Research Of NADH Protecting Against Radiation Enteritis And Its Mechanism

ObjectiveTo verify whether NADH has a protective effect on radiation-induced enteritis,one of the side effects of radiation therapy,and to try to explore its mechanism of action at the level of cellular processes and molecular signaling pathways.Methods1.Use IEC-6 rat intestinal mucosal cells,culture in complete culture medium,pass them at appropriate time,wait until enough bottles are dispensed,add cells with different concentrations(0,100,200,300,400,500,600,700,800ug/ml)of NADH,and cell count.The kit analyzes cell viability.2.The IEC-6 cells were placed on a multiwell plate at a concentration of 2.5 x 105 and randomly divided into 3 groups and the cells were treated with 4 Gy X-rays.However,Oug/ml,100ug/ml,and 200ug/ml NADH were added to each group.Annexin V-APC/7-AAD staining was used to determine the apoptosis of cells by flow cytometry.The other cells were lysed to extract total protein.Western blot was used to determine the apoptosis-inducing Bax protein content of anti-apoptosis protein BCL2.3.For ROS NADH-treated cells,10 ?M 2,7-dichlorofluorescein diacetate(DCFDA)was used to determine ROS levels.Western blot was used to determine inflammatory cytokines IL-1?,P65 and TNFa,and autophagy-related signaling molecules PI3K.And expression of phosphorylated AKT4.Cells cultured with IEC-6 were further divided into 4 groups:radiotherapy,Oug/ml,100ug/ml,200ug/ml NADH and 100ug,/ml+3-MA autophagy inhibitor,annexin V-APC/7 Apoptosis was detected by flow cytometry using AAD staining.The expression of reactive oxygen species,PI3K and phosphorylated AKT were measured by Western blot,and IL-1?,P65,TNFa and DCFDA were used to determine the expression of inflammatory cytokines.5.Sixteen male C57BL mice aged 6 weeks were selected for animal experiments.They were randomly divided into 4 groups:control group,NADH group,RT group,RT + NADH group.4 in each group.After 1 week of adaptation,lethal doses of radiation were administered to the RT and RT + NADH groups,and 10 mg/kg/day of NADH was administered to the NADH and RT + NADH groups.After 7 days,body weights were recorded,animals were sacrificed,and intestinal tissue samples were taken.The cells were stained with HE and immunohistochemistry was used to determine the expression of inflammatory factors.Results1.NADH with different concentrations(0,100,200,300,400,500,600,700,800ug/ml),cell viability were 1.73,2.08,2.06,1.74,1.78,1.71,1.61,1.52,and 500ug/ml NADH had no obvious side effects on IEC-6 cell viability.However,the concentration of NADH above 600ug/ml had a certain inhibitory effect on cell viability.In the subsequent cell experiments,the amount of NADH was within the safety range of 100,200 ug/ml.2.After 4Gy irradiation of IEC-6 cells,compared with the blank control group,the apoptosis rate in the NADH treated group was significantly reduced by 9.85.2.4.1.Western blotting also showed that the expression of anti-apoptotic protein BCL2 in NADH-treated cells was increased.Reduced pro-apoptotic Bax protein expression,these differences are all related to the dose of NADH addedImmunofluorescence results showed that the expression of LC3 in NADH-treated IEC-6 cells was increased,the content of intracellular reactive oxygen species was significantly decreased,and the expression of inflammatory factors IL-1?,P65 and TNFa in Western blot was also inhibited.In NADH-treated IEC-6 cells,the expression of PI3K and phosphorylated AKT signaling molecules in the autophagy down-regulation pathway was reduced.NADH dose related4.On the basis of NADH treatment,3-MA inhibited autophagy,PI3K and phosphorylated AKT expression recovery,increased reactive oxygen species content by 7.089 4.5 3.6,increased apoptotic rate by 17.6 7.9 6.4 14.35.The weight loss of RT group revealed that NADH significantly inhibited intestinal congestion,edema and weight loss.Overexpression of autophagy by inflammatory factors is inhibited.HE shows that NADH can protect the height of fluff.Intestinal mucosal damage is less.Conclusionsour experiments show that NADH can effectively inhibit the production of ROS and inflammation through activation of autophagy in RT.Animal studies showed that after RT,NADH + RT treated animals had better conditions than the only RT group.Intestinal inflammation was lighter than the control group,and autophagy increased.At the same time,inhibition of the PI3K/AKT signaling pathway involves NADH-induced autophagy.