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1.
Chinese Journal of Dermatology ; (12): 747-752, 2019.
Article in Chinese | WPRIM | ID: wpr-796842

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.

2.
Braz. oral res. (Online) ; 31: e16, 2017. tab, graf
Article in English | LILACS | ID: biblio-839530

ABSTRACT

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Gingival Crevicular Fluid/chemistry , Receptor, PAR-2/analysis , Chronic Periodontitis/pathology , Reference Values , Severity of Illness Index , Cysteine Endopeptidases/analysis , Biomarkers/analysis , Case-Control Studies , Gene Expression , Periodontal Index , Dental Plaque Index , Periodontal Attachment Loss , Porphyromonas gingivalis , Statistics, Nonparametric , Adhesins, Bacterial/analysis
3.
Article in English | WPRIM | ID: wpr-184744

ABSTRACT

BACKGROUND/AIMS: There have been no reports on the effect of chronic psychological stress on colonic immune cells or the regional differences. We aimed to investigate the effect of chronic psychological stress on the number of mast cells and protease-activated receptor (PAR)-2-positive cells in the rat colonic mucosa. METHODS: Six-week-old and 14-week-old Ws/Ws rats, which lack mast cells after 10 weeks, were used as control and mast cell-deficient groups, respectively. The rats were divided into stress and sham-treated groups. Rats in the stressed group were exposed to water avoidance stress (WAS, 1 hour/day) for 13 days. Fecal pellet output and the number of mast cells and PAR-2-positive cells in colonic mucosa were compared between the WAS and sham groups. RESULTS: In 6-week-old rats, the WAS group showed a significantly higher number of mast cells compared to the sham group. In 14-week-old rats, mast cells were nearly absent in the colonic mucosa. WAS significantly increased PAR-2-positive cells in 14-week-old rats, but not in 6-week-old rats. Indirect estimation of PAR-2-positive mast cells in 6-week-old rats suggested that the majority of increased mast cells following WAS did not express PAR-2. WAS increased mast cells and PAR-2-positive cells mainly in the proximal colon. Fecal pellet output was continuously higher in the WAS group than in the sham group, and the difference was significant for both 6-week-old and 14-week-old rats. CONCLUSIONS: Chronic psychological stress increased the number of mast cells and PAR-2-positive cells in rat colonic mucosa, and these increases were more prominent in the proximal colon.


Subject(s)
Animals , Rats , Cell Count , Colon , Mast Cells , Mucous Membrane , Receptor, PAR-2 , Stress, Psychological
4.
Article in English | WPRIM | ID: wpr-59650

ABSTRACT

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a lethal pulmonary fibrotic disease. In general, the exaggerated activation of the coagulation cascade has been observed during initiation or maintenance of the fibrotic disease. In our recent study, immunohistochemical expression of protease-activated receptor-2 (PAR-2), which plays a key role in coagulation cascade, was observed in surgical specimen of IPF patients, and associated with poor clinical outcome. The aim of this study was to evaluate the overexpression of PAR-2 in inflammatory cells from peripheral blood and bronchoalveolar lavage fluid in IPF patients. METHODS: From May 2011 to March 2012, IPF patients and controls were enrolled in Seoul National University Hospital. Peripheral blood and bronchoalveolar lavage fluid were collected for analysis of PAR-2 expression. Flow cytometry and reverse transcription polymerase chain reaction were used for PAR-2 receptor and mRNA assessment. RESULTS: Twelve IPF patients and 14 controls were included in this study. Among them, flow cytometry analysis was conducted from 26 peripheral blood (patient group, 11; control group, 13) and 7 bronchoalveolar lavage fluid (patient group, 5; control group, 2). The expression of PAR-2 receptor was not different between patient and control groups (p=0.074). Among all 24 population, PAR-2 mRNA assessment was performed in 19 persons (patient group, 10; control group, 9). The mRNA expression of PAR-2 was not significant different (p=0.633). CONCLUSION: In IPF patients, PAR-2 receptor and mRNA expression were not different from control group.


