Summary: The role of protease activated receptor-2 (PAR-2) in the renal tubulointerstitial lesion induced by unilateral ureteral obstruction (UUO) was explored. Mice were sacrificed on the day 1, 3, 5, 7, 10, 14 ...Summary: The role of protease activated receptor-2 (PAR-2) in the renal tubulointerstitial lesion induced by unilateral ureteral obstruction (UUO) was explored. Mice were sacrificed on the day 1, 3, 5, 7, 10, 14 and 21 after UUO. The expression of PAR-2 mRNA and protein and a-smooth muscle actin (α-SMA) protein in tubuloin,terstitium was detected by RT-PCR and immunohistochemistry at each time point, respedtively. The results showed that the PAR-2 expression in renal tubulointerstitium was increased progressively starting from 24 h to the day 14 post-ligation, and it was significantly associated with the relative volume of interstitium and the positive area of α-SMA. PAR-2 was mainly expressed in renal tubule epithelial cells, especially in proximal tubular cells. It also located in renal capillary ansa, interstitial infiltrate cells and fibroblasts. It was concluded that PAR-2 was active in interstitial and tubular cells in the early phase of fibrotic process and played an important role in mediating the tubulointerstitial lesion after UUO.展开更多
Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK)...Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.展开更多
Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced infl...Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells. Methods Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay (ELISA). Data were analysed by the S-N-K one-way ANOVA test. Results The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.展开更多
文摘Summary: The role of protease activated receptor-2 (PAR-2) in the renal tubulointerstitial lesion induced by unilateral ureteral obstruction (UUO) was explored. Mice were sacrificed on the day 1, 3, 5, 7, 10, 14 and 21 after UUO. The expression of PAR-2 mRNA and protein and a-smooth muscle actin (α-SMA) protein in tubuloin,terstitium was detected by RT-PCR and immunohistochemistry at each time point, respedtively. The results showed that the PAR-2 expression in renal tubulointerstitium was increased progressively starting from 24 h to the day 14 post-ligation, and it was significantly associated with the relative volume of interstitium and the positive area of α-SMA. PAR-2 was mainly expressed in renal tubule epithelial cells, especially in proximal tubular cells. It also located in renal capillary ansa, interstitial infiltrate cells and fibroblasts. It was concluded that PAR-2 was active in interstitial and tubular cells in the early phase of fibrotic process and played an important role in mediating the tubulointerstitial lesion after UUO.
基金This work was supported by the grants from Jiangsu Province Key Medical Center (No. ZX200608), the National Nature Science Foundation of China (No. 30672140, No. 81071451), the Colleges and Universities Natural Science Foundation in Jiangsu Province (No. 10KJB320019), the Key Project Surpported by the Medical Science and Technology Department Foundation, Jiangsu Province, Department of Health (No. H201012) and the Social Development Projects in Suzhou (No. SS08020).
文摘Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.
基金This study was supported by grants from National Natural ScienceFoundation of China(No.30470689), and the Science DevelopingFoundation of Shanghai Medical Healthy Bureau (No.034087)
文摘Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells. Methods Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay (ELISA). Data were analysed by the S-N-K one-way ANOVA test. Results The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.