PM2.5’s effect on the autophagy level of human keratinocytes

1. PM2.5’s introduction

In the process of cell metabolism, there are the synthesis and degradation of proteins and other macromolecules as well as the update of organelles. There are two ways to degrade substances in cells: proteasome and autophagy.Some scholars have proposed that autophagy is the basic stress response of cells to ensure their survival under harsh conditions, and it can be used as a protective mechanism of cells to adapt to the environment, preventing or mitigating the occurrence of damage.

PM2.5 refers to PM whose aerodynamic diameter is less than 2.5 microns. At present, a large number of studies have shown that PM2.5 can induce autophagy in alveolar macrophages and vascular endothelial cells.In this study, the expression of biological markers related to autophagy was observed by treating human keratinocytes with PM2.5 of different concentrations and components, and the possible mechanism of autophagy induction was studied.

2.PM2.5’s skin impact test materials and methods

2.1Materials and reagents

Human immorphic epidermal cell line Ha Ca T was purchased from China center for the preservation of typical cultures (wuhan university).PM2.5 location: 6 meters above the ground, on the roof of the institute of atmospheric research, school of environment, China university of geosciences (wuhan).The specific collection work was completed with the assistance of the school of environment, China university of geosciences (wuhan).Microtubule-related protein 1 light chain 3(LC3) rabbit anti-human polyclonal antibody (Cell Signaling, USA), mouse anti-beta-actin (wuhan baode biological engineering co., LTD.), BCA protein concentration assay kit, protease inhibitor and RIPA lysate (jiangsu biyuntian institute of biotechnology);Immunofluorescence: goat anti-rabbit Ig G, goat anti-rabbit HRP labeled secondary antibody (wuhan bode biological engineering co., LTD.), BX53 fluorescence microscope (Olympus, Japan).

2.2Preparation and grouping of contaminated venom

A certain weight of PM2.5 powder was weighed and added to 0.9% normal saline to prepare mother liquor with a concentration of 1000 mu g/mL, which was fully mixed and centrifuged at 13000g/min for 30 min. The supernatant was taken as the reserve solution of PM2.5 water-soluble components with a concentration of 1000 mu /mL.By the same method, the sediment was taken as a reserve solution of PM2.5 insoluble components with a concentration of 1000 mug /mL.The preset concentrations of the dye venom are :0.1 micron /mL, 1 micron /mL, 10 micron /mL and 100 micron /mL.The cells contaminated with venom were divided into two groups: group A (PM2.5 water-soluble components) and group B (PM2.5 water-soluble components).According to the concentration, they were divided into A1 group (0.1 mug /mL), A2 group (1 mug /mL), A3 group (10 mug /mL), A4 group (100 mug /mL), B1 group (0.1 mug /mL), B2 group (1 mug /mL), B3 group (10 mug /mL) and B4 group (100 mug /mL).

2.3Cell culture and grouping

Ha Ca T cells were conventionally cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 37℃, 5% CO2, and cell incubator with relative saturated humidity. The solution was changed for 2 ~ 3d times, and conventional passage culture was conducted. Logarithmic cells with good growth state were selected for the experiment.In the experimental group, Ha Ca T cells with good growth were respectively treated with PM2.5 of different concentrations in group A and group B and incubated in sterile incubator at 37℃ for 24h.Meanwhile, a blank control group was set up.

2.4The expression of LC3 was detected by immunofluorescence staining

Cells of the above experimental groups were taken, and the slides with good climbing cells were soaked and washed in PBS for 3 times in the culture plate, 3min each time, fixed with 4% paraformaldehyde, and then at room temperature for 15min.PBS containing 0.5% Triton x-100 was added and permeated at room temperature for 20min.After three times of PBS immersion and washing, they were sucked dry, and normal goat serum was added onto the glass slide, sealed at room temperature for 30min.The sealing solution was absorbed by absorbent paper without washing. Each glass slide was added with enough diluted primary antibody LC3(1:500) and placed in a wet box, incubated at 4℃ overnight.Remove primary antibody, rinse with PBS for 5min, repeat 3 times.Goat anti-rabbit Ig G secondary antibody (1:100) was incubated at room temperature for 1h.Remove the secondary antibody, rinse with PBS for 3min, and repeat 3 times.DAPI solution (1:400) was stained, incubated at room temperature under dark for 5min, rinsed with PBS for 5min, repeated for 4 times, and removed.The closed orifice plates of PBS were placed in Olympus BX53 inverted fluorescence microscope in the laboratory to take photos and imaging and observe the experimental results.