Subject(s)
Humans , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid , Flow Cytometry , Idiopathic Pulmonary Fibrosis , Polymerase Chain Reaction , Receptor, PAR-2 , Reverse Transcription , RNA, Messenger
5.
Chinese Journal of Dermatology ; (12): 886-890, 2012.
Article in Chinese | WPRIM | ID: wpr-430883

ABSTRACT

Objective To assess the expression pattern of protease-activated receptor 2 (PAR2) in human keratinocytes and to characterize its biological functions in the regulation of skin barrier.Methods Primary human keratinocytes and human N/TERT keratinocytes were used as the subject of this study.The expression and distribution of PAR2 in the keratinocytes were analyzed by using immunoflorescence staining and Western blot.Two different PAR2 agonists,trypsin and a PAR2-activating peptide (AP),as well as a PAR2-antagonistic peptide (H2N-FSLLRY-COOH) and a control peptide were used to induce the activation of PAR2 in the keratinocytes.Then,a fluorescence-based calcium mobilization assay was performed to evaluate the biological function of PAR2.Data were statistically analyzed by one-factor analysis of variance.Results Under normal culture conditions,PAR2 was weakly expressed in keratinocytes,and the expression was unaffected by culture medium composition or culture duration.Calcium mobilization was induced by trypsin of 50-250 nmol/L and the PAR2-activating peptide in a dose-and time-dependent pattern.The maximal activation of PAR2 was observed in keratinocytes treated with the PAR2 agonist HAN-SLIGKV-COOH of 75-250 μmol/L.The PAR2-antagonistic peptide (H2N-FSLLRY-COOH) obviously suppressed the increase in calcium mobilization induced by trypsin,while the control peptide PAR-RAP showed no inductive effect on the PAR2 activation based on the absence of calcium mobilization.The substrate-induced calcium release was complete within 250 seconds,and peaked at 50 seconds after the initial trypsin or PAR-AP stimulation.Moreover,the activation of PAR2 was accompanied by an increase in ERK phosphorylation and elicitation of MAPK signaling pathway in keratinocytes.Conclusions Human keratinocytes positively express PAR2,which can be activated by trypsin and PAR2-activating peptides,and the activation of PAR2 may influence the physiological function of keratinocytes by inducing intracellular calcium release.

6.
Article in Chinese | WPRIM | ID: wpr-417750

ABSTRACT

Objective To detect the expression of proteinase activated receptor 2 (PAR-2) in the skin of patients with atopic dermatitis (AD) and to evaluate the effects of tacrolimus on the expression.Methods Six patients with acute moderate or severe AD were enrolled in this study and topically treated with tacrolimus 0.1% ointment twice daily for 3 weeks.Tissue samples were obtained from the lesions and non-lesional skin at least 10 cm away from the lesions before and after the 3-week treatment.Skin specimens from 6 normal human controls served as the control.Patients were evaluated at the baseline,1 and 3 weeks after the beginning of treatment for clinical symptoms and signs by visual analogue scale (VAS),eczema area and severity index (EASI) and investigator's global assessment (IGA).The expression of PAR-2 in tissue specimens were determined by immunohistochemistry.Results PAR-2 was expressed throughout the whole epidermis,especially in the granular layer,hair follicles,sweat glands,endothelial cells and nerve fiber-like structures.Before treatment,the expression level (mean optical density) of PAR-2 in keratinocytes was 4339.6 ± 115.8 in lesional skin of AD patients,significantly higher than that in non-lesional skin (4189.0 t 228.9,t =2.85,P <0.05) and in normal skin (3864.0 ± 237.3,t =4.31,P < 0.05).After the 3-week treatment with tacrolimus ointment,the expression level of PAR-2 significant decreased to 3942.4 ± 176.6 in keratinocytes from lesional skin of patients with AD (t =4.55,P < 0.05).The expression level of PAR-2 was positively correlated with VAS score for itch,EASI and IGA score in the patients.Conclusions The expression of PAR-2 is enhanced in keratinocytes of lesions from AD patients,and is positively correlated with itch and lesion severity.Topical tacrolimus may suppress the overexpression of PAR-2 in keratinocytes in lesional skin of AD.

