The Studies On The Role Of Autophagy In Photo-damaging And Photoaging Of Cultured Human Dermal Fibroblasts

The cell homeostasis is maintained by a precisely regulated balance between synthesis and degradation of cellular components. The constitutive degradation of proteins by the proteasome . However, this process is limited to cytoplasmic short-lived proteins, and the majority of cellular components are long lived. Therefore, there must be another route for large-scale macromolecular recycling. This route is autophagy. This process has been observed in all eukaryotic cells, and play an important role in the maintenance of homeostasis. Autophagy pathways can be classified into three different types: macroautophagy, microautophagy and chaperone-mediated autophagy . Macroautophagy is the major lysosomal route for the turnover of cytoplasmic components and will hereafter be referred as autophagy. Extensive evidence has been reported that macroautophagy process declines with increasing age. This impairment, probably due to ad libitum feeding, may cause accumulation of altered structures leading to the age-related decline in cell functions. On the contrary, a limited feeding can delay the decline of autophagy. According to some researches, autophagy plays an important role in the aging of organisms. As a part of organisms, the aging of skin may have some matter with autophagy. For skin, photoaging poses large effects on the aging process besides intrinsic aging. Analyzed from signal transduction pathways, ultraviolet radiation can down-regulate the autophagy through activating the pathway of PI3K/AKT/mTOR or inhibiting the expression of UVRAG.. This paper initially elucidates the effects of UVB on the autophagy of human skin fibroblast, and observes the changes of the activity and the level of MMP-1 and MMP-3 of fibroblast by regulating the level of autophagy., then to discuss the role of autophagy in the photoaging process and explore the new mechanisms of photoaging. The paper consists of three sections.SectionⅠThe relationship between the autophagy of human skin fibroblast and photo-damaging by UVB Through MDC assay, it was detected that in the groups of 100 m J/cm~2 and 200 m J/cm~2, acute photo-damaging of human skin fibroblast can up-regulate the autophagy, having significant difference from normal control group (p<0. 01) . The appearance of fibroblast became deformed and shrink in the groups of 300 m J/cm~2 and 400 m J/cm~2, the level of autophagy of was lower than that of the former two groups, but still significantly higher than normal control group yet (p<0. 01). The expression of autophagy of fibroblast was also observed in different time such as 12h, 24h and 48h after UVB irradiation. The results showed that the activated autophagy decreased gradually with the lapse of time, and there was significant difference among the three groups (p<0. 01) .Some conclusions can be drawn from the research. Mild to moderate UV-damaging can increase the expression of autophagy, but serious UV-damaging can deform the shape of fibroblast and down-regulate the autophagy within some degree. The autophagy activated by UV-damaging becomes normal with the passage of time.SectionⅡThe relationship between the photoaging of human skin fibroblast induced by UVB and autophagyA cell model of photoaging fibroblast was developped by repeat irradiation of UVB. The shape of photoaging fibroblast was obviously different from the normal, and the cell proliferation stagnated. The results ofβ-galactosidase assay showed that the photoaging fibroblast was more prematurely senescent than the controlled cells and the difference between the two groups had statistical significance (p<0. 01) . Meanwhile, the autophagy of photoaging fibroblast was deactivated obviously, and had significant difference with normal group (p<0. 01). The degree of the decreasing of autophagy was in accordance with senescence. So the conclusion is that the autophagy of photoaging human skin fibroblast is expressed lower than the normal fibroblast, which is related to the degree of senescence.SectionⅢThe relationship between the changes of function of autophagy and photoagingThe results of autophagy measurement showed that the autophagy of fibroblast which was pretreated with rapamycin and 3-MA was up-regulated by the increasing of concentration of rapamycin at equal pace, and showed corresponding down-regulation with the increasing of concentration of 3-MA. The activity of fibroblast was measured by MTT method, which was increasing when the autophagy is mildly up-regulated, and had significant difference from the normal group (p<0. 01) . However, the activity of fibroblast was decreasing when the autophagy was intensively activated, which was same when the autophagy is obviously deactivated, and significantly lower than the normal group (p<0.01) . The expression of MMP-1 and MMP-3 was increasing with the down-regulation of autophagy, which is measured by ELISA method, and significantly higher than normal group (p<0. 05) . There was no significant difference from the normal group in the level of MMP-1 and MMP-3 after mild up-regulation of autophagy (p>0. 05) . The level of MMP-1 and MMP-3 also increased when autophagy of fibroblast was intensively activated, and had significant difference from the normal group (p<0. 01) . Conclusion: The results show that the inhibition of autophagy could down-regulate the activity of fibroblast and exacerbate the senescence. The activity of fibroblast could be up-regulated when autophagy was mildly activated, in which the effect of which on the senescene was not clear yet. When the autophagy of fibroblast is dramatically activated, the activity of fibroblast decreases and the aging process is accelerated.