| PrefaceDramatic destruction of the ozone layer and frequent occurrences of the solar storms result in more and more ultraviolet (UV) reaching the earth surface. Exposure to UV excessively may cause some biological effects,such as cutaneous erythematosus,photo aging,irnmunosuppression,even tumorigensis and so on. Long wave ultraviolet (UVA) accounts for the proportion in the solar spectrum in a big way and UVA penetrates more deeply into human skin. More and more attentions were paid to the research of immunosuppression induced by UVA.Keratinocytes (KC) are the richest in the epidermis, accountting for more than 90%. KC may affect the immunological function indirectly through affecting the release of cytokines in KC. However, the pathway UVA induces the expression of cytokines in KC are still unclear.In the present study, the human KC cell line HaCaT were cultured in vitro, and TNF-α, IL-1β, IL-10 expressions in UVA-irradiated cells were detected. Then HaCaT cells were treated with oxidant, JNK inhibitor respectively, in order to exploring the effects of oxidative stress and JNK transduction pathways on expressions of cytokines mentioned above in KC induced by UVA.Materials and Methods1. Cell cultureThe spontaneous transformed human epidermal cell line HaCaT was cultured in modified Eagle's medium supplemented with l‰nonessential amino acid, 10% heat-inactivated fotal bovine serum, and containing 100 U/ml penicillin, 100 mg/ml streptomycin, in a fully humidified atmosphere with 5%CO2 at 37℃. Cells were trypsined and subcultured when grown in logarithmic course.2. Viability of cellsCells were cultured in 96-well plastic Petri dishes. When the confluency of the cultures reach approximately 60%, the growth medium was aspired and cells was washed three times with phosphate-buffer saline (PBS) warmed to 37℃. The cultures were overlayed with 0.1ml PBS and irradiated without the petri dish lid to different doses of UVA (0～3.6J/cm2). Viability of cells in each group was determined by methyl thiazolyl tetrazolium (MTT) method.β-carotene was dissolved in dimethyl sulphoxide (DMSO), then added to the growth medium of cells 1h before irradiation at final concentration 3μM. The same volume DMSO was added to the growth medium of sham-irradiated (control) and irradiated cells. Viability of cells in each group was examined by the method mentioned above.3. JNK levelsCells were cultured on glass cover slips in 6-well plastic Petri dishes. Two days later, the growth medium was aspired and cells was washed three times with PBS warmed to 37℃. The cultures were overlayed with 1.0ml PBS and irradiated without the petri dish lid to 2.4 J/cm2 UVA. After irradiation, the PBS was aspired and fresh culture medium was added to the cultures for further incubation. Sham-irradiated cells were treated in the same manner, except that they were not irradiated. Samples were collected at different time points post-irradiation (0, 2, 4, 12, 24, 48h, respectively) and the levels of phospherated JNK and nonphospherated JNK in cells were detected by immunocytofluorescent method.β-carotene was dissolved in DMSO, then added to the growth medium of cells before irradiation at final concentration 3μM. The same volume DMSO was added to the growth medium of sham-irradiated and irradiated cells, lh later, cells were irradiated with 2.4 J/cm2 UVA, except sham-irradiated cells. Samples were harvested and the levels of JNK were detected by the method mentioned above.4. Cytokines mRNA levels and protein expressions Cells were cultured in 90mm flat plate. After cell subconfluence, the growth medium was removed and cells was washed three times with PBS wanned to 37℃. The cultures were overlayed with 5.0ml PBS and irradiated without the petri dish lid to 2.4J/cm2 UVA (according to the results of cells viability). After irradiation, the PBS was aspired and fresh culture medium was added to the cultures for further incubation. Cells in the control group were the same treated but not irradiated. Cells and the growth medium were collected at 0, 2, 4, 12, 24, 48h post-irradiation, respectively. TNF-α, IL-1β, IL-10 mRNA levels and protein expressions were detected by reverse transcriptional polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).β-carotene or anthrapyrazolone (SP600125) was dissolved in DMSO, then added to the growth medium of cells before irradiation at final concentration 3μM and 10μM respectively. The same volume DMSO was added to the growth medium of sham-irradiated and irradiated cells, 1h later, cells were irradiated with 2.4J/cm2 UVA, except sham-irradiated cells. Samples were collected and cytokines mRNA levels and protein expressions were detected by the methods mentioned above.5. Statistical analysesThe data were analyzed with SPSS12.0 software and were considered significant if P＜0.05.Results1. Cell ViabilityThe viability of cells irradiated with more than 3.0J/cm2 UVA decreased significantly (P＜0.05). No significant decrease (P＞0.05) were detected on the viability of cells irradiated with 2.4J/cm2 UVA or below. There was no significant decrease (P＞0.05) of viability in each group cells with P-carotene intervention.2. JNK levelsBoth phosphorylated JNK and nonphosphorylated JNK levels in irradiated cells gradually increased from 0 to 24h post-irradiation, and reached the peak