2.5Western blot was used to detect lc3-ii /I changes

Take the above experimental cells, 4 ℃ precooling, PBS rinsed three times, adding suitable amount of cell lysis solution (RIPA) and protease inhibitors (PMSF), the proportion according to configuration, 1:100 ultrasonic crack on the ice for 30 min, learned that moved to 1.5 mL centrifugal supernatant fluid tube, the BCA kit to determine protein concentration, sds-page electrophoresis separation, transferred to nitrocellulose membrane, closed 2 h at room temperature, the resistance to LC3 (1:1000) and beta actin (1:200) 4 ℃ incubation overnight, HRP labeling two resistance (1:50 000) at room temperature 2 h incubation,Finally, the PVDF film was scanned by ECL chemiluminescence.Each experiment was repeated three times.The expression of beta actin was used as the internal reference, and the grayscale value of the film was analyzed by Band Scan.

2.6statistical treatment

All data were analyzed with SPSS20.0 statistical software. The measurement data in this study were expressed as mean standard deviation (x±s). T-test was used for comparison of mean between the two samples, while one-way analysis of variance was used for comparison of mean between multiple samples.

3.PM2.5’s skin impact test results

3.1The results of immunofluorescence staining were shown in figure 1

In the control group, LC3 showed very weak and little expression, while cells in the experimental group treated with 0.1, 1.0, 10.0 and PM2.5 of different concentrations and components showed positive or even strong positive expression after fluorescence staining, and the red fluorescence intensity increased with the increase of venom concentration, and the autophagy level increased significantly.At the same concentration, there was no statistically significant difference in red fluorescence intensity between groups A and B.

3.2The results of western blot were shown in table 1 and figure 2 and 3

After 24h of PM2.5 intervention cells, lc3-ii protein expression levels in A1 ~ A4 group and B2 ~ B4 group increased with the increase of PM2.5 concentration, and were significantly higher than those in control group (t=5.38, 11.06, 23.07, 11.84 in group A, 2.77, 4.24, 8.83, 14.88 in group B).P < 0.05);Group B1 was also higher than the control group, but the difference was not statistically significant (t=2.77, P = 0.05).At the same concentration, the protein expression level of group A1 was higher than that of group B1 (F = 0.42, P > 0.05).The protein expression level of group A2 was higher than that of group B2 (F=1.85, P > 0.05).The protein expression level of A3 group was higher than that of B3 group (F= 3.96, P > 0.05).The protein expression level of group A4 was higher than that of group B4 (F=5.19, P > 0.05).

4.PM2.5’s skin impact test discussion

Autophagy is a conserved protein degradation pathway in cells, which refers to the process in which the isolation membrane wraps cytoplasm and/or organelles to form autophagosomes, and then fuses with lysosomes to form autophagosomes and degrades in them.Its occurrence is mediated by a group of autophagy-relatedproteins (Atg), in which Beclin 1, LC3 beta (or LC3 beta-ii) and P62 protein levels are representative indicators of autophagy occurrence and intensity.When cells are under stress conditions such as starvation, nutritional deficiency, hypoxia, structural reconstruction during cell development and organelle damage, autophagy level will increase.Studies have shown that autophagy can not only promote body health, but also has a close relationship with tumors, infectious diseases, lung diseases, neurodegenerative diseases, cardiomyopathy, aging and senile diseases.

Fig. 1 LC3 immunofluorescence staining ( × 400)

Tab. 1 The Western blotting results of LC3Ⅱ / Ⅰ each group ( x ± s)

A large number of studies have found that PM2.5 can have toxic effects on human respiratory, cardiovascular, endocrine and other systems.As the largest organ of the human body, skin is directly exposed to the body surface and in contact with air pollutants, which is an important target organ for the effects of exogenous toxic substances. However, there are few studies on the effects of PM2.5 on skin toxicity.Magnani et al. studied the interaction between CAPs and the ultrastructure of skin tissues with transmission electron microscope. After 24h of 100mg CAPs/mL intervention, CAPs were observed to appear in the upper layer of recon structed human epidermis (RHE), and then appeared in deeper cells 48h later, demonstrating that PM2.5 can be directly absorbed by the skin.So, when human epidermal keratinocytes are exposed to PM2.5 environment, will it induce the occurrence of epidermal autophagy?If so, what are the effects of changes in autophagy levels on human keratinocytes and what mechanisms might occur?To solve these problems, this study used immunofluorescence staining and west-ern blot detection technology to qualitatively analyze the changes in the number of autophagosomes in human epidermal keratinocytes, and quantitatively analyze the changes in the expression of autophagy-related protein LC3.

At present, there are many methods available to detect autophagy, among which LC3(microtubule-related protein l light chain 3) is the marker protein for the detection of autophagy. The molecular weight of lc3-i is 18kD, distributed in the cytoplasm.The molecular weight of lc3-ii is 16kD. When autophagy occurs, autophagosome formation increases, lc3-i is specifically cut into lc3-ii and transferred to autophagosome.Therefore, the conversion of lc3-i to lc3-ii can usually be detected to determine the occurrence of autophagy.Through qualitative analysis of immunofluorescence staining, it was found that LC3 protein in the control group showed very weak and little expression, while cells in the experimental group induced by PM2.5 showed positive or even strong positive expression after fluorescence staining, and showed an increase in concentration dependence.The author further conducted quantitative analysis by western blot method and found that the lc3-ii /I protein ratio in a1-a4 group and B2 ~ B4 group increased with the increase of PM2.5 concentration, and was significantly higher than that in the control group (P < 0.05).At the same concentration, there is no significant difference in the action intensity of PM2.5 water-soluble components and water-insoluble components.These experimental results not only confirm that PM2.5 can induce autophagy in human epidermal keratinocytes, maintaining the homeostasis of intracellular environment by decomposing its own organelles and proteins, but also enhance the autophagy activity of human keratinocytes with the increase of its concentration.Compared with the control group, lc3-ii protein level in group B1 was not statistically significant, but the expression was slightly increased, suggesting autophagy in keratinocytes after PM2.5 induction.At the same time, we observed through fluorescence microscope that the number of cells in the experimental group was significantly reduced after the treatment of PM2.5 venom at the maximum concentration (100 mug /mL), with incomplete cell membrane accompanied by nuclear fragmentation, poor cell state and cell tendency to apoptosis.This is similar to the characteristics of autophagy of human keratinocytes irradiated by UV observed by Milan et al. with confocal electron microscope, and proposed that the occurrence of this phenomenon may be a self-protection mechanism of keratinocytes against uv-induced apoptosis.

Li, a new study has found that in exposure to PM2.5 especially the metal component A1 and Pb, can cause human alveolar epithelial cells in the model of miR – 4516 and its target gene expression level RPL37 change and ribosomes cell dysfunction and autophagy occurs at the same time, in addition, also put forward in the cell RPL37 consumption increased the expression of LC3 – II, thus infer that miR – 4516 / RPL37 pathway may be mediated the alveolar epithelial cells of PM2.5 exposure – inducing autophagy.Deng et al. confirmed through transmission electron microscope, PCR and western-blot that PM2.5 may induce ROS production and loss of antioxidant activity, leading to ROS accumulation and cell death in human alveolar epithelial cells, and concluded that PM2.5- oxidative stress injury plays an important role in inducing cell autophagy.Although there are few studies on the effect of PM2.5 on skin autophagy and its mechanism, the results of this study suggest that PM2.5 may induce autophagy in human keratinocytes, which may be a self-protective mechanism of cells against oxidative stress injury and apoptosis.As for the mir-4516 /RPL37 pathway studied abroad, whether it is also involved in the occurrence of autophagy mechanism of human keratinocytes remains to be determined by further studies.

Fig. 2 LC3Ⅱ / Ⅰprotein expression

Fig. 3 Comparison of LC3-Ⅱ/Ⅰprotein levels between the ex-perimental group and the control groupNote: Compared with control group，* P＜0. 05; Compared with thesame concentration of PM2. 5 water-soluble extracts，#P＞0. 05

5.PM2.5’s skin impact test conclusion

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