7.
Article in English | WPRIM | ID: wpr-187909

ABSTRACT

This study was performed in order to assess whether acute stress can increase mast cell and enterochromaffin (EC) cell numbers, and proteinase-activated receptor-2 (PAR2) expression in the rat colon. In addition, we aimed to investigate the involvement of corticotrophin-releasing factor in these stress-related alterations. Eighteen adult rats were divided into 3 experimental groups: 1) a saline-pretreated non-stressed group, 2) a saline-pretreated stressed group, and 3) an astressin-pretreated stressed group. The numbers of mast cells, EC cells, and PAR2-positive cells were counted in 6 high power fields. In proximal colonic segments, mast cell numbers of stressed rats tended to be higher than those of non-stressed rats, and their PAR2-positive cell numbers were significantly higher than those of non-stressed rats. In distal colonic segments, mast cell numbers and PAR2-positive cell numbers of stressed rats were significantly higher than those of non-stressed rats. Mast cell and PAR2-positive cell numbers of astressin-pretreated stressed rats were significantly lower than those of saline-pretreated stressed rats. EC cell numbers did not differ among the three experimental groups. Acute stress in rats increases mast cell numbers and mucosal PAR2 expression in the colon. These stress-related alterations seem to be mediated by release of corticotrophin-releasing factor.


Subject(s)
Animals , Male , Rats , Colon/metabolism , Corticotropin-Releasing Hormone/antagonists & inhibitors , Enterochromaffin Cells/cytology , Mast Cells/cytology , Peptide Fragments/pharmacology , Rats, Wistar , Receptor, PAR-2/metabolism , Restraint, Physical , Stress, Physiological
8.
Article in Korean | WPRIM | ID: wpr-40766

ABSTRACT

BACKGROUND: Serine protease promotes desquamatation of the stratum corneum and this is controlled by serine protease inhibitors (SPI). After disruption of the skin barrier, signals for barrier recovery are started with the activation of cytokines and a migration of calcium ions. On the other hand, the protease-activated receptor-2 (PAR-2) pathway is initiated as a negative signal. As the pH of the stratum corneum become neutral, activated serine protease and PAR-2 inhibit the secretion of lamellar bodies and the formation of the lamellar structure. OBJECTIVE: We wanted to screen noble synthetic peptides and identify the efficacy of a selected peptide, Palmitic acid-Lysine Threonine Threonine Lysine Serine (Pal-KTTKS), on PAR-2 in vitro and in vivo, and a clinical study was performed. METHODS: in vitro:Changes of the intracellular calcium ion concentration were measured in cultured HaCaT cells by fluorescence imaging according to treatment with sample peptides and trypsin. in vivo animal study:The efficacy of 2% Pal-KTTKS cream as a selected noble peptide was evaluated in an oxazolone-induced atopic dermatitis animal model. Clinical study:A total of twenty three atopic dermatitis patients applied 2.5% Pal-KTTKS peptide-containing cream on the one side of their extremities and pseudo-ceramide containing moisturizer on the other side of the extremities as a control twice a day for 4 weeks. Clinical improvement was evaluated by the Eczema Area Severity Index (EASI) score, a subject questionnaire and comparison of photographs. RESULTS: Suppression of the intracellular calcium concentration via PAR-2 inhibition was noted in the Pal-KTTKS peptide treated cultured HaCaT cells. In the oxazolone-induced atopic dermatitis hairless mice model, 2% Pal-KTTKS peptide containing lotion was more effective than vehicle lotion only. In the atopic dermatitis patients, the sites treated with 2.5% Pal-KTTKS peptide-containing cream showed better improvement for the EASI score, the subject questionnaire and the clinical photographs as compared to that of the control sites. There were no remarkable side effects related to the treatment. CONCLUSION: A PAR-2 inhibitor-containing topical agent would be an effective and safe modality for treating atopic dermatitis.


Subject(s)
Animals , Humans , Mice , Calcium , Cytokines , Dermatitis, Atopic , Eczema , Extremities , Hand , Hydrogen-Ion Concentration , Ions , Lysine , Mice, Hairless , Models, Animal , Oligopeptides , Optical Imaging , Peptides , Serine , Serine Proteases , Serine Proteinase Inhibitors , Skin , Threonine , Trypsin , Surveys and Questionnaires
9.
Chinese Journal of Dermatology ; (12): 818-820, 2008.
Article in Chinese | WPRIM | ID: wpr-397501

ABSTRACT

Objective To study the role of cutaneous nerve and protease activated receptor 2 (PAR2)in the development of pruritus in atopic dermatitis(AD).Methods Dermal sheets were prepared from chronically pruritic skin lesions of 7 patients with AD,as well as from the normal skin of 7 healthy human controls.Double labeled immunofluorescence was performed using mouse anti-protein gene product 9.5(PGP9.5)monoclonal antibody and rabbit anti-substance P(SP)polyclonal antibody to observe the morphological changes in cutaneous nerve fibers,and Image-Pro Plus 6 software was used to semiquantitively assess the length,diameter of nerve fibers,integral optimal density of PAR2 and SP in dermal sheets.Results Immunofluoresence double staining showed that PAR2 co-expressed with PGP9.5 or SP in cutaneous nerve fibers.Compared with the normal control skin,both the total length and average diameter of PGP 9.5-expressing nerve fibers were increased(11051.8±1900.9 μm vs 7264.0±2659.9 μm,4.23±0.15 μm vs 3.95±0.15 μm,both P<0.01)in pruritic lesions,while only the average diameter of SP-expressing nerve fibers was up-regulated(3.99±0.20 μm vs 3.80±0.07 μm,P<0.05),and the total length of them remained unchanged(4304.7±1455.0 μm vs 3380.0±1735.4 μm,P>0.05).Also,increased integral optimal density was observed for SP and PAR in pruritic lesions in comparison with the normal control skin (27.71±16.52 vs 12.63±4.31.35.99±8.63 vs 22.69±9.56.both P<0.05).Conclusion Our results indicate a hyper-plasia of cutaneous nerve fibers in chronic itchv skin lesions of AD and an increase in the expression of PAR2 and SP in the cutaneous nerve fibers,suggesting that the signal enhancement in PAR2 pathway may be related to the mechanism of pruritus in patients with AD.

10.
Article in Chinese | WPRIM | ID: wpr-574901

ABSTRACT

Objective To explore the potential relationship between the mast cells (MCs) in renal interstitium and the renal interstitial fibrosis in lupus nephritis (LN). Methods Renal biopsy specimens from patients with types Ⅲ,Ⅳand Vof LN (n=10, respectively), and with minimal change diseases (n=11,as control) were evaluated. Immunohistochemistry staining and immunofluorescence double-staining were used to detect the amount of MCs, the expression of proteinase-activated receptor-2 (PAR-2), transforming growth factor-?1 (TGF-?1) and collagen type I (Col I ) in the renal tissues. Results The amount of MCs in renal interstitium, the positive areas of PAR-2 and TGF-?1 in the renal tubular epithelial cells (RTECs), the amount of PAR-2-positive cells and TGF-?1-positive cells in renal interstitium, and the positive areas of Col I in the renal inter stitium were all higher in three LN groups compared with those in control. Furthermore, among the three LN groups, the above-mentioned parameters were the highest in type Ⅳ and second in type Ⅲ.There were significant positive correlations between the amount of MCs in renal interstitium and the positive areas of PAR-2, TGF-?1 in RTECs as well as the positive areas of Col I in renal interstitium (r=0.513, 0.508, 0.611, respectively, P

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