PD combined with radiotherapy can reduce tumor cell proliferation and promote tumor cell apoptosis,and PD has a significant inhibitory effect on Lgr5~+CSCs proliferation.BMP signaling pathways may be involved in radiosensitization of PD.