The Study On The Mechanism That Betaine Regulates ROS Through Wnt/β-catenin Pathway In The ZF4 Cells Of Zebrafish

Reactive Oxygen Species（ROS）is a normal product of cell metabolism,but excessive ROS can cause oxidative damage.Betaine is a kind of alkaloid widely existing in animals and plants,which has a variety of physiological regulation functions,but its antioxidant regulation mechanism is less studied.Wnt/β-catenin signaling pathway is involved in the regulation of embryonic development,immunity,metabolism,apoptosis and necrosis,and is closely related to ROS.This project aims to study the mechanism of Wnt/β-catenin pathway involved in betaine regulating ROS level in zebrafish ZF4 cells.The regulation mechanism of betaine on ROS level in zebrafish cells will be studied from the cellular level,so as to provide theoretical reference for the research of fish and aquatic products.The detailed research results are as follows:（1）The effect of betaine on ROS level in zebrafish ZF4 cells was studied.ZF4 cells were treated with betaine for 24 hours,3-（4,5-dimethylthiazol-2）-2,5-diphenyltetrazolium bromide（MTT）was used to detect the effect of betaine on the proliferation rate of ZF4 cells.It was found that 0.01～100 m M of betaine had significant proliferation effect on ZF4 cells.Furthermore,2’,7’-dichlorofluorescein diacetate（DCFH-DA）was used to label the total ROS in the cells.It was found that betaine treatment had no significant effect on the level of total ROS in the cells.The content of ROS and the activity of antioxidant enzymes were detected by kit.It was found that betaine treatment could significantly reduce the content of hydrogen peroxide（H₂O₂）and increase the activity of catalase（CAT）.Real time fluorescent quantitative PCR（q PCR）was used to detect the expression levels of related genes,and it was found that betaine could significantly increase the gene expression levels of SOD and CAT in cells.The results showed that betaine had antioxidant effect on ZF4 cells.（2）The effect of betaine on Wnt/β-catenin signaling pathway in zebrafish ZF4 cells was studied.ZF4 cells were treated with betaine for 24 hours,the result of q PCR showed that 10 m M betaine could significantly promote the expression of Wnt8a and PPARγgenes,and significantly inhibit the expression of Wnt3a,Wnt10b and GSK-3βgenes.Western blot showed that betaine could significantly inhibit the expression of GSK-3βand P-β-catenin（ser33）,and significantly promote the expression ofβ-catenin.Furthermore,the distribution ofβ-catenin protein in cells was observed by immunofluorescence technique.It was found that betaine could promote the expression ofβ-catenin protein and the phenomenon ofβ-catenin protein accumulated around the nucleus was observed.These results suggest that betaine activates Wnt/β-catenin signaling pathway in normal cultured ZF4 cells,which may play an important role in cell proliferation.（3）The zebrafish ZF4 cell oxidative stress model was constructed.In order to better study the antioxidant effect of betaine in ZF4 cells,we used 50μM hydrogen peroxide to treat ZF4 cells for 24 hours.After hydrogen peroxide treatment of ZF4 cells,it was found that the survival rate of ZF4 cells was 76%～86%of that of the control group.The fluorescence intensity of ROS was 26%higher than that of normal ZF4 cells.The content of H₂O₂ increased by 34%,the ability of anti superoxide anion（O₂.^-）decreased by 14%,and the ability of inhibiting hydroxyl radical（·OH）decreased by 14%.The activities of CAT、SOD and GSH-PX were decreased by 11%,9%and 16%,respectively.Therefore,the oxidative stress model of zebrafish ZF4 cells was successfully constructed by adding 50μM hydrogen peroxide into zebrafish ZF4 cells.（4）The effects of betaine on ROS level and Wnt/β-catenin signaling pathway in the oxidative stress model of zebrafish ZF4 cells were studied.We used 0.1～70 m M betaine to treat the oxidative stress model of ZF4 cells.It was found that 1～70 m M betaine could significantly reduce the ROS level in the oxidative stress model of ZF4 cells.When the concentration of betaine was 10 m M,the level of ROS in ZF4 cells decreased by 20%.Therefore,we further used 10 m M betaine for the following experiments.After betaine treatment,it was found that the content of H₂O₂ in cells decreased by 15%,and the ability to inhibit·OH increased by 14%.10 m M betaine significantly increased the gene expression and enzyme activity of CAT and SOD.In addition,we further detected the gene and protein levels of Wnt/β-catenin signaling pathway related molecules.It was found that betaine significantly promoted the expression of GSK-3βgene and protein in ZF4 cell oxidative stress model,but significantly inhibited the expression ofβ-catenin gene and protein.Betaine significantly promoted the production of P-β-catenin（ser33）protein,reduced the phenomenon ofβ-catenin protein accumulated around the nucleus,and induced PPARγgene expression level.The results show that betaine has a good antioxidant effect in ZF4 cell oxidative stress model,and betaine has inhibitory effect on Wnt/β-catenin signaling pathway in this model.（5）The effect of betaine on ROS in the over-expressedβ-catenin cells was studied.In order to further study the relationship between Wnt/β-catenin signaling pathway and the antioxidant effect of betaine,we constructed p EGFP-N1-β-catenin eukaryotic expression vector and transfected ZF4 cells with lipo2000.Then ZF4 cellsa were added 10 m M betaine,and further treated the cells with 50μM hydrogen peroxide for 24 hours.It was found that PPARγ、SOD and CAT gene expression decreased significantly in theβ-catenin over-expression group,which indicated thatβ-catenin played an important role in the antioxidant effect of betaine.In conclusion,betaine has no significant effect on the total ROS in the normal cultured ZF4 cells,but it has significant antioxidant effect in the oxidative stress model of ZF4 cells.The results show that betaine can inhibit Wnt/β-catenin signaling pathway,and then promote the gene expression levels of PPARγ、SOD and CAT,so as to play an antioxidant role.