Abstract: 【Abstract】 Objective To evaluate the in vitro effect of tacrolimus on the expression and function of protease-activated receptor 2 （PAR-2） in cultured human keratinocytes. Methods After 24-hour co-culture of human keratinocytes with 10-9 - 10-5 mol/L tacrolimus, semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）was performed to determine the mRNA expression of PAR-2, immunofluorescence（IF）staining and Western blot analysis were performed to determine the protein expression of PAR-2 in the keratinocytes, and the fluorescent calcium probe fluo-4 was used to evaluate the effect of tacrolimus at different concentrations on the intracellular calcium concentration after the activation of PAR-2 in the keratinocytes. The group treated without tacrolimus served as control group. One-way analysis of variance was used to compare the PAR-2 expression and calcium concentration in the human keratinocytes in different groups, and least significant difference （LSD）-t test was carried out for multiple comparisons. Results PAR-2 was expressed on both the membrane and cytoplasm of keratinocytes. After 24-hour co-culture of keratinocytes with 10-9 - 10-5 mol/L tacrolimus, the PAR-2 mRNA expression significantly decreased in these cells compared with the control group （all P < 0.05）, and was negatively correlated with the tacrolimus concentration （r = -0.962, P = 0.009）. IF staining and Western blot analysis showed that the PAR-2 protein expression was significantly lower in the 10-5- and 10-6-mol/L tacrolimus groups than in the control group （both P < 0.05）, and decreased to a certain extent in the 10-7-mol/L tacrolimus group （IF staining: P < 0.05; Western blot analysis: P > 0.05）. No significant difference in the PAR-2 protein expression was observed between the 10-8- or 10-9-mol/L tacrolimus group and the control group （both P > 0.05）. After 24-hour co-culture, the peak concentration of intracellular calcium after PAR-2 activation was significantly lower in the 10-5-, 10-6- and 10-7-mol/L tacrolimus groups （peak absorbance: 1 463 ± 283, 1 455 ± 270, 1 423 ± 291 respectively） than in the control group （1 602 ± 407; t = 2.582, 2.821, 2.923, P = 0.032, 0.022, 0.019, respectively）, while there was no significant difference between the 10-8- or 10-9-mol/L tacrolimus group （1 649 ± 379, 1 633 ± 415 respectively） and the control group （t = 0.846, 0.462, P = 0.422, 0.657, respectively）. Conclusion Tacrolimus can inhibit PAR-2 expression and suppress calcium mobilization induced by a PAR-2 agonist in keratinocytes.

Key words: Tacrolimus, Keratinocytes, Calcium signaling, Receptors, thrombin, Receptor, PAR-2