Effect Of Four Traditional Chinese Medicine Monomer On Mechanical Barrier Damage Of IPEC-J2 Cells Caused By Soybean 7S Globulin

Soybean protein is the main protein source of animal feed,and the higher content of soybean 7S globulin is the main allergen causing diarrhea in weaned piglets.Numerous studies have shown that traditional Chinese medicine monomer such as turmeric,spikenard,bilberry and forsythia have good effects in preventing diarrhea in piglets.Therefore,this experiment was conducted to investigate whether four traditional Chinese medicine monomer,namely curcumin,eleutheroside E,anemoside B4 and forsythoside A,could alleviate the mechanical barrier damage of intestinal epithelial cells caused by soybean 7S globulin,and to lay the foundation for the development of green additives to prevent diarrhea in weaned piglets caused by soy protein diets.In this experiment,IPEC-J2 cells were used as the test subjects,and the effective proliferative activity concentrations of four traditional Chinese medicine monomer,namely curcumin,eleutheroside E,anemoside B4 and forsythoside A,were screened by the CCK-8method,and then the above four traditional Chinese medicine monomer with effective proliferative activity concentrations were applied to investigate their effects on the mechanical barrier of IPEC-J2 cells damaged by soybean 7S globulin using ELISA,RTqPCR and Western blot assays,and the optimal traditional Chinese medicine monomer and their concentrations were screened;then this traditional Chinese medicine monomer was used to initially investigate whether it could alleviate the mechanical barrier damage of IPEC-J2 cells caused by soybean 7S globulin by modulating the Rho/ROCK signaling pathway.The following results were obtained:(1)Effects of four traditional Chinese medicine monomer on soybean 7S globulininduced permeability and integrity damage in IPEC-J2 cells: 0.01-1.28 μg/mL of curcumin,3.125-400 μg/mL of eleutheroside E,3.125-400 μg/mL of anemoside B4,and 5-640 μg/mL of forsythoside A alone on IPEC-J2 cells for 24 h.Compared with the control group,the relative cell viability of all four traditional Chinese medicine monomer at the above concentrations increased,comparison of each traditional Chinese medicine monomer individually: 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A had the best proliferative effect on the cells,so 0.02,0.04,0.08 μg/mL of curcumin,12.5,25,50 μg/mL of g/mL of eleutheroside E,12.5,25,50μg/mL of anemoside B4 and 10,20,40 μg/mL of forsythoside A were selected for the followup experiments.The screened traditional Chinese medicine monomer were treated with soybean 7S globulin at the same time.As a result,compared with the 7S-treated group,0.04 and 0.08μg/mL of curcumin,25 and 50 μg/mL of eleutheroside E,25 and 50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A significantly increased relative cell viability(P < 0.05),significantly increased TEER values(P < 0.05),significantly decreased sodium fluorescein permeability(P < 0.05),and significantly decreased LDH,DAO,ALP,and IFABP1 levels(P < 0.05),and 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4,and 40 μg/mL of forsythoside A had the best effect.It was shown that 0.08μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A could effectively protect cell integrity and alleviate the cellular mechanical barrier damage caused by soybean 7S globulin.(2)To investigate the effects of traditional Chinese medicine monomer on soybean7 S globulin-induced inflammation and tight junction-related proteins in IPEC-J2 cells based on the Rho/ROCK signaling pathway: compared with the 7S group,0.02,0.04,0.08μg/mL of curcumin,25,50 μg/mL of eleutheroside E,12.5,25,and 50 μg/mL of anemoside B4 and 10 and 40 μg/mL of forsythoside A significantly increased the levels of antiinflammatory cytokines IL-4 and IL-10(P < 0.05)and significantly decreased the levels of pro-inflammatory cytokines TNF-α,IL-1β and IL-6(P < 0.05),and 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A had the best effect.It was shown that 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A can effectively exert antiinflammatory effects and protect the cellular mechanical barrier.The results showed that all four traditional Chinese medicine monomer significantly increased the m RNA and protein expression of ZO-1,Occludin and Claudin-1 after treating the cells with soybean 7S globulin at optimal action concentrations(P < 0.05).This indicates that 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A could increase the expression of tight junction proteins and protect the mechanical barrier of cells,and 50 μg/mL of anemoside B4 had the best effect.Finally,when IPEC-J2 cells were treated with 50 μg/mL of anemoside B4 and soybean 7S globulin,the expression of Rho/ROCK signaling pathway-related proteins Rho A,ROCK1,ROCK2,and MLCK was significantly reduced compared with the 7S-treated group(P <0.05),indicating that anemoside B4 can affected the Rho/ROCK signaling pathway and thus protect the intestinal mechanical barrier.Conclusion: 0.08 μg/mL of curcumin,50 μg/mL of eleutheroside E,50 μg/mL of anemoside B4 and 40 μg/mL of forsythoside A can alleviate the mechanical barrier damage of IPEC-J2 cells caused by soybean 7S globulin,indicating that these four traditional Chinese medicine monomer are expected to be used in weaning piglets with diarrhea caused by soy protein diets,and laying the foundation for the subsequent development of green antibiotic-free feed additives.The mechanism by which 50 μg/mL of anemoside B4 alleviated the mechanical barrier damage caused by soybean 7S globulin was that it reduced the expression of Rho A,ROCK1,ROCK2 and MLCK and affected the Rho/ROCK signaling pathway.