TRPA1 in the spinal dorsal horn is involved in post-inflammatory visceral hypersensitivity: in vivo study using TNBS-treated rat model.

INTRODUCTION: The transient receptor potential ankyrin-1 (TRPA1) channel, a pain transducer and amplifier, is drawing increasing attention in the field of visceral hypersensitivity, commonly seen in irritable bowel syndrome and inflammatory bowel disease. However, the role of TRPA1 in visceral nociception during post-inflammatory states is not well defined. Here, we explore the correlation between TRPA1 expression in the spinal dorsal horn (SDH) and persistent post-inflammatory visceral hypersensitivity. METHODS: We injected rats intracolonically with 2,4,6-trinitrobenzene sulfonic acid (TNBS) or vehicle (n=12 per group). Post-inflammatory visceral hypersensitivity was assessed by recording the electromyographic activity of the external oblique muscle in response to colorectal distension. TRPA1 expression and distribution in the spinal cord and colon were examined by Western blotting and immunohistochemistry. RESULTS: Animals exposed to TNBS had more abdominal contractions than vehicle-injected controls (P<0.05), which corresponded to a lower nociceptive threshold. Expression of TRPA1 in the SDH (especially in the substantia gelatinosa) and the colon was significantly greater in the TNBS-treated group than in controls (P<0.05). In the SDH, the number of TRPA1-immunopositive neurons was 25.75±5.12 in the control group and 34.25±7.89 in the TNBS-treated group (P=0.023), and integrated optical density values of TRPA1 in the control and TNBS-treated groups were 14,544.63±6,525.54 and 22,532.75±7,608.11, respectively (P=0.041). CONCLUSION: Our results indicate that upregulation of TRPA1 expression in the SDH is associated with persistent post-inflammatory visceral hypersensitivity in the rat and provides insight into potential therapeutic targets for the control of persistent visceral hypersensitivity.

[Role and mechanism of NKp44+NK cells in the proliferation and inflammation of synovium of rheumatoid arthritis patients].

OBJECTIVE: To explore the effects of NKp44+NK cells from rheumatoid arthritis (RA) patients on the proliferation and monocyte chemotactic protein 1 production of fibroblast-like synoviocytes (FLS). METHODS: The proportions of natural killer (NK) p44 NK cells in peripheral blood (PB) of 50 RA patients and 50 healthy individuals were detected by flow cytometry. Synovial fluid (SF) samples from 30 RA patients were also detected. NKp44+NK cells in RA SF were sorted by flow cytometry for 5 times. The supernatant level of interleukin (IL)-22 was measured by enzyme-linked immunosorbent assay (ELISA). The proliferation of FLS after an addition of culture supernatant of NKp44+NK cells was detected by methyl thiazolyl tetrazolium (MTT) at 24, 48 and 72 h. Monocyte chemotactic protein (MCP)-1 production of RA FLS after an addition of rhIL-22 was detected by ELISA. RESULTS: The proportion of NKp44+NK cells in PB of RA patients was significantly higher than that of normal controls while the proportion of NKp44+NK cells in SF of RA patients was higher than that in PB of matched RA patients (1.270% vs 0, 15.190% vs 2.425%, P < 0.01). The supernatant level of IL-22 in NKp44+NK cell culture was (1 603 ± 332) ng/L. Rapid proliferation of RA FLS was observed at 24, 48, 72 h after an addition of culture supernatant (P < 0.01). IL-22 antibody obviously inhibited the proliferation of RA FLS induced by NKp44+NK cells. MCP-1 production of RA FLS was detected at 72 h after an addition of rhIL-22 (P < 0.01). CONCLUSION: NKp44+NK cells can promote the proliferation and MCP-1 production of RA FLS through the production of IL-22 so as to play an important role in the synovial proliferation and inflammation of RA.

HSP27 as a biomarker for predicting skin irritation in human skin and reconstructed organotypic skin model.

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessing parameters or endpoints are still not sufficient. To discover novel endpoints for skin irritation responses, 2DE-based proteomics was used to analyze the protein expression in human skin exposed to sodium lauryl sulfate (SLS) following the test protocol of the European Centre for the Validation of Alternative Methods (ECVAM) in the present study. HSP27 was up-regulated most significantly among the eight identified proteins, consistent with our previous reports. Acid and basic chemicals were applied on human skin for further validation and results showed that the up-regulated expression of HSP27 was induced in 24h after the exposure. Skin-equivalent constructed with fibroblasts, basement membrane and keratinocytes was used to investigate the potential of HSP27 as a biomarker or additional endpoint for the hazard assessment of skin irritation. Our skin-equivalent (Reconstructed Organotypic Skin Model, ROSM) had excellent epidermal differentiation and was suitable for the skin irritation test. HSP27 also displayed an up-regulated expression in the ROSM in 24h after the irritants exposure for 15min. All these results suggest that HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

[Role of NK-22 cells and interleukin-22-related molecules in proliferation of fibroblast- like synoviocytes in patients with rheumatoid arthritis].

OBJECTIVE: To investigate the role of natural killer-22 (NK-22) cells in the synovial fluid in the proliferation of fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA) and explore the possible signal pathway involved. METHODS: NK-22 cells in the SF of RA patients were sorted by flow cytometry. NK-22 cells were cultured for two weeks and the purity was detected by flow cytometry before stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 µmol/L ionomycin for 4 h. The level of interleukin-22 (IL-22) in the culture medium supernatant was then measured with ELISA. The proliferation of FLS in the presence of the culture supernatant of NK-22 cells was assessed with MTT assay at 24, 48 and 72 h, and the effect of IL-22 antibody on FLS proliferation was also observed. Real-time PCR and Western blotting were used to detect Stat3 mRNA and p-Stat3 protein levels, respectively, in the FLS exposed to rhIL-22 and AG490. RESULTS: NK-22 cells were successfully sorted by flow cytometry with a purity exceeding 90%. The levels of IL-22 in the supernatant of NKp44(+)NK cell culture averaged 1273.42∓254.48 pg/ml. The FLS proliferated rapidly 24, 48, and 72 h after the addition of culture supernatant of NK-22 cells (P<0.05). IL-22 antibody obviously inhibited the proliferation of FLS induced by NK-22 cell culture supernatant (P<0.05). Exposure of the FLS to rhIL-22 obviously increased cellular Stat3 expression levels, which were significantly lowered by the addition of AG490 (P<0.05). CONCLUSION: NK-22 cells in the SF of RA patients can produce high concentrations of IL-22 to promote the proliferation of FLS through the STAT3 signal pathway.

Identification of potential biomarkers for predicting acute dermal irritation by proteomic analysis.

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessment parameters or endpoints are still not sufficient for analysis. To discover novel endpoints for skin irritation responses, a proteomics approach was used to analyze the protein expression in human keratinocytes exposed to sodium lauryl sulfate in the present study. Among the 20 identified proteins with altered expression, small heat shock protein 27 (HSP27) and superoxide dismutase [Cu-Zn] were down-regulated while cofilin-1 was up-regulated significantly in response to the chemical challenge. Keratinocytes were exposed to acid and basic chemicals for further validation of the proteins. HSP27 displayed the most significant alteration both in mRNA and protein levels, accompanied by nuclear translocation. The irritation also induced an increased production of interleukin-1α in keratinocytes. These findings suggest that these proteins may be combinational biomarkers or additional endpoints for skin hazard assessment. Further investigation into the protein alterations would be helpful for the mechanistic understanding of skin irritation.