Hydroxytyrosol Regulates Autophagy Against Oxidative Stress Damage In Hair Dermal Papilla Cells

Background and objectiveHair loss is a polygene disorder under the influence of not only internal（e.g.,X-linked androgen receptor gene）but also external（e.g.,oxidative stress）.Oxidative stress（OS）plays a significant role in the pathogenesis of androgenetic alopecia,especially the male pattern alopecia.Dermal papilla cells（DPCs）regulate hair follicles（HFs）formation,development and cycling,which are more susceptible to oxidative stress by presenting diminished proliferation and increasing higher secretion of the hair growth inhibitory factor.Autophagy is physiologic process of eukaryotic systems,which have significant role in adaptation to oxidative stress by degradation of metalloproteins and oxidatively damaged macromolecules.Autophagy has also been identified as a protective process during the development of hair loss,which is critical to stop stem cell exhaustion.Hydroxytyrosol（HT）,a major phenolic antioxidant found in olive oil,can afford protection from OS in several types of nontumoral cells,including Skin cells.In particular,it regulates several inflammation-associated processes as well as in improving antioxidant defense system.A key clinic research has showed that Mediterranean diet may reduce the risk of alopecia.However,there are no information about HT used in the treatment of hair loss.This work was aimed at exploring the potential protective actions of HT against OS in rat DP cells.Methods1.The effect of HT on the rat DPCs under oxidative stress.DPCs were isolated from 5 weeks rat vibrissae.The third passage of primary cells were pretreated with or without HT 75μM for 30min and then stimulated with different concentration of H₂O₂（50,100,100 and 400uM）.Cell viability was determined by CCK-8 assay.The DPCs were stimulated with H₂O₂ 100uM for 2 h.The intracellular reactive oxygen species（ROS）levels was detected by fluorescent DCFH-DA.These data detennine that H₂O₂.treatment causes damage to DPCs.To further observe the protective effect of HT on DPCs,DPCs were pretreated with HT 75umol/ml for 30 minutes.The effects of HT on the biological activity and characteristics of DPCs after H₂O₂ treatment were detected by PCR,ELISA and DCFH-DA.2.The mechanism of HT on the rat DPCs under oxidative stress.DPCs were treated with 100 uM H₂O₂ and 100 uM H₂O₂ +HT75uM for 2 hours,and the autophagy-associated protein LC3 and Beclin-1 gene were detected by western blot and PCR,respectively.To further clarify the mechanism by which autophagy regulates the action of HT on DPCs,the autophagy inhibitor chloroquine（CQ）was used to detect the relevant biological activities and characteristics.Results1.The effect of HT on the rat DPCs under oxidative stress.The CCK-8 results showed that the activity of H₂O₂ decreased the cell viability of DPCs in a dose-dependent manner compared with the blank control group.After H₂O₂ treatment,ROS levels and inflammatory factors（IL-6,TNF-a）level were significantly increased,and the secretion of hair growth factors（FGF,PDGF,VEGF）were decreased.Thus,H₂O₂ is cytotoxic to DPCs.In contrast,the pretreatment of HT significantly inhibits H₂O₂-induced oxidative stress damage to DPCs.2.The mechanism of HT on the rat DPCs under oxidative stress.The H₂O₂ treatment significantly decreased the expression of LC3 and Beclin-1,suggesting that H₂O₂ inhibits the expression of autophagy.In contrast,the pretreatment of HT in DPCs reversed this trend and significantly enhanced autophagy expression.To further verify the protection in the HT-mediated autophagy against oxidative stress,autophagy inhibitor chloroquine（CQ）was used:the protection of HT in DPCs was significantly reversed,further demonstrating that regulatling autophagy is an important mechanism for HT to protect DPCs from oxidative stress damage.ConclusionHT enhanced the autophagy against oxidative stress damage by inhibiting inflammation and upregulated the expression of hair growth factors in DPCs.