“Bystander Effects”of Cell Supernatant From Premature Human Dermal Fibroblasts Induced By UVB On The Oxidative Damage,proliferation,aging And Autophagy Of The Normal Human Dermal Fibroblasts

BackgroundPremature ageing can be caused by various endogenous and exogenous factors. Since UV radiation(Solar radiation) happens to be the major factor involved in skin ageing, it can also be referred to as photo-ageing. Premature ageing may appear in in-vitro cultured normal cells after being exposed to UV radiation(Stress Induced Premature Senescence). Human dermal fibroblasts are involved in maintaining the integrity of the skin and replenish the extracellular matrix by producing a variety of cytokines and adhesion factors. Hence we can assume that they are the key cells involved in the process of skin aging.In vitro, premature senescent fibroblasts model can be used to research skin photoaging and stress-induced premature senescence is the most popular. Various research groups have proved that continuous exposure of human dermal fibroblasts to ultraviolet B radiation can successfully create(replicate) premature senescence model. Upon repeated exposure to small doses of UVB irradiation, the replicating HDF’s exhibit the characteristic features of senescence.In recent years, many scholars have proved that senescence can be induced in fibroblasts upon a single exposure to the sun’s ultraviolet rays, by certain drugs and tumors. Premature fibroblasts secrete certain soluble substances within the cell supernatant, then through the bystander effect they can induce certain biological effects in the surrouding fibroblasts’ s such as hypoproliferation, DNA damage, cell cycle arrest and so on. The essence of the bystander effect is: that certain cells can bring about biological changes within the surrounding cells after interacting with them via molecular signalling.However the effects of cell supernatant from the post UVB irradiated premature HDF’s on the surrounding normal fibroblasts through bystander effect has not been reported yet. Object“Bystander effects ”of cell supernatant from premature human dermal fibroblasts induced by UVB on the oxidative damage,proliferation, aging and autophagy of the normal Human dermal fibroblasts. Methods 1. Cell separation and culture Removal of the foreskin from healthy, young and middle-aged males via circumcision, separation of the epidermis and dermis to get the HDFs. Take 4th ~10th generation(sub-passage) cells in logarithmic growth phase for the experiment. 2. UVB irradiation Human dermal fibroblasts in logarthmic growth phase were exposed to 10mJ/cm2 UVB irradiation one time a day for five consecutive days and then were normal incubated for three days. 3. Collecting conditioned medium 2-3 days later cell supernatant was collected from each of the groups of the controlled group fibroblasts and premature fibroblasts and were frozened in a-80℃ refrigerator. 4. Conditioned medium fr om premature fibroblasts’ oxidative damage on normal fibroblasts Fluorescence microscopy and flow cytometry were used to detect the changes of ROS and mitochondrial membrane potential in cells. 5. Conditioned medium fr om premature fibroblasts’ effect on pr oliferation of normal human dermal fibr oblasts CCK-8 was used to detect proliferation activity of fibroblasts, flow cytometry and EDU were used to detect retardation rate of fibroblasts during the S phase of the cell cycle. 6. Conditioned medium fr om premature fibroblasts’ effect on pr emature of normal human dermal fibr oblasts Senescene associated- β-galactosidase chemical dyeing method was used to detect senescent cells. 7. Conditioned medium fr om premature fibroblasts’ effect on autophagy of human dermal fibr oblasts Acridine orange,indirect immunofluorescence and Western blot were used to detect the autophagy level of fibroblasts. Result 1. Conditioned medium fr om premature fibroblasts can incr ease oxidative damage of fibr oblasts Fluorescence microscopy and flow cytometry results indicated that the Ros and mitochondrial membrane potential of fibroblasts of the PH-CM group were relatively higher than the control group. Hence the difference between the two groups have statistical significance. 2. Conditioned medium fr om premature fibroblasts can decrease proliferation activity of fibroblasts CCK-8 results showed that the cellular proliferation activity of the PH-CM group was relatively lower than the control group. Hence the difference between the two groups have statistical significance(P<0.05);Flow cytometry results showed that the percentage of cells of PH-CM group in the G1 phase were significantly lower than the control group, while the percentage of cells in S phase were relatively higher in the PH-CM group in comparison with the control group. Hence difference between the two groups have statistical significance(P<0.05). 3. Conditioned medium fr om premature fibroblasts can accelerate premature senescence of fibroblasts Senescene associated-β-galactosidase chemical dyeing showed that the group of PH-CM positive rate 25.710%±0.3042 higher than the group of FB-CM 5.257%±1.023,difference between the two groups have statistical significance(t=19.17,P<0.05). 4. Conditioned medium from premature fibroblasts can decrease autophagy of fibroblasts Acridine orange staining results showed red fluorescence in the PH-CM group fibroblasts to be 29.61±2.6500 which was relatively lower than the FB-CM group. Western blot and indirect immunofluorescence results showed that the LC3-B expression of PH-CM group was significantly lower than the FB-CM group. Hence the differences between the two groups have statistical significance. ConclusionCell supernatant from the UVB induced premature human dermal fibroblasts can decrease the proliferation activity, increase the oxidative damage,accelerate premature senescence,decrease autophagy of normal human dermal fibroblasts through “bystander effects”..