The Effect Of Calcipotriol On The Expression Of S100A8in Human Keratinocytes

1. Background and ObjectivePsoriasis is a chronic inflammatory skin disease which has high prevalence. The unregulated proliferation and differentiation of keratinocytes plays an important role in the pathophysiology of psoriasis. Vitamin D is synthesized in human skin. Vitamin D and its analog Calcipotriol in turn regulate the proliferation, differentiation and apoptosis of keratinocytes. It has been reported that antimicrobial S100A7, S100A8and S100A9could interactive with inflammatory cytokines such as IL-17, IL-1, TNF-a, et al. It resulted in the onset of psoriasis. Vitamin D could lighten the inflammation of skin via down-regulation of the expression of S100A7in kerationcytes. As a result, it’s possible that Vitamin D and its analogs may also regulate the expression of S100A8and participate in the pathogenesis of psoriasis. In our study we mainly aim at how the Vitamin D analog Calcipotriol affect the expression of S100A8in cultured human immortal keratinocytes (HaCaT cells) in vitro.2. Methods(1) Culture of HaCaT:HaCaT cells were cultured in MEM-EBSS medium with10%FBS and antibiotics, and passaged routinely.(2) Intervention of HaCaT by TNF-a and Calcipotriol:Cells were divided into four groups when cell dishes were80～90%covered:the blank group with no interventions, the TNF-a group stimulated by1OOng/ml TNF-a, the Calcipotriol group with10-5M～10-9M Calcipotriol added and the Calcipotriol+TNF-a group with both1OOng/ml TNF-a and10-5M～10-9M Calcipotriol added. The entire groups all incubated for24hours.(3) Detection of S100A8expression in HaCaT:Total RNA was extracted from cultured cells and reverse-transcripted to cDNA. S100A8and endogenous β-actin fragments were amplified and electrophoresed with1%agarose gel. The same gene fragments were amplified by Real-time PCR devices. Relative expression of S100A8was calculated using2-△△Ct method. Statistical differences among groups were analyzed via ANOVA method.3. Results(1) The S100A8RT-PCR product was about250bps in length while the β-actin RT-PCR product was about130bps in length.(2) The relative expression of S100A8was up-regulated to19.62times (P<0.01) when stimulated by100ng/ml TNF-a for24h compared with blank.(3) The relative expression of S100A8was up-regulated to5.029and2.848times (P<0.01) when cultured with10-7M,10-8M Calcipotriol for24h compared with blank respectively.(4) The relative expression of S100A8showed no statistic difference when cultured with10-5M,10-6M and10-9M Calcipotriol for24h compared with blank (P>0.05).(5) The relative expression of S100A8was down-regulated to0.5951time (P<0.05) when cultured with10-5M Calcipotriol+100ng/ml TNF-a for24h compared with cells stimulated by TNF-a.(6) The relative expression of S100A8was up-regulated to1.873times (P<0.01) when cultured with10-7M Calcipotriol+100ng/ml TNF-a for24h compared with cells stimulated by TNF-a.(7) The relative expression of S100A8showed no statistic difference when cultured with10-6M,10-8M and10-9M Calcipotriol for24h compared with cells stimulated by TNF-a (P>0.05).4. Conclusion(1) TNF-a could induce the high expression of S100A8in cultured HaCaT cells in vitro. It meant that TNF-a might regulate the proliferation and differentiation of keratinocytes via high expression of S100A8with the help of IL-1, IL-8, IL-17, etc. As a result, the inflammation of skin was strengthened.(2) Calcipotriol affected the expression of S100A8and regulated the proliferation of keratinocytes bi-directionally:High concentration (about10-5M) Calcipotriol could down-regulate the expression of S100A8. It suppressed cell proliferation and promoted apoptosis. At the same time, interaction between S100A8and inflammatory cytokines was broken. Psoriasis skin lesions were relieved. On the contrary, low concentration (10-7-10-8M) Calcipotriol could up-regulate the expression of S100A8and promote cell proliferation.