The Protective Effect And Mechanism Of Baicalin Against Ipec-J2 Cell Damage Which Caused By H₂O₂-Induced Oxidative Stress

The study aims to illustrate the protective effect and mechanism of baicalin aganinst IPEC-J2 cell damage which caused by H₂O₂-induced oxidative stress.In this experiment,the IPEC-J2 cell oxidative stress model was established by 500μM H₂O₂,and the model was treated with baicalin at concentrations of 10 and 20m M.The cells were divided into 6groups:Control Group,10μM Bai Group,20μM Bai Group,500μM H₂O₂ Group,10μM Bai+500μM H₂O₂ Group and 20μM+500μM H₂O₂ Group.The samples were collected after 4 hours of the treatment.The samples were tested for cell viability,and the antioxidant status were judged by detecting the content of ROS,the expression of antioxidant-related gene m RNA,oxidant and antioxidant index.Detecting the apoptosis rate and the m RNA transcription and protein expression of apoptosis-related genes to analyze the status of apoptosis.In addition,the expression of Claudin1,Occludin and ZO-1protein was detected to evaluate the function of the intestinal mechanical barrier.Then,an AMPK knockdown cell line was established by RNA interference,and a new cell group was constructed with the Nrf-2 inhibitor ML385（5μM）.The above experimental process was repeated.The experimental results are as follows:1.IPEC-J2 cells were treat with different concentrations of baicalin for 12 h and it produced significant cytotoxicity when the concentration reaches 500μM.And 10μM and20μM dose of baicalin were selected within the safe concentration range for the following tests.The cell vitality decreased to 60-70%when the concentration was 500μM when IPEC-J2 cells were treated with different concentrations of H₂O₂ for 4 h.Thus,the oxidative stress model was established with 500μM H₂O₂ in this trial.Two doses of baicalin significantly reduced cytotoxicity caused by H₂O₂ in a dose-dependent manner.2.Compared to the control group,the cell viability of the 500μM H₂O₂ group was significantly decreased（P<0.05）while the level of ROS was significantly increased（P<0.05）.The activities of antioxidant enzymes SOD,CAT,GSH-Px were significantly increased while the MDA contents were significantly decreased（P<0.05）.Apoptosis rate was significantly enhanced,and the levels of apoptosis-related proteins like Cleaved-Caspase3 and Bax increased significantly（P<0.05）.The expression of Occludin and ZO-1 protein decreased significantly（P<0.05）but Claudin1 did not（P>0.05）.H₂O₂induces oxidative stress in IPEC-J2 cells,resulting in decreased levels of cell antioxidants,increased apoptosis levels,and damage to mechanical barriers.3.Compared to the control group and 500μM H₂O₂ group,the addition of baicalin significantly improved cell viability,the activities of SOD,CAT,and GSH-Px（P<0.05）.At the same time,ROS levels and MDA contents were significantly reduced（P<0.05）.Bax m RNA and protein were significantly down-regulated（P<0.05）.The cleaving of Caspase3and Caspase9 was inhibited（P<0.05）and apoptosis rate decreased（P<0.05）.The expressions of ZO-1 and Occludin were up-regulated in a concentration-dependent manner.Baicalin effectively relieves cell damage caused by H2O2-induced oxidative stress.4.The 20μM Bai+500μM H₂O₂ group in the AMPK knockout cell line and the ML385 added cell line had no significant changes in ROS levels and cell viability compared with the 500μM H₂O₂ group（P<0.05）.Compared to the control group,the activities of SOD,CAT,and GSH-Px were significantly decreased（P<0.05）and there was no change in the baicalin-added groups,while the MDA content was significantly increased（P<0.05）,especially in the ML385 supplement group.Apoptosis rates,Bax m RNA and protein expression and Cleaved-Caspase3 protein expression levels were increased（P<0.05）.ZO-1 expression levels increased significantly（P<0.05）.Knockdown of the AMPK weakens the effect of baicalin,while the use of Nrf-2 inhibitors makes baicalin lose most of its functions.Conclusion:Baicalin alleviates the reduction of cellular antioxidant capacity,the enhancement of cell apoptosis and the damage of intestinal mechanical barrier caused by H₂O₂-induced oxidative stress via activating Nrf-2/AMPK signaling pathway in IPEC-J2cells.