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Objective:To preliminary study the effects of long noncoding Ribonuclei Acid (RNA) small nucleolar RNA host gene 1 (SNHG1) on proliferation of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and the possible mechanism.Methods:The FLS from RA and trauma group were primarily cultured by the explant culture, and the expression of SNHG1 were detected by quantitative polymerase chain reaction (qPCR). Transfection of siRNA was used to interfere the expression of SNHG1. Cell viability was measured using CCK-8 assay and cell cycle distribution was detected by flow cytometry. And the protein level of cyclinD1 was detected by Western blotting. Independent sample t test was used for the comparison between two groups, and the one-way analysis of variance (ANOVA) analysis was used to compare the samples of multiple groups. Results:Compared with FLS from trauma group, SNHG1 expression in RA-FLS was up-regulated [(2.13 ±0.55) vs (1.00 ±0.01)] ( t=-5.87, P=0.004). In RA-FLS, after silencing the expression of SNHG1, the cell viability of SNHG1-siRNA treatment group was down-regulated [(0.930 ±0.033) vs (0.759 ±0.027)]( t=6.879, P=0.002), the proportion of G 2/M+S cells was down-regulated [(28.2 ±1.5)% vs (9.7 ±2.6)%]( t=10.715, P<0.01), and thelevelof cyclinD1 protein was down-regulated ( t=6.168, P=0.004) compared with the negative control group. Conclusion:The SNHG1 is abnormally expressed in RA-FLS, and SNHG1 may participate in the regulation of FLS proliferation by affecting the expression of cyclinD1 protein, thereby contributing to synovial hyperplasia.
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Objective To evaluate the relationship between spinal Sonic hedgehog (Shh) signaling pathway and inflammatory responses in rats with neuropathic pain (NP).Methods Forty-eight cleangrade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were divided into 4 groups (n=12 each) using a random number table method:sham operation group (group S),group NP,dimethyl sulfoxide (DMSO) group (group D) and specific Shh signaling pathway inhibitor cyclopamine group (group CP).Spared nerve injury was produced by exposing the sciatic nerve and branches followed by ligation and transection of tibial and common fibular nerves in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before establishing the model and 1 and 7 days after establishing the model.,The animals were sacrificed after measurement of pain threshold at 7 days after operation,and the lumbar segment L4-6 of the spinal cord was obtained for determination of the expression of Shh,Patched homolog (Ptch),Smoothened (Smo) and zinc finger-containing transcription factors 1 (Gli1) (by Western blot) and contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay).Results Compared with group S,the MWT was significantly decreased,the expression of Shh,Patched,Smo and Gli1 was up-regulated,and the contents of TNF-α and IL-1β were increased in NP,D and CP groups (P＜0.05).Compared with group NP,the MWT was significantly increased,the expression of Shh,Patched,Smo and Gli1 was down-regulated,and the contents of TNF-α and IL-1β were decreased in group CP (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＜0.05).Conclusion The mechanism by which Shh signaling pathway is involved in the development and mainterance of inflammatory responses is related to inhibiting inflammatory responses of the spinal cord in rats with NP.
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Objective To investigate the expression of fibroblast growth factor 4 (FGF4) in serum of active rheumatoid arthritis (RA) and its role in RA synoviocyte proliferation. Methods The serum level of FGF4 were detected by protein arrays in 20 patients with RA, and 20 age and gender matched healthy controls. FLSs were isolated from RA synovium,and were co-cultured with recombinant human FGF4 (rhFGF4). Cell proliferation was quantified by Cell Counting Kit-8 assay and cell cycle distribution was evaluated by flow-cytometry. The protein levels of cyclin D1, phospho-Akt (p-Akt) and phospho-p38 (p-p38) were measured by western blot. Results The serum expression of FGF4 in RA group was higher than that in control group (P=0.041). After being treated with different concentrations of rhFGF4 (12.5, 25, 50, and 100 ng/ml), RA-FLS showed significant increase in cell proliferation, with different rates of [(121 ±8)％], [(126 ±12)％], [(129 ± 12)％], a nd [(134 ±14)％] respectively, comparing with that of the controls [(100 ±0)％, (P12.5=0.049, P25=0.009, P50=0.004, P100=0.001).]. Among them, the percentage of G2/M+S phase cells were [(12.6±3.6)％], [(15.3±4.5)％], [(17.1±5.1)％], [(19.6±4.1)％] respectively, and except the lowest rhFGF4 concentration treatment group of 12.5 ng/ml, G2/M+S phase cells in other groups was significantly increased compared with the controls [(5.4±2.4)％] (P12.5=0.159, P25=0.042, P50=0.018, P100=0.005). And the protein expression of cyclin D1 was up-regulated after being treated with 50 ng/ml and 100 ng/ml rhFGF4 (P50=0.035, P100=0.027). FGF4 transiently increased the expression of p-Akt and p-p38 protein at the concentration of 50 ng/ml. Comparisons of data between groups were performed by independent sample Student's t-test. Statistical significant differences among groups were tested by one-way analysis of variance (ANOVA) or the Kruskal-Wallis test. The Dunnett's t-test was used for multiple comparisons. A P-value of <0.05 was considered statistically significant. Conclusion Our results suggest that FGF4 is highly expressed in the serum of active RA patients. FGF4 may promote the proliferation of RA-FLS via modulating PI3K/Akt and p38-MAPK signaling pathways, which subsequently contributs to synovial hyperplasia.
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Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.
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Objective To evaluate the role of Sonic hedgehog (Shh) signaling pathway in the spinal cord in neuropathic pain (NP) in the rats.Methods Seventy-two male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly divided into 2 groups (n=36 each) using a random number table:sham operation group (S group) and NP group.Spared nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured before operation and at 1,4,7,14 and 21 days after operation.After measurement of the pain threshold at 1,4,7,14 and 21 days after operation,the animals were then sacrificed,and the lumbar segment (L46) of the spinal cord was obtained for determination of Shh,Patched (Ptch),Gli1 and glial fibrillary acidic protein (GFAP)expression (by Western blot),Shh,Ptch and Gli1 mRNA expression (by fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction),and Shh and GFAP expression (by immunohistochemistry).Results Compared with group S,the MWT was significantly decreased,and the expression of Shh,Ptch and Gli1 protein and mRNA and GFAP in spinal cord tissues was up-regulated in group NP (P＜ 0.05).Shh was mainly expressed in the cytoplasm of spinal dorsal horn neurons and in the gap around glial cells.Conclusion Shh signaling pathway in spinal cord is involved in the development and maintenance of NP in the rats.