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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.
ABSTRACT
Objective To investigate the effect of breviscapine on the proliferation and the expression of thrombin receptor mRNA of vascular smooth muscle cells (VSMCs) . Methods Rat thoracic aortic VSMCs cultivated in vitro were randomly assigned to control,breviscapine 0.5 μg/mL, 5 μg/mL and 50 μg/mL groups, The proliferation was induced by thrombin, The proliferative effect of VSMCs was measured by the3H-thymidine (3H-TdR) incorporation method; the expression intensity of thrombin receptor mRNA relative to β-actin mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results The incorporation rate (cpm/1. 5 × 105 cells) of3H-TdR were 1 216. 00±241.57,673.25±12.63,602.50±80.59, and 522.00±103.99 respectively in the control, and breviscapine 0. 5 μg/mL, 5 μg/mL and 50 μg/mL groups. As compared with the control group, the prolifera-tion of rat thoracic aortic VSMCs was inhibited significantly in all breviscapine groups (all P<0.05). RT-PCR showed that the expressions of thrombin receptor mRNA relative to β-actin mRNA were 0. 614, 0. 389, 0. 310, and 0. 280 respectively in the control, and breviscapine 0. 5μg/mL, 5μg/mL and 50 μg/mL groups, The expression ratios of TR/β-actin mRNA in thoracic aortic VSMCs in all the breviscapine groups were lower than those in the control group, which suggesting that the expression of thrombin receptor mRNA was inhibited. Conclusions Breviscapine inhibits the proliferation of rat VSMCs. Its mechanism may he associated with the inhibition of the thrombin receptor gene expression of VSMCs.
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Objective To determine the changes in expressions of thrombin receptor and fibrin deposit in glomeruli during the process of senility. Method Rats were divided into 3 groups (8 rats in each group): 3-month-old group (3m), 12-month-old group (12m) and 24-month-old group (24m). Fibrin deposition was detected by Martius-Scarlet-Blue staining and direct immunofluorecence method. Immunohistochemical studies were performed to detect the expression of thrombin receptor (PAR-1) and transforming growth factor-? (TGF-?). Semi-quantitative PCR was performed to detect the changes in PAR-1 mRNA expression. A quantitative analysis of the expressions was performed by image analysis system. Result Significant pathological changes were found in glomeruli during the process of senility. Fibrin deposition was not observed in glomeruli in different groups. Significant expression of PAR-1 was found in glomerular endothelial cells, mesangial cells and epithelial cells in 3m rats. On the contrary, in 24m rats, PAR-1 expression in glomeruli was significantly decreased. Expression of TGF-? was increased with senility in glomeruli. PAR-1 gene expression, barely detectable in control tissue, was strikingly increased in 24m rats. Conclusion Thrombin receptor activation could be found in glomeruli of senile rat, and it is independent of fibrin deposition. Activation of PAR-1 may play an important role in the process of renal senility.
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To determine thrombin activation and fibrin deposition in the development of lupus nephritis in MRL lpr/lpr mice and the inhibitory effects of "shenle". "Shenle" (4g/(kg?d) orally) was administered daily to MRL lpr/lpr mice at the age of 8 weeks. After treatment for 20 weeks, we compared thrombin receptor (Protease Activated Receptor-1, PAR-1) expression with immunohistochemistry and fibrin deposition with MSB(Martius-Scarlet-Blue)staining in renal sections. PAR-1 mRNA expression was analyzed with RT-PCR method in the two groups. With the development of murine lupus nephritis, we observed an increase in thrombin receptor mRNA and severe fibrin deposition in renal tissue in the control group, while thrombin receptor protein expression was strikingly downregulated, suggesting its continuous activation and degradation. "Shenle" inhibited PAR-1 activation significantly and it was correlated with reduced fibrin deposition. These results suggested that thrombin activation may play an important role in the development of glomerulonephritis in MRL-lpr mice. "Shenle" ameliorated the murine renal lesions probably by inhibiting thrombin receptor activation and fibrin deposition.