Based On The AGEs/RAGE Pathway To Explore The Protective Mechanism Of Taohong Siwu Decoction On Oxidative Stress In HDMEC Cells Damaged By Light

Purpose:This study by in vitro experiments,aims to observe the expression of skin Photodamage HDMECs oxidative stress injury related biochemical indexes caused by UVA irradiation in simulated solar light,as well as AGEs/RAGE signal transduction pathways in the role of skin Photoaging oxidative stress mechanism;According to the research of"blood stasis-aging"pathogenesis in TCM,choose Taohong Siwu Decoction as the experimental carrier,to explore the protective mechanism of oxidative stress in photodamage HDMECs,in order to find effective drugs and methods of prevention and treatment of skin photoaging,provide new ideas and scientific theory basis for the application of TCM in skin Photoaging.Material and Methods:HDMEC cells were irradiated with UVA ultraviolet light source,detected the proliferation activity changes of HDMEC cells by CCK-8 method,and determined the optimal radiation dose of UVA in HDMEC cell model;prepared drug serum with different concentrations of Taohong Siwu Decoction,detected the proliferation activity of the drug serum of Taohong Siwu Decoction on HDMECs by CCK-8 method and determined the optimal intervention dose of drug serum with Taohong Siwu Decoction.Select 50 bottles of HDMECs raised in normal cells,the cells were randomly divided into 5 groups:normal control group,UVA model group,blank serum group,containing drug serum group,vitamin E group,each group of ten bottles.Detection following indicators in each group cells:detection of active oxygen fluorescent probe method（ROS）;Colorimetry to detect the ultra oxygen anion（O₂^-）,hydrogen peroxide（H₂O₂）,hydroxyl radical（-OH）,malondialdehyde（MDA）,and superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）activity.HDMECs cells were irradiated by UVA with different irradiation doses of 2.5J/cm²,5J/cm²,10J/cm²,enzyme-linked immunosorbent（ELISA）method to detect the contents of AGEs,RT-PCR method to detect RAGE mRNA expression,Western-blot method to detect RAGE protein expression;And prepare AGEs in vitro to induce HDMEC cells,detection following indicators in each group cells:detection of active oxygen fluorescent probe method（ROS）;RT-PCR method to detect RAGE mRNA and NADPH oxidase P22^PhoxmRNA expression;Western-blot method to detect RAGE and NADPH oxidase P22^Phox protein expression.Select well-grown endothelial cells suspension(1×10⁸·L^-1),were seeded in 96 well plates,cultured to fusion state,joined cultures of 10%Taohong Siwu Decoction of rats serum5mL,at 37℃and 5%CO₂ incubator culture 12h,then change in serum-free DMEM culture,AGEs model group and drug group,were added 200μg/mL AGE-BSA,normal control group with 200μg/mL BAS stimulation12 h.The inhibitor intervention group was preloaded with anti-rage neutralizing antibody,and then the concentration was 200μg/mLAGE-BSA incubation cell 12h.Detection following indicators in each group cells:detection of active oxygen fluorescent probe method（ROS）;Colorimetry to detect malondialdehyde（MDA）,and superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）activity.RT-PCR method to detect RAGE mRNA expression;Western-blot method to detect RAGE protein expression;Results:1.Changes in proliferation activity of HDMECs irradiated by different doses of UVACompared with the 0J/cm² dose group,the cell activity of 1.25J/cm²,2.5J/cm²,5J/cm²,10J/cm²,20J/cm²,40J/cm² was significantly reduced,with statistical difference（P<0.01）;At the time of UVA 5J/cm²,OD value in the irradiation group decreased significantly,and cell proliferation rate was significantly inhibited at this time.2.Containing serum Taohong Siwu Decoction against changes of light damage HDMECs OD valueCompared with the normal control group,the cell activity of the blank serum group and the serum group with different concentrations of Taohong Siwu Decoction group decreased significantly,and the difference was statistically significant（P<0.01）.Compared with the blank serum group,the cell proliferation activity of the serum group with different concentrations of Taohong Siwu Decoction increased significantly（P<0.01）.Compared with the low dose serum group,the cell activity of the medium and high dose serum group increased significantly（P<0.01）.There was no statistically significant difference in cell activity in the middle and high dose serums（P>0.05）.3.Detection of oxidative stress index Taohong Siwu Decoction in photodamage HDMECsROS expression:compared with the normal control group,ROS expression level of UVA model group was significantly increased,with statistical difference（P<0.01）;Compared with the UVA model group,ROS expression in serum group and VE group was significantly reduced,with statistical difference（P<0.01）;Compared with VE group,ROS expression in serum group had a significantly increase,with statistical difference（P<0.05）.O₂^-,H₂O_2,-OHexpression:compared with the normal control group,O₂^-,H₂O_2,-OH expression level of UVA model group was significantly increased,with statistical difference（P<0.01）;Compared with the UVA model group,O₂^-,H₂O_2,-OHexpression in serum group and VE group was significantly reduced,with statistical difference（P<0.01）;Compared with VE group,O₂^-,H₂O₂ expression in serum group had a significantly increase,with statistical difference（P<0.01）;Compared with VE group,-OH expression in serum group had a relatively increase,with statistical difference（P<0.05）.MDA content:compared with the normal control group,MDA content of UVA model group was significantly increased,with statistical difference（P<0.01）;Compared with the UVA model group,MDA content in serum group and VE group was significantly reduced,with statistical difference（P<0.01）;Compared with VE group,MDA content in serum group had a significantly increase,with statistical difference（P<0.01）.SOD,CAT,GSH-Px activity:Compared with the normal control group,SOD,CAT,GSH-Px activity of UVA model group was significantly reduced,with statistical difference（P<0.01）;Compared with the UVA model group,SOD,CAT,GSH-Px activity in serum group and VE group was significantly increased,with statistical difference（P<0.01）;Compared with VE group,SOD,CAT,GSH-Px activity in serum group was relatively reduced,with statistical difference（P<0.05）.4.Effect of UVA irradiation on expression levels AGEs and RAGE in HDMECsAGEs expression:compared with the normal control group,AGEs expression in UVA low,middle and high dose group was significantly increased,with statistical difference（P<0.01）;Compared with UVA low dose group,AGEs expression in UVA high-dose group was significantly increased,with statistical difference（P<0.01）.RAGE mRNA and protein expression:compared with the normal control group,RAGE mRNA and protein expression in UVA low,middle and high dose group was significantly increased,with statistical difference（P<0.01）;Compared with UVA low dose group,RAGE mRNA and protein expression in UVA high-dose group was significantly increased,with statistical difference（P<0.01）.5.Detection RAGE and oxidative stress index in AGEs-induced HDMECsRAGE mRNA and protein expression:Compared with the normal control group,RAGE mRNA and protein expression in AGE-BSA group was significantly increased,and concentration dependence（P<0.05）,When the concentration of AGE-BSA is 200ug/mL,it reaches the peak value（P<0.05）,RAGE mRNA and protein expression no longer increases with the increase of concentration.P22^PhoxmRNA and protein expression:Compared with the normal control group,P22^phoxmRNA and protein expression in AGE-BSA group was significantly increased,and concentration dependence（P<0.05）,When the concentration of AGE-BSA is 200ug/mL,it reaches the peak value（P<0.05）,P22^phoxmRNA and protein expression no longer increases with the increase of concentration.ROS expression:Compared with the normal control group,ROS expression in AGE-BSA group was significantly increased,and concentration dependence（P<0.05）,When the concentration of AGE-BSA is 200ug/mL,it reaches the peak value（P<0.05）,ROS expression no longer increases with the increase of concentration.6.The inhibitory effect of Taohong Siwu Decoction on the AGEs/RAGE pathway and oxidative stress in HDMEC cellsRAGE mRNA and protein expression:compared with the normal control group,RAGE mRNA and protein expression level of AGEs model group was significantly increased,with statistical difference（P<0.01）;Compared with the AGEs model group,RAGE mRNA and protein expression in serum group and anti-RAGE inhibitor group was significantly reduced,with statistical difference（P<0.01）;Compared with anti-RAGE inhibitor group,RAGE mRNA and protein expression in serum group without obvious statistical significance（P>0.05）.ROS expression:compared with the normal control group,ROS expression level of AGEs model group was significantly increased,with statistical difference（P<0.01）;Compared with the AGEs model group,ROS expression in serum group and anti-RAGE inhibitor group was significantly reduced,with statistical difference（P<0.01）;Compared with anti-RAGE inhibitor group,ROS expression in serum group without obvious statistical significance（P>0.05）.MDA content:compared with the normal control group,MDA expression level of AGEs model group was significantly increased,with statistical difference（P<0.01）;Compared with the AGEs model group,MDA expression in serum group and anti-RAGE inhibitor group was significantly reduced,with statistical difference（P<0.01）;Compared with anti-RAGE inhibitor group,MDA expression in serum group without obvious statistical significance（P>0.05）.SOD、CAT、GSH-Px activity:compared with the normal control group,SOD、CAT、GSH-Px activity of AGEs model group was significantly reduced,with statistical difference（P<0.01）;Compared with the AGEs model group,SOD、CAT、GSH-Px activity in serum group and anti-RAGE inhibitor group was significantly increased,with statistical difference（P<0.01）;Compared with anti-RAGE inhibitor group,SOD、CAT、GSH-Px activity in serum group without obvious statistical significance（P>0.05）.Conclusions:1.When the UVA dose was 5J/cm²,the model of photodamage HDMEC cells was successfully replicated.Taohong siwu decoction can significantly improve the proliferation activity of photodamage HDMEC cells.The medium dose group of Taohong siwu decoction containing drugs serum was selected as the follow-up experimental intervention group.2.UVA irradiated HDMECs,which could induce expression of AGEs and RAGE in the cells significantly increased.Through binding with receptor RAGE,AGEs promotes the production of reactive oxygen species and the increase of MDA content in HDMECs,and decreases the activities of SOD,CAT and GSH-Px in HDMECs,resulting in the imbalance of oxidation/antioxidant system,which induces HDMECs cells to be in oxidative stress state.3.Taohong Siwu Decoction by inhibiting HDMECs which combined AGEs and RAGE,blocking the activation of AGEs-RAGE signaling pathway,reduced the incidence of ROS and the content of MDA,increased antioxidant enzymes activity of SOD,CAT and GSH-Px in HDMECs,correct imbalance of oxidation/antioxidation system,so as to achieve the protective effect of photodamage HDMECs on oxidative damage.