at 24h, then declined slightly at 48h. Both phosphorylated JNK and nonphosphorylated JNK levels in control cells increased gradually from 0 to 48h post-irradiation, and peaked at 48h. Compared with the control cells, the phosphorylated JNK levels in irradiated cells increased significantly (P＜0.05) over a 48h period, and the nonphosphorylated JNK levels significantly increased (P＜0.05) from 0 to 48h except 4h post-irradiation.The phosphorylated JNK in irradiated cells with P-carotene intervention was the same levels as that in irradiated cells at Oh after irradiation, which were significant higher (P＜0.05) than that in control cells. The phosphorylated JNK levels in irradiated cells withβ-carotene intervention were significant lower (P＜0.05) than those in irradiated cells from 2 to 48h post-irradiation and those in control cells from 24 to 48h. The nonphosphorylated JNK levels in irradiated cells with p-carotene intervention were lower significantly (P＜0.05) than those in irradiated cells througout the 48h period following irradiation and those in control cells from 24 to 48h.3. Cytokines mRNA levels and protein expressions3.1 Cytokines mRNA levelsTNF-α, IL-1βmRNA levels in control cells were relatively constant over a 48 period. The levels of TNF-α, IL-1βmRNA in irradiated cells increased immediately after irradiation, and peaked at 4h, then returned to the control levels at 12h and remain fairly constant until 48h. Compared with the control cells, TNF-α, IL-1βmRNA levels in irradiated cells were higher significantly (P＜0.05) from 0 to 4h post-irradiation.TNF-α, IL-1βmRNA in irradiated cells withβ-carotene intervention were the same levels as those in control cells throughout the 48h period post-irradiation. Both aboved groups of TNF-α, IL-1βmRNA levels in cells were lower significantly (P＜0.05) than those in the control group from 0 to 4h after irradiation.The levels of TNF-αmRNA in irradiated cells with SP600125 intervention were lower than those in the irradiated cells thoughout the 48h period post-irradiation, and there were significant lower (P＜0.05) from Oh to 48h except 4h after irradiation, and lower significantly (P＜0.05) compared with the control cells from 12 to 48h post-irradiation. The IL-1βmRNA levels in irradiated cells with SP600125 intervention were significant lower (P＜0.05) than those in the control cells throughout the 48h period following exposure, and lower significantly (P＜0.05) compared with the control cells from 12 to 48h post-irradiation. No IL-10 mRNA expression was detected in each group cells except that slight expression of IL-10 mRNA was detected only at 12h post-irradiation in cells irradiated with 2.4J/cm2 UVA radiation.3.2 Cytokines protein expressionsTNF-α, IL-1βmRNA levels and TNF-α, IL-1βprotein expressions in control cells were relatively constant over a 48 period. TNF-α, IL-1βprotein expressions in irradiated cells were the same levels as those in control cells from 0 to 4h after irradiation, and increasede at 12h, then returned to the control levels at 24h and remain fairly constant over the next 24h. Compared with the control cells, TNF-α, IL-1βprotein expression in irradiated cells were higher significantly (P＜0.05) at 12h after irradiation.TNF-α, IL-1βprotein expression in irradiated cells withβ-carotene intervention were the same levels as those in control cells throughout the 48h period post-irradiation, and both of the two groups of TNF-α, IL-1βprotein expression were lower significantly (P＜0.05) than that in the control group at 12h after irradiation.The expression of TNF-αprotein in irradiated cells with SP600125 intervention was lower than that in the irradiated cells thoughout the 48h period post-irradiation, and there were significant difference (P＜0.05) between two groups from 12h to 48h after irradiation, and lower significantly (P＜0.05) compared with the control cells from 24 to 48h post-irradiation. IL-1βprotein expression in irradiated cells with SP600125 intervention were the same levels as those in control cells and irradiated cells from 0 to 4h post-irradiation, then higher significantly (P＜0.05) at 12h after irradiation compared with the control cells, but lower significantly (P＜0.05) compared with the irradiated cells, however, significantly higher (P＜0.05) from 24 to 48h after irradiation compared with the control and irradiated cells.No IL-10 protein expression was detected in each group cells except that lower level of IL-10 protein was detected only at 24h post-irradiation in cells irradiated with 2.4 J/cm2 UVA radiation.Conclusions1. HaCaT cell viability decreased significantly in vitro irradiated with 3.0 J/cm2 UVA or above.β-carotene can antagonize the effect on HaCaT cell viability decrease induced by UVA.2. JNK activity increased in HaCaT cells irradiated with 2.4J/cm2 UVA.β-carotene can antagonize the effect on HaCaT cell JNK activity increase induced by UVA.3. TNF-α, IL-1βmRNA levels up-regulated and their protein expressions increased in HaCaT cells irradiated with 2.4 J/cm2 UVA, IL-10 may not be expressed in HaCaT cells, but UVA can induce the expression of IL-10 gene and protein.4. Bothβ-carotene and SP600125 can antagonize the up-regulated effect on TNF-α, IL-1βand the expression of IL-10 induced by UVA radiation.
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