A Preliminary Study On The Role Of Autophagy In The Formation Of Lamellar Bodies

Background Skin permeability barrier locates in stratum corneum of epidermis and its main function is to prevent the loss of water and electrolytes.Lamellar bodies(LB)exist in the cells of the upper spinous layer and granular layer of the epidermis and play an important role in maintaining the stability of the skin permeability barrier.Their absence or dysfunction will lead to a series of skin barrier disorders,such as psoriasis and ichthyosis.However,the specific source of LB is not yet clear.Recent studies have shown that autophagy may be involved in the formation of LB.Narrow band ultraviolet radiation B(NB-UVB)is an effective treatment for psoriasis,besides ultraviolet radiation can induce autophagy.Therefore,we speculated that autophagy may play an important role in the formation of LB.Objective To study the role of autophagy in the formation of LB,so as to provide a new treatment for dermatosis with skin barrier disorders.Methods Human immortalized keratinocyte lines HaCaT were treated with rapamycin 50nmol/L for 0h,12 h,24h and 48 h respectively.The expression of four autophagyrelated proteins,including mammalian target of rapamycin(mTOR),microtubule associated protein 1 light-chain 3(LC3),autophagy-related protein Sequestosome(P62),Rab11 a were detected by Western blot.The number and diameter of LB were observed by transmission electron microscopy(TEM).After HaCaT cells were treated with autophagy inhibitor Baflomycin A1 100nmol/L for 0h,12 h,24h and 48 h respectively,the changes of autophagy-related proteins mTOR,LC3,P62 and Rab11 a were detected by immunoblot,and the changes of the number and diameter of LB were observed by TEM.Results After HaCaT cells treated with 50nmol/L rapamycin,the Western blot results showed that compared with the control group at 0h,the expression of LC3-?/? increased significantly at 12 h,24h and 48h(t=22.87,34.03,10.13,P<0.05),the expression of LC3-? increased significantly at 12 h and 24h(t=8.54,9.82,P<0.05),the expression of P62 increased significantly at 12 h,24h and 48h(t=15.35,29.12,1.38,P<0.05),and the expression of mTOR decreased significantly at 12 h,24h and 48h(t=6.22,8.42,8.89,P<0.05);there was no significant difference in Rab11 a between the four time periods(F=0.13,P>0.05).TEM results showed that compared with 0h control group,the number of LB increased significantly at 48h(q=10.00,P<0.05),and there was no significant difference in the diameter of LB in the other four periods(F=2.17,P>0.05).After HaCaT cells treated with 100nmol/L baflomycin A1,the Western blot results showed that compared with the control group at 0h,the expression of LC3-?/? decreased significantly at 12 h,24h and 48h(t=18.91,24.86,25.68,P<0.05),the expression of LC3-? increased significantly at 12 h,24h and 48h(t=5.94,9.66,7.71,P<0.05),and the expression of P62 increased significantly at 12 h,24h and 48h(t=3.83,8.54,11.3,P<0.05);while there was no significant difference in mTOR and Rab11 a between the four time periods(F=0.21,0.64,P>0.05).TEM results showed that compared with 0h control group,the number of LB decreased significantly at 48h(q=5.00,P<0.05),and the diameter of LB decreased significantly at 12 h and 24h(t=2.09,2.26,P<0.05).Conclusion Autophagy signal may be involved in the regulation of the formation of LB.The number of LB increased when the autophagy signal was enhanced;the formation of LB was inhibited when the autophagy signal was weakened,and the number and diameter of LB decreased.LB may be formed by the appearance or degradation of autophagosomes.