Preventive Effect Of Moringa Oleifera Seed Protease Hydrolysate On Intestinal Mucositis In Mice

5-fluorouracil(5-FU)is a broad-spectrum anticancer drug,often used to treat digestive system cancer,breast cancer,etc.,but in the course of 5-FU treatment,about 80% of people will develop intestinal mucositis.Moringa oleifera seed protein(MSP)and Moringa oleifera seed protease hydrolysate(MSPH)have been found to have antibacterial,antioxidant,anticancer and cardioprotective effects,but their effects in intestinal mucosa have not been reported.Based on this,5-FU injection was used to establish intestinal mucositis mice in this study,and the effect of MSPH on intestinal mucositis in mice was discussed.MSP was extracted from moringa oleifolia seed,enzymatic hydrolysis was performed,and MSPH was obtained by freeze-drying.The molecular weight of MSPH was determined by SDS-PAGE gel electrophoresis.The effect of MSPH on survival rate of IEC-6 cells was determined.Male BALB/c mice were randomly divided into blank control group,model group(5-FU,30 mg/kg·BW),MSPH low-dose,medium-dose and high-dose groups(200,400,800mg/kg·BW),5-FU was used to construct intestinal mucositis model,and the preventive effect of MSPH on intestinal mucositis was studied.Physiological state of mice was recorded and observed every day.After death,the organs of mice were weighed and the organ index was calculated.HE staining was used to observe the morphological changes of intestinal tissue,and villus height/crypt depth was calculated.The mRNA expression of ZO-1 and Occludin in small intestine was detected by RT-qPCR.The contents of INF-γ,SIgA,ROS,G-CSF,ET and D-LA in serum and IL-1β,IL-6,MDA,MPO and TNF-α in small intestine were determined by enzyme-linked immunosorbent assay(ELISA).Microbial diversity was analyzed by 16 S r RNA gene sequencing.The results are as follows:(1)The molecular weight of MSPH was determined to be between 23 KDa and 3.3～14KDa.(2)In vitro experiments,the IEC-6 cells were cultured with different concentrations of MSPH for 36 h,and the concentration of 50 μg/m L could significantly increase the viability of IEC-6 cells(P<0.001).(3)Compared with experimental mouse model group,the body weight of mice in MSPH medium and high dose groups was significantly increased(P<0.01);Compared with blank control group,spleen and thymus indexes in model group were significantly decreased(P<0.01).High dose of MSPH significantly increased the spleen and thymus indices(P<0.01).(4)HE staining results showed that the intestinal villi in the blank control group were arranged neatly with regular shape and no atrophy,while those in the model group were atrophied and absent.Compared with blank control group,villus height in model group was significantly decreased(P<0.05)and crypt depth was significantly increased(P<0.05).Compared with model group,villus height was significantly increased(P<0.01)and crypt depth was significantly decreased(P<0.05)in high-dose MSPH group.(5)RT-qPCR results showed that compared with blank control group,mRNA expression of ZO-1 and Occludin in intestinal tissue in model group significantly decreased(P<0.01,0.05).Compared with model group,high-dose MSPH group significantly increased mRNA expression of ZO-1 and Occludin in intestinal tissue(P<0.05,0.001).(6)Compared with blank control group,serum ET and D-LA levels in model group were significantly increased(P<0.01,0.001);Compared with model group,ET(P<0.05)and D-LA(P<0.05)levels were significantly decreased in high-dose MSPH group.(7)In model group,IL-1β(P<0.05),IL-6(P<0.05),TNF-α(P<0.001),MPO(P<0.01)and MDA(P<0.05)were significantly increased;Compared with model group,the contents of IL-1β(P<0.05),IL-6(P<0.01),TNF-α(P<0.05),MPO(P<0.05)and MDA(P<0.01)in high-dose MSPH group were significantly decreased.(8)The serum SIgA content of model group was significantly decreased(P<0.05),and the contents of IFN-γ(P<0.01),G-CSF(P<0.05)and ROS(P<0.05)were significantly increased;Compared with model group,the contents of SIgA were significantly increased(P<0.001),while the contents of IFN-γ(P<0.001),G-CSF(P<0.05)and ROS(P<0.001)were significantly decreased in high-dose MSPH group.(9)To some extent,MSPH can change the abundance of pathogenic and beneficial microorganisms in the intestinal microbes of mice with intestinal mucositis caused by 5-FU,reduce the abundance of pathogenic bacteria and increase the abundance of beneficial bacteria,such as reducing the abundance of Helicobacter pylori and increasing the abundance of lactobacillus in the intestine.In summary,the results of this study show that MSPH has a preventive effect on intestinal mucositis in mice,which can provide a theoretical basis for the prevention of intestinal mucositis caused by chemotherapy.