OBJECTIVE: To investigate the role of IL-17A in promoting the activation of lung fibroblasts and the secretion of chemokine CXCL12, and to explore the possible mechanism. METHODS: Lung tissues of BALB/c mice were collected after intraperitoneal injection of recombinant mouse IL-17A (rmIL-17A). Real-time RT-PCR and Western blotting were used to detect the expression levels of α-smooth muscle actin (α-SMA) and collagen I in lung tissues, and immunohistochemical staining and real-time RT-PCR were used to determine the expression of CXCL12. Normal mouse primary lung fibroblasts were isolated and cultured, and identified by immunofluorescence staining with optical microscopy. Cells and supernatant of culture medium were collected after stimulation with rmIL-17A at different concentrations. mRNA levels of α-SMA, collagen I, and CXCL12 in the cells were determined by real-time RT-PCR, and the levels of collagen I and CXCL12 in the supernatant of culture medium were determined by ELISA. RESULTS: The mRNA and protein levels of α-SMA and collagen I in the lung tissue of mice injected with rmIL-17A were significantly increased compared with the control group (all CONCLUSIONS s: IL-17A can promote the activation of lung fibroblasts and translation into myofibroblast. The secretion of collagen is increased, which promote the deposition of extracullular matrix, and leads to the occurrence and development of lung fibrosis. CXCL12, a chemokine secreted by activated fibroblasts, may be involved in this process.
Actins/genetics* ; Animals ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Fibroblasts/metabolism* ; Interleukin-17/pharmacology* ; Lung/metabolism* ; Mice ; Mice, Inbred BALB C

OBJECTIVETo observe the effect of shikonin on the proliferation of human keratinocytes induced by IL-17 and secretion of chemokines, in order to discuss the mechanism of Shikonin in the treatment of psoriasis.

METHODIn vitro cultured HaCaT cells were stimulated by IL-17A (200 μg x L(-1)) and mixed with different concentrations (2, 1 mg x L(-1)) of shikonin for 24 hours. The cell proliferation was detected by CCK-8 assay. Cell secretion inflammatory factor interleukin-23 (IL-23) was detected by ELISA. The expressions of intracellular chemokines CXCL1, CXCL2, CCL20 and 6-defensin 4 (DEFB4) were detected by Real-time PCR.

RESULTShikonin (2,1 mg x L(-1)) could distinctly inhibit HaCaT cell proliferation induced by IL-17A, with statistical difference (P < 0.01). Each shikonin group showed decreases in the secretion of IL-23 and inhibition in expressions of intracellular CXCL1, CXCL2, CCL20 and DEFB4.

CONCLUSIONShikonin could inhibit HaCaT cells proliferation induced by IL-17 and secretion of relevant cytokines and recruit leukocytes by inhibiting chemokines, so as to show the effect in treating psoriasis.

Cell Line ; Cell Proliferation ; drug effects ; Chemokines ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interleukin-17 ; genetics ; metabolism ; Keratinocytes ; cytology ; drug effects ; metabolism ; Naphthoquinones ; pharmacology

OBJECTIVETo investigate the effects of fulvotomentoside (Ful) on inflammatory factors and antiinflammatory factors in intestine of ovalbumin (OVA)-sensitized BALB/c mice, and to explore the mechanisms of its anti-food allergy effect.

METHODTwenty-four female BALB/c mice aged 6 weeks fed with ovalbumin-free feed were randomly divided into 3 groups, food allergy (FA) group, Ful group and normal saline (NS) group. Mice in FA and Ful groups were sensitized intraperitoneally two times with OVA and challenged intