Protective Effect And Mechanism Of Lactoferrin On Radiation Induced Intestinal Injury

Radiation-induced gastrointestinal injury is a common complications of radiotherapy for abdominal or pelvic neoplastic conditions.It mainly manifests as nausea,vomiting,abdominal pain,diarrhea,tenesmus,intestinal obstruction,and intestinal perforation.In severe cases,it can lead to death.Emerging evidence has implicated iron death in the pathogenesis of radiation-induced intestinal injury.Irradiation can generate many reactive oxygen species and upregulate the expression of key enzyme acyl-CoA synthetase long-chain family member 4(ACSL4),promoting lipid peroxidation and ultimately leading to iron death.In addition,research has shown that gut microbiota also plays an important role in radiation-induced intestinal injury.Lactoferrin(Lf)is a non-heme iron-binding glycoprotein.Lf plays a crucial role in regulating iron absorption and immune responses.Additionally,it possesses anti-inflammatory and antioxidant properties.Recent research has indicated that Lf may ameliorate radiation-induced intestinal injury,although its underlying mechanisms remain unclear.Objective:To explore the protective effect of Lf on radiation-induced intestinal injury and its mechanism in apoptosis,ferroptosis,and gut microbiota.Methods:The mice were divided into three groups(control,10Gy,and Lf+10Gy).The Lf±10Gy group received a dose of 250mg/kg of Lf via oral gavage once daily for 7 days prior to irradiation.The mice in the 10Gy group and Lf+10Gy group were subjected to abdominal irradiation(WAI)with a dose of 10Gy.The mice were euthanized 3.5 days after irradiation,and blood samples,liver,small intestine,and other tissues were collected.The peripheral blood cell count,antioxidant indicators of the liver,and levels of inflammatory cytokines in the serum were measured.Intestinal tissue damage was assessed through hematoxylin and eosin staining and immunohistochemistry.TUNEL,electron microscopy,and Western blot techniques were employed to analyze apoptosis and ferroptosis in mouse intestinal tissue.Additionally,16S rDNA analysis was conducted to determine the gut microbiota composition.Results:1.The weight of the mice subjected to radiation continued to decrease,causing damage to their intestinal tissues,immune organs,and hematopoietic systems,and triggering inflammatory responses and oxidative stress.2.General vital signs:Compared to the 10Gy group,the Lf+10Gy group of mice experienced less weight loss and intestinal damage.Additionally,their spleen,thymus,and liver coefficients demonstrated signs of recovery.3.Hematopoietic system:Compared to the 10Gy group,the Lf+10Gy group demonstrated recovery in white blood cell count,lymphocyte count,and monocyte count.4.Antioxidant index and inflammatory index:Compared with the 10Gy group,the levels of glutathione(GSH)and catalase(CAT)were increased,and the tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were decreased in the Lf+10Gy group.5.The intestinal tissue structure and function:In comparison to the 10Gy group,the Lf+10Gy group demonstrated significant restoration of villus length,a slight increase in goblet cell count,and a marked increase in the number of Lgr5 and Ki67 positive cells.6.Intestinal tissue apoptosis level:Irradiation caused apoptosis of small intestinal tissue.Compared to the 10Gy group,the Lf+10Gy group showed a significant decrease in the number of TUNEL-positive cells in each crypt,a significant increase in the expression of anti-apoptotic B-cell CLL/lymphoma 2 protein(Bcl-2),and a significant decrease in the expression of pro-apoptotic BCL2-Associated X protein(Bax).7.Intestinal tissue ferroptosis level:Irradiation caused ferroptosis of small intestinal tissue.Compared to the 10Gy group,the Lf+10Gy group showed a significant improvement in mitochondrial morphology.Regarding iron metabolism,Lf significantly downregulated the expression of divalent metal transporter 1(DMT1)protein and Fe2+content in the small intestine.In the GSH/GPX4-lipid peroxidation pathway,Lf significantly upregulated the protein expression of solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4),downregulated ACSL4 protein expression,and reduced malondialdehyde(MDA)levels.8.Intestinal microbiota:Irradiation caused dysbiosis of the intestinal microbiota.Compared to the 10Gy group,the Lf+10Gy group showed a significant increase in the Shannon,ACE,and Chao1 indices,which are measures of α-diversity.Compared to the 10Gy group,the Lf+10Gy group showed a significant increase in bacteria such as Anaerofustis,Dehalobacterium,Bacteroides,Oscillospira,and Ruminococcus and a significant decrease in bacteria such as Prevotella.Conclusions:1.Lactoferrin enhances the proliferation and regeneration capacity of intestinal cells in irradiated mice,mitigating radiation-induced intestinal damage,and ameliorating radiation-induced oxidative stress;2.Lactoferrin potentially mitigates radiation-induced intestinal damage by inhibiting apoptosis and ferroptosis;3.Lactoferrin improves radiation-induced dysbiosis of the gut microbiota,thereby ameliorating radiation-induced intestinal damage.