Protective Effect Of Lactoferrin On Radiation-induced Intestinal Injury And Its Mechanism

As the main means of cancer treatment,about 60%of cancer patients need radiotherapy at different stages of the disease.Because of the special position of small intestine,it is unavoidable that the radiotherapy of pelvic and abdominal tumors will be accidentally irradiated.Intestinal tissue is rich in highly proliferative cells,which are very sensitive to radiation.The dose of radiation used by clinical patients is very close to the dose that causes small intestinal injury.The above three points determine that radiation intestinal injury is one of the most common complications after radiotherapy for pelvic and abdominal malignant tumors,which not only affects the therapeutic effect of tumors,but also seriously affects the quality of life and mental health of patients.At present,there is a lack of effective prevention and treatment for radiation-induced intestinal injury in clinic.Therefore,searching for effective and low toxicity protective drugs to prevent and treat radiation-induced intestinal injury has become a hotspot in radiation medicine research and a clinical problem that needs to be solved urgently.Objective:To study the protective effect of lactoferrin on radiation-induced intestinal injury and its mechanism,so as to provide theoretical basis for clinical prevention and treatment ofradiation-induced intestinal injury.Methods:Eight-week-old male Balb/c mice of SPF grade were randomly divided into 8Gy,2mg Lf+8Gy,4mg Lf+8Gy and 6mg Lf+8Gy groups,10 mice in each group,and 9 mice in control,5Gy,2mg Lf+5Gy and 4mg Lf+5Gy groups.The former was used to study the effect of lactoferrin intervention on the survival of mice exposed to lethal dose radiation,while the latter was used to study the protective effect and mechanism of lactoferrin intervention on mice exposed to sublethal dose radiation.A special plastic fixed box for mice was used.Six MeV high-energy X-ray linear accelerator(Siemens)was used for single whole body irradiation.The dose rate was 2Gy/min,and the irradiation field was 40 cm x 40 cm.Lactoferrin is produced by Hilmar Company of the United States.Its purity is 92.5%and it is powder-like.Lactoferrin was dissolved in sterilized saline and mixed into lactoferrin solution of 10 mg/ml,20 mg/ml and 30 mg/ml.Lactoferrin solution was injected intraperitoneally 4 hours before irradiation and 1,2 and 3 days after irradiation.The vital signs,mortality,body weight,food intake and drinking water of mice were observed every day in the survival observation experiment.In the experiment of observing the effect and mechanism,blood was collected by eyeball removal 1,3,9 days after irradiation.One part of the blood was used for routine blood tests,the other part was centrifuged and stored at-80℃ for measuring inflammatory factors and antioxidant indexes of blood;thymus and spleen were taken for weighing;A portion of small intestinal tissue was fixed with formalin for HE staining.The other part of small intestine tissue was stored at-80℃ and the proteins were detected by Western blotting.Results:Experiment 1:The effect of lactoferrin intervention on the survival time of mice exposed to lethal dose of irradiationAfter 8Gy lethal dose irradiation,the mice’s activity decreased,the intake and drinking water decreased,the body weight decreased,and diarrhea occurred to varying degrees.At the same time,the response to external stimuli became slower,and the hair fell off or gathered.The average survival days of 8Gy,2mg Lf+8Gy,4mg Lf+8Gy and 6mg Lf+8Gy groups were 4.30±1.34,4.20±0.71,5.75±2.44 and 6.70±2.54 days.Compared with 8Gy,the survival time of 4mg Lf+8Gy and 6mg Lf+8Gy groups was significantly prolonged(P<0.05).Survival analysis showed that the survival rates of mice in 4mg Lf+8Gy and 6mg Lf+8Gy groups were significantly higher than those in 8Gy groups(P<0.05).Experiment 2:Protective effect and mechanism of lactoferrin intervention on sublethal dose irradiated mice(1)Establishment of radiation-induced intestinal injury model:After irradiation,the intake and drinking water of mice decreased,weight loss,hair gloss and activity decreased.The general situation of mice after dissection showed that the intestinal contents of irradiated mice were reduced and the texture was sparse.HE staining of small intestine showed that after irradiation,the structure of small intestine was destroyed,villus height was reduced,shedding and breaking appeared,capillaries were dilated and congested,inflammatory cells were infiltrated,and the number of small intestinal crypts was reduced.These phenomena confirm that the model of radiation-induced intestinal injury in mice is successful.(2)Weight change:After irradiation,the weight of mice decreased to the lowest level on the 3rd day after irradiation.The weight of mice in 5Gy,2mg Lf+5Gy and 4mg Lf+5Gy groups decreased by 3.16g,2.73g and 2.97g,respectively.From the 4th day after irradiation,the body weight gradually recovered.Weight recovery of mice in lactoferrin intervention group was faster than that in irradiation group.On the 9th day,the weight of mice in 4mg Lf+5Gy group was close to that of control group.(3)Spleen index and thymus index:On the first day after irradiation,spleen index of mice in 2mg Lf+5Gy and 4mg Lf+5Gy groups was 20.8%and 26.9%higher than that in 5Gy group(P<0.05).On the third day after irradiation,the spleen index of mice in 4mg Lf+5Gy group was 25.0%higher than that in 5Gy group(P<0.05).There was no significant difference on the 9th day.On the 1st,3rd and 9th day after irradiation,There was no significant difference in thymus index between lactoferrin intervention group and irradiation group.(4)White blood cells,red blood cells and platelet levels:On the first day after irradiation,the number of white blood cells in 2mg Lf+5Gy and 4mg Lf+5Gy groups was 43.7%and 54.2%higher than that in 5Gy group(P<0.05).On the third day after irradiation,the number of leukocytes in 2mg Lf+5Gy and 4mg Lf+5Gy groups was 66.4%and 67.4%higher than that in 5Gy group(P<0.05).On the 9th day after irradiation,there was no significant difference.On the 1st and 3rd day after irradiation,the number of red blood cells and platelets did not change significantly.On the 9th day after irradiation,the number of red blood cells and platelets decreased significantly,especially the number of platelets was significantly lower than that of the control group.(5)Serum levels of inflammatory factors:On the 9th day after irradiation,the level of IL-6 in serum of mice increased significantly.On the 3rd and 9th day after irradiation,the level of serum TNF-alpha in mice increased significantly.On the 3rd and 9th day after irradiation,the serum levels of IL-6 and TNF-alpha in lactoferrin intervention group were significantly lower than those in simple irradiation group(P<0.05).(6)Serum SOD level:There was no significant difference in serum SOD levels between groups 1,3 and 9 days after irradiation.(7)HE staining of intestinal tissue:Compared with the control group,the intestinal mucosa surface of the irradiated mice was incomplete,the epithelium of the mucosa fell off,the length and number of villi were significantly reduced,the number of crypts was significantly reduced,accompanied by edema and congestion.However,the intestinal injury of mice in the lactoferrin intervention group was less than that in the irradiation group.The intestinal villus height and the number of crypts in the intervention group were better than those in the irradiation group,and the difference was statistically significant(P<0.05).(8)Changes of expression level of NF-kappa B signaling pathway protein in small intestine:Compared with the control group,the expression of IKKα,IKKβ and NF-κB protein in small intestine tissue increased after irradiation.On the 9th day after irradiation,the expression levels of the three proteins in the 4mg lactoferrin intervention group were 35.71%,43.75%and 47.83%lower than those in the simple irradiation group respectively(P<0.05).Conclusions:1.Lactoferrin intervention can prolong the survival time of mice exposed to lethal dose of radiation and improve the survival rate.2.Lactoferrin has a protective effect on the injury of blood system and intestinal tissue induced by radiation.Its mechanism may be related to the inhibition of inflammatory factors and regulation of NF-kappa B signaling pathway.