Autophagy And Oxidative Stress Afected By Ultraviolet-B In Keratinocytes:A Pilot Study

Abstract:Autophagy is a conserved physiological mechanism for the maintenance of cellular homeostasis through cytoplasmic and organelle turnover in response to various harmful stimuli. Ultraviolet irradiation induces human skin to produce reactive oxygen species (ROS), followed by generating lipid peroxidation and DNA damage in the tissue and cells. In order to explore the molecular mechanism between autophagy and oxidative stress and their role in diseases development, we studied autophagy and oxidative phenomenon in human keratinocytes related to Ultraviolet-B (UVB) radiation, preliminary investigated the basic signal pathway between autophagy and oxidative stress by introducing an antioxidant alfa-lipoic acide (LA).The first part of this study compared the proliferation activities of keratinocyte cell line HaCaT and primary human keratinocyte after stimulated by different doses of UVB and different concentrations of hydrogen peroxide (H2O2). The second part compared the autophagic vesicle expression levels of the two cells lines after irradiated by UVB. The third part tested the oxidative stress changes in the primary keratinocytes after irradiated by UVB, H2O2treatment group served as a positive control. Furthermore introduced the antioxidant LA and tested the antioxidant capacity in UVB related oxidative stress. The last part verified the autophagic level changes in the keratinocytes after incubated with LA or H2O2and irradiated by UVB.Objective:1. Study the tolerance capacity of two keratinocyte cell lines exposed to UVB or H2O2.2. Compare the autophagosome level between two keratinocyte cell lines after irradiated by UVB.3. Verify whether antioxidante LA is able to change the oxidative stress and autophagic level in keratinocytes induced by UVB, and then discuss the relationship and possible mechanism between them.Methods:(1) The oxidative stress cell models induced were established after UVB irradiadion and H2O2incubation. The Cell morphology and microstructure change were observed under inverted phase contrast microscope. The cell proliferation activity was measured by MTT method after UVB and H2O2treatment.(2) MDC staining method was adopted to dye autophagosome and late autophagic vesicles. Then inverted fluorescence microscope was used to take photos and count the autophagosome positive rate in each visual field.(3) Intracellular lipid peroxide MDA and total antioxidative capacity (TAC) were detected by chemical method.(4) The intracellular ROS was stained by DCFH-DA, and inverted fluorescence microscope was used to detect fluorescence intensity, and flow cytometer instrument was to quantify the green fluorescent.(5) LC3, p62and mTOR proteins were semiquantified by western blot method.Results:(1) HaCaT cells and primary keratinocytes expressed different proliferation inhibition capacity after UVB and H2O2treatment, and primary keratinocytes were more resistant to UVB or H2O2injury.(2) MDC staining method showed that the two cell lines express significantly different levels of autophagic vesicles under the same dose of UVB irradiation.5,10and20mJ/cm2UVB were able to promote autophagic vesicles levels with dose dependent manner in HaCaT cells, but had no significant impact in primary keratinocytes.40mJ/cm2UVB irradiation produced more autophagic vesicles than control group but less than that of20mJ/cm2UVB. Howerer,40mJ/cm2UVB may significantly inhibit the autophagic vesicles expression in primary keratinocytes.(3) Both10mM H2O2and40mJ/cm2UVB were able to induce ROS and MDA generation markedly in primary keratinocytes, especially in4hours group after irradiated by UVB, but almost back to normal in12hours group. LA incubation was capable of decreasing both ROS and MDA levels in4hours group.(4) MDC staining revealed the autophagosome positive rate of UVB radiation plus LA treatment group was larger than that of simple UVB radiation group, remarkably in LA treated12hours group. The autophagosome positive rate and LC3level were up regulated in12hours UVB radiation group compared with4hours group.(5) Both antioxidant LA and pro-oxidant H2O2could up-regulate the primary keratinocyte autophagome formation and LC3level through non-mTOR pathway.Conclusions:(1) HaCaT cells and primary keratinocytes had inconsistent proliferation inhibition and autopagy expression ability under UVB radiation. The primary keratinocytes were more resistant to UVB injury, and UVB radiation inhibited autophagosome formation in them.(2) Both UVB and H2O2stimulation were able to induce oxidative stress in primary keratinocytes. UVB induced ROS and MDA level were recovered and the inhibition effect was alleviate by self-repair ability of cells.(3) LA was capable of reducing oxidative stress and autophagy inhibition phenomena related to UVB. The protective effect of LA may act through the induction of autophagy.(4) Both antioxidant LA and pro-oxidant H2O2could regulate the primary keratinocyte autophagy level through non-mTOR pathway, but p62may not be involved in their autophagic regulation process.