Explore The Protective Role And Mechanism Of Orientin In Human Bronchial Epithelial Cells Of Oxidative Stress

Background and objective:Oxidative stress which refers to the body's cells in vivo leads to a state of imbalance between oxidation and antioxidation in various unfavorable factors to stimulate,also the host innate immune response against the pathogen outside of important defense systems.But excessive oxidative stress can lead to systemic inflammatory response and tissue damage[1-4].Related research shows that,Oxidative stress and lung diseases have a clear relationship[1.7.17.18]:?1?Oxidizing agent can weaken the ability of neutrophils to promote its accumulation in the microcirculation,activation,activation of neutrophils release ROS,leading to tissue damage;?2?Oxidative stress can activate intracellular signaling pathways?such as:nuclear factor-KB etc.?regulating release of inflammatory mediators,activation of neutrophils,further amplifies the inflammatory injury[5].?3?By activating Rho protein,oxidative stress can cause macrophage remodeling,damage the function of macrophages,that leading the macrophages can not normally clear pathogens,foreign bodies and apoptotic cells[6-7].?4?Oxidative stress can lead to a decrease in HDAC2 activity,leading to a decrease in glucocorticoid sensitivity in patients With chronic obstructive pulmonary disease[8-9].?5?ROS increased induction of neutrophil release protease,destruction of lung tissue elastic fibers,prompting protease and anti-protease imbalance,leading to the occurrence of emphysema.Orientin of the traditional Chinese medicine is an active ingredient of bamboo,flavonoids belonging monomer[10],It has significant antioxidant anti-apoptotic,analgesic anti-lipid formationand anti-inflammatory[11-16]and so on.Its main ingredients have been put into clinical use of Hong Ye Xin Tong soft capsule and oriental freeze-dried powder preparation.The research of orientin in human bronchial epithelial cells under oxidative stress is rare,Therefore,through in vitro cytology experiments,this study used hydrogen peroxide to stimulate human bronchial epithelial cells,Given different concentrations of orientin intervention,The survival rate,apoptosis rate,morphology and related protein expression of the drug were examined.The effect of orientin on the expression of human bronchial epithelial cells under oxidative stress was demonstrated by antioxidant effect.Methods:1.Cell culture:Human bronchial epithelial cells?16HBE cells?were cultured in RPMI-1640 supplemented with 10%fetal bovine serum and maintained in a humidified atmosphere consisting of 5%CO₂ at 37 0C.2.The effects of hydrogen peroxide on survival rate of bronchial epithelial cells:The survival rate of the cells was measured by MTT assay at different concentrations?20,100,200 ?M?of H₂O₂ for 2 h.3.The survival rate after different concentrations of orientin intervention:The bronchial epithelial cells were divided into the following groups:Control group,DMSO40?M,Orientin5?M,Orientin100?M,Orientin40?M,H₂O₂200?M,H₂O₂+Orientin5?M,H₂O₂+Orientin10?M,H₂O₂+Orientin 40?M.The total time of intervention was 2h,and the survival rate was detected by MTT.4.Morphological changes of apoptotic cells were observed by fluorescence microscopy:Hoechst33342 was used to observe the morphology of bronchial epithelial cells.5.Flowcytometry was used to detect the apoptotic rate through Annexin-FITC/PI double staining.6.Detection of intracellular SOD production by microplate reader.7.Effects of orientin on the expression of apoptotic protein and P-AKT pathway:Extraction of cellular proteins,the expression of Cleaved-caspase3 and P-AKT/AKT protein was detected by Western Blot.Results:1.16HBE cells were stimulated with different concentrations of H₂O₂:16HBE cells were stimulated with different concentrations?20,100,200?M?H₂O₂ for 2h,and the cell survival rate was detected by MTT assay.With the increase of H₂O₂ concentration,the cell survival rate decreased gradually,Compared with Con group?97.88±1.87%?,20?M H₂O₂ stimulated 16HBE cell survival rate was 95.75 ± 3.90%,there is no significant difference.When the concentration of H₂O₂ stimulation100?M,the cell survival rate was 75.86 ± 1.01%,When the concentration of H₂O₂ reached 200?M,the survival rate decreased to 57.76 ±2.10%,the difference was statistically significant?P<0.05?;2.Orientin can increase the survival rate of 16HBE cells under oxidative stress:The experiments were divided into the following groups:blank control group,DMSO 40?pM,Orientin 5?M,Orientin 10?M,Orientin 40pM,H₂O₂?200?M?,H₂O₂ +Orientin?5?M?,H₂O₂+ Orientin?10?M?,H₂O₂ + Orientin?40?M?.MTT assay was used to detect the survival rate.There was no significant difference compared the blank control group with DMSO 40?M,Orientin 5?M.10?M and Orientin 40?M?Figure 2?.The survival rate of H₂O₂ group was significantly lower than that of blank control group?P<0.05?.H₂O₂+ Orientin 5?M compared with H₂O₂ group,the cell survival rate increased,but the difference was not statistically significant;H₂O₂ + Orientin 10?M significantly increased the cell survival rate,the difference was statistically significant?P<0.05?;H₂O₂+ Orientin 40?M compared with H₂O₂,cell survival increased,but the difference was not statistically significant?P>0.05??Figure 3,Table 2?;3.Observation of apoptotic cell morphology by Hoechst33342 staining:Fluorescence microscope to observe the blank control group 16HBE cell activity is good,the dye can enter a normal cell,the nuclear color is low blue,nuclear staining uniform;H₂O₂ group had more apoptotic cells,membrane permeability increased,the nucleus was bright blue,some nuclear rupture,shrinkage,uneven staining;The number of apoptotic cells in the group of Orientin 10?M + H₂O₂ decreased,the number of nuclei was less than that of blue and blue,and most of the nuclei were stained more uniformly.Compared with the H₂O₂ group,the apoptotic cells in the Orientin 40?M + H₂O₂group were lower than those in the H₂O₂ group.?Fig.4?.4.Flow cytometry detected the Apoptosis Rate by Annexin-FITC/PI:Compared the apoptotic rate with control group 0.21 ±0.05%,The apoptotic rate of H₂O₂ group was 66.74 ± 7.53%,and the number of early withered and late withered cells was significantly increased?P<0.05?.The apoptotic rate of H₂O₂ ?Ori10?M group?49.02 ± 6.20%?was significantly lower than that of H₂O₂ group?P<0.05?;H₂O₂ + Ori 40?M group?51.21 ± 6.98%?compared with H₂O₂ group,the number of early withered and late withered cells decreased,but there was no significant difference?P>0.05?;5.Orientin can antagonize the expression of Caspase3 apoptotic protein in oxidative stress state:Western blot showed that the expression of Cleaved-caspase3 in the 16HBE cells with oxidative stress group was significantly higher than that in the blank control group?P<0.05?;Compared with H₂O₂ group,the expression of Cleaved-caspase3 protein was decreased and the level of Caspase3 was not changed after H₂O₂+ Orientin10?M intervention,the difference was statistically significant?P<0.05?;The expression of apoptotic protein in H₂O₂ + Orientin40pM group was lower than that in H₂O₂ group?P<0.05?;6.Detection of intracellular superoxide dismutase SOD activity by microplate reader:Abscissa for the experimental group:Control group,H₂O₂,H₂O₂ +Ori10?M,H₂O₂+ Ori40?M,Y-axis was the activity of SOD?U/mg?.Fig.7,Table 5 shows:the comparison with the control group?68.76±2.99 U/mg?,The activity of SOD in H₂O₂ group?34.71 ±2.55 U/mg?decreased significantly,the difference was statistically significant?P<0.05?.The activity of SOD?45.09 ±2.19 U/mg?in Orientin?10?M?+ H₂O₂ group was higher than that in H₂O₂ group?P<0.05?.The activity of SOD?41.05 ± 0.75 U/mg?in Orientin?40?M?+ H₂O₂ group was higher than that in H₂O₂ group?P<0.05??Figure 7,Table 5?;7.P-AKT signaling pathway is involved in the protective effect of orientin on bronchial epithelial cells under oxidative stress:The expression of P-AKT/AKT protein in 16HBE cells was detected by Western blot.As shown in Figure 8 and Table 6:Compared with Control group,the protein expression level of P-AKT in H₂O₂ group was significantly decreased,The expression level of P-AKT protein in H₂O₂ +orientin?100?M?group was significantly higher than that in oxidative stress group?P<0.05?.H₂O₂+ orientin?40?M?group increased the expression of P-AKT/AKT in H₂O₂ group,but the difference was not statistically significant?P>0.05?.Conclusion:1.Hydrogen peroxide stimulation can lead to decrease survival rate of human bronchial epithelial cells in vitro;2.A certain concentration of orientin caused the apoptotic rate of 16HBE cells to decrease under oxidative stress,apoptosis-related proteins decreased,indicating that the protective effect of orientin on oxidative damage is caused by inhibition of apoptosis;3.The inhibitory effect of orientin on apoptosis is to protect the 16HBE cells by increasing the activity of T-SOD;4.The antioxidant effect of orientin may be related to P-AKT/AKT signaling pathway;5.When the concentration of orientin was 5?M and 10?M,the antioxidant capacity was enhanced,but when the concentration reached 40?M,the effect is weakened.