Effects Of Resveratrol,Quercetin And Chincetin On Inflammatory Injury And Apoptosis Of IPEC-J2 Cells Induced By Soybean 7S Globulin

As the main dietary allergen of weaner piglets,soybean 7S globulin can cause allergic enteritis in weaner piglets,resulting in decreased digestion and absorption function and even death due to diarrhea.Resveratrol,Quercetin and Chincetin are all clinically used in the treatment of inflammatory intestinal diseases.They have good anti-inflammatory and antioxidant effects and can improve intestinal immunity and maintain intestinal health.The purpose of this study was to explore whether Resveratrol,Quercetin and Cincetin could be used in the treatment of allergic enteritis of weaned piglets caused by soybean 7S globulin,and to initially explore the mechanism,which is of great significance for the clinical application of traditional Chinese medicine monomeric in the prevention and control of allergic enteritis of weaned piglets caused by soybean protein.Experiment 1 Effects of three traditional Chinese medicine monomers on inflammatory damage of IPEC-J2 cells induced by soybean 7S globulin(1)The effects of different concentrations of Resveratrol,Quercetin and Cincetin on the survival rate of IPEC-J2 cells were determined by CCK-8 method,and three concentrations of each Chinese medicine were screened to detect the effects on the survival rate of IPECJ2 cells induced by 5 mg/m L soybean 7S globulin.The results showed that 0.156 mg/L,0.313 mg/L,0.625 mg/L Resveratrol,0.625 mg/L,1.25 mg/L,2.5 mg/L Quercetin,10 mg/L,15 mg/L and 20 mg/L Cinquetin can significantly improve the survival rate of IPEC-J2 cells under the treatment of 5 mg/m L soybean 7S globulin,which was used for follow-up tests.(2)After different concentrations of the three drugs were co-cultured with 5 mg/m L soybean 7S globulin for 24 h,the contents of IL-1β,IL-6,TNF-α,IL-4,IL-10 and i NOS in the supernatant of the cells were detected by ELISA.The contents of cytokine NO,oxidative product MDA and the activities of antioxidant enzymes GSH and SOD were determined by microassay.The results showed that 5 mg/m L soybean 7S globulin significantly increased the contents of proinflammatory factors and peroxide products,and significantly decreased the contents of anti-inflammatory factors and the activity of antioxidant enzymes(P <0.01).Compared with soybean 7S globulin group,the above three drugs at different concentrations could significantly improve the cell survival rate,significantly reduce the contents of pro-inflammatory factors and peroxide products,and significantly increase the contents of anti-inflammatory factors and the activity of antioxidant enzymes(P < 0.01),suggesting that 5 mg/L soybean 7S globulin could reduce the activity of cells,Regulating the secretion of inflammatory factors in IPEC-J2 cells,synthesis of oxidative products and activity of antioxidant enzymes can cause inflammatory damage to IPEC-J2 cells,while the above three drugs in different concentrations can alleviate the inflammatory damage of IPEC-J2 cells induced by 5 mg/m L soybean 7S globulin.Among them,0.625 mg/L Resveratrol,2.5 mg/L Quercetin and 20 mg/L Cincetin had the best effect.Experiment 2 The effects of three traditional Chinese medicine monomers on the apoptosis and inflammatory damage of IPEC-J2 cells induced by soybean 7S globulin were investigated based on the NF-κB/MAPK pathwayThe experiment was divided into control group,7S model group(5 mg/m L)and different drugs(0.625 mg/L Resveratrol,2.5 mg/L Quercetin,20 mg/L Cincetin)and soybean7 S globulin group.(1)To investigate the effects of Resveratrol,Quercetin and Chincetin on the apoptosis of IPEC-J2 cells induced by soybean 7S globulin.ROS levels and apoptosis rate were detected by flow cytometry.The expressions of pro-apoptotic genes Caspase-3 and Bax and anti-apoptotic genes Bcl-2 were detected by RT-q PCR.The results showed that compared with the control group,5 mg/m L soybean 7S globulin could significantly increase the apoptosis rate and intracellular ROS levels of IPEC-J2 cells,significantly promote the expression of apoptosis gene and inhibit the expression of anti-apoptosis gene(P < 0.01).In addition,0.625 mg/L Resveratrol,2.5 mg/L Quercetin and 20 mg/L Cincetin could significantly down-regulate intracellular ROS levels and apoptosis rate,and significantly inhibit the expressions of pro-apoptosis genes and anti-apoptosis genes(P < 0.01).(2)To explore the regulatory effects of Resveratrol,Quercetin and Cincetin on the NF-κB/MAPK signaling pathway in IPEC-J2 cells induced by soybean 7S globulin.m RNA levels,protein expression and phosphorylation levels of P65 NF-κB,P38 MAPK and JNK were detected by RT-q PCR and Western Blot.The results showed that 5 mg/m L soybean 7S globulin could activate NF-κB/MAPK signaling pathway and significantly up-regulate the m RNA,protein expression and phosphorylation levels of P65 NF-κB,P38 MAPK and JNK(P < 0.01).Adding 0.625 mg/L Resveratrol,2.5 mg/L Quercetin and 20 mg/L Cincetin could significantly down-regulate m RNA levels,protein expression levels and phosphorylation levels of the above proteins(P < 0.01).Therefore,5 mg/m L soybean 7S globulin may induce inflammatory damage of IPC-J2 cells through activation of NF-κB/MAPK signaling pathway.However,0.625 mg/L Resveratrol,2.5 mg/L Quercetin and 20 mg/L Cincetin can alleviate the inflammatory damage of IPEC-J2 cells induced by 5 mg/m L soybean 7S globulin by inhibiting the activation of NF-κB/MAPK signaling pathway.Conclusion: 5 mg/m L soybean 7S globulin can induce the oxidative stress of IPEC-J2 cells,activate the NF-κB/MAPK signaling pathway and regulate the expression of apoptotic gene,and cause the inflammatory injury and apoptosis of IPEC-J2 cells.0.625 mg/L Resveratrol,2.5 mg/L Quercetin and 20 mg/L Cincetin can significantly relieve inflammatory damage and apoptosis induced by soybean 7S globulin by influencing the activation of NF-κB/MAPK signaling pathway.These three traditional Chinese medicines are expected to be used in the prevention and control of diarrhea of weaned piglets caused by soybean protein diet.