The Effects And Mechanism Of Cannabidiol Treatment On Exogenous Factor-Induced Senescence-Damage Of Human Skin Fibroblasts

Objective:To investigate the intervention of senescence damage by canabidiol(CBD)on hydrogen peroxide(H2O2)or lipopolysaccharide(LPS)induced human skin fibroblasts(HSF)and to explore the potential mechanism.Methods:In the HSF senescence model induced by H2O2:The HSF cells in logarithmic growth phase were selected and divided into different groups,including control group,H2O2 model group which was treated with 600μM H2O2,and CBD administration groups which were induced by 600μM H2O2 and treated with different concentrations of CBD.The cell viability was detected by CCK-8.The assays of ROS,CAT,SOD,MDA and GSH-Px were respectively proceeded with corresponding kit,while the contents of MMP-1 and TIMP-1 were analyzed by ELISA.RT-qPCR was then used to quantify the mRNA expression level of p53,p21,p16,BCL-2,Bax,p38,COX-2 and iNOS.In addition,the nuclear staining was observed with addition of Hoechst 33342.In the HSF senescence model induced by LPS:The HSF cells in log-phase were selected and divided into different groups,including control group,LPS model group which were induced by 1μg/mL LPS,and CBD administration groups which were induced by 1μg/mL LPS and then treated with different concentrations of CBD.ROS,CAT,MDA and GSH-Px were separately detected by respective kit.The level of IL-6,TNF-α,NF-κB p65 pS536 and NF-κB p65 Total were measured by ELISA,while the mRNA expression level of p53,p21,p16,BCL-2,Bax,p38,COX-2 and iNOS were determined by RT-qPCR.Hoechst 33342 staining was used to examine the damage on nucleus.Results:The cell viability significantly decreased after H2O2 stimulation compared to control group,but 1-10μM CBD can increase that compared with H2O2 model group(p<0.01).CBD can inhibit the production of ROS and MDA induced by H2O2,and can relieve the decrease of CAT,SOD and GSH-Px activity induced by H2O2(p<0.05).CBD can alleviate the increase of MMP-1 induced by H2O2(p<0.05),but had no significant influence on the reduction of TIMP-1.0.1 and 0.5μM CBD could limit the upregulation of p53,p21 and p16 mRNA expression level after stimulation of H2O2(p<0.01),while 10μM CBD can increase the ratio of BCL-2/Bax(p<0.01).Compared to H2O2 model group,0.1 and 0.5μM CBD can effectively inhibit the upregulation of p38 mRNA expression level(p<0.01),and 0.1-10μM CBD can reduce the upregulation of COX-2 mRNA expression level at the same time(p<0.01).Hoechst 33342 staining results showed that CBD had no obvious effect on nuclear DNA damage resp(?)se induced by H2O2.After stimulation of LPS,compared with control group,the production of ROS and MDA increased(p<0.05),while the viability of CAT and SOD decreased in the LPS model group(p<0.05).However,compared with LPS model group,CBD could limit the production of ROS and MDA,and restore the viability of SOD in different degrees(p<0.05).CBD can attenuate the LPS-induced increased secretion of IL-6 and TNF-α(p<0.05),and inhibit the hyperphosphorylation of NF-κB p65 at S536 induced by LPS(p<0.05),but there was no dose-effect relationship with concentration.Moreover,CBD can inhibit the significant upregulation of p38 and COX-2 mRNA expression level induced by LPS(p<0.01).In addition,CBD can reduce the upregulation of p53,p21 and p16 mRNA expression level after stimulation of LPS(p<0.01),while 1 and 10μM CBD can partially restore the decrease of BCL-2/Bax ratio induced by LPS(p<0.01).The results of Hoechst 33342 suggested that the fluorescence intensity of the model group increased compared with the control group,but CBD did not significantly alleviate the DNA damage response induced by LPS.Conclusions:1.CBD could alleviate oxidative stress and inflammatory response in HSF cells induced by H2O2 or LPS,but there is a threshold range of CBD concentration and its effect is related to the type of stressors.2.In H2O2 or LPS-induced HSF cell models,CBD may exhibit anti-inflammatory and antioxidant effects by inhibiting the activation of the NF-κB pathway and related cytokines,and finally plays a role in anti-aging.3.In H2O2 or LPS-induced HSF cell models,CBD may alleviate senescence due to cell cycle arrest accumulation by down-regulating the expression level of genes in p53/p21 and p16 related signal pathway.