Assessment Of The Toxicity Related To Celiac Disease Of Xinjiang Wheat Varieties Based On Epitopes Contents Of Gluten And Caco-2 Cell Model

Celiac disease(CD)is an autoimmune disorder that occurs in genetically predisposed individuals and is a chronic inflammation of the small intestine caused by gluten ingestion.The prevelance of CD is about 1% in the world and a strict gluten-free(GF)diet is the only available therapeutic treatment of CD.However,complete avoidance of gluten proteins could seriously affect the life quality of celiac patients.It is very important to screen and cultivate wheat varieties with low CD toxicity as raw material for producing gluten-free foods.At present,there are many methods for screening wheat varieties with low CD toxicity.How to choose the more simply and accurate methods is the key in the screening work.Therefore,this study will evaluate the gluten toxicity related to CD of 9 Xinjiang wheat varieties based on 4 kinds of methods for epitopes quantitation and Caco-2 cell model.The consistency and difference between different methods were analyzed and compared to provide the theoretical basis for the methods selection in subsequent research related to CD.The main methods,results and conclusions of this study are addressed as follows:1.The levels of CD epitopes in different wheat gluten proteins were determined by Western blot.The results showed that the total content of Glia-?9 and Glia-?20 epitopes in Xinshuguang.1 was the highest while Xinchun 20 was the lowest.The epitope contents of other varieties were in order from high to low 04-Yinjian 105,Tuchun.6,98-14,Moba 66,Achun.2,01-25 and Lanmai.2.R5 ELISA was also used to evaluate the CD toxicity of Xinjiang wheat varieties.The gluten content of different varieties were from high to low as follows: 04-Yinjian 105(112,333 ppm),Xinshuguang.1(91,333 ppm),01-25(70,400 ppm),Moba 66(57,680 ppm)/Lanmai(56,107 ppm),98-14(44,200 ppm),Xinchun 20(40,786 ppm)/ Tuchun.6(40,653 ppm)/Achun.2(39,800 ppm).3.A total of 21 ?-gliadin fragments(UPFs)were deduced from RNA transcriptome analysed by high-throughput sequencing,and the abundance of UPFs ranged from 0.27 to 35.32.The frequency of major CD epitopes and their variants(multiplied by 100)in 9 wheat varieties rank(from high to low)was: Moba 66(321.48),Achun.2(312.06),Tuchun.6(310.98),01-25(300.49),Lanmai(292.93),Xinchun 20(283.13),Xinshuguang.1(279.89),04-Yinjian 105(271.77)and 98-14(257.86).4.The standard curves of marker peptides were successfully prepared in LC-MRM/MS mass spectrometry.And then the contents of CD peptides in the gluten proteins were calculated.The order of the CD peptides contents in 9 wheat varieties from high to low was Xinshuguang.1(19,145.51 ng),01-25(14,699.60 ng),Xinchun 20(12,855.20 ng),Lanmai(10,935.20 ng),98-14(10,837.70 ng),Achun.2(7,131.97 ng),Moba 66(6,019.50 ng),04-Yinjian 105(5,779.55 ng)and Tuchun.6(5,002.95 ng).5.Among the 4 detection methods mentioned above,the high-throughput sequencing based on gene level was greatly different from the other 3 methods based on protein level,while the results of detection methods based on protein level were relatively consistent.6.Caco-2 cell monolayers was successfully constructed.The peptides derived from in vitro gastrointestinal digestion of gliadin stimulated Caco-2 cells at the final concentration of 1.5 mg/mL,which resulted in the increase of monolayer permeability.The ability of different wheat varieties to increase monolayer permeability from high to low was Moba 66,01-25,Xinchun 20,98-14/Lanmai,Achun.2/Xinshuguang.1,Tuchun.6 and 04-Yinjian 105.Gliadin digests can also stimulate oxidative stress in Caco-2 cells.The results showed that the ability of different wheat varieties to induce oxidative stress in Caco-2 cells from high to low was: Xinchun 20,01-25,Xinshuguang.1,98-14,Achun.2,Moba 66,Lanmai,Tuchun.6 and 04-Yinjian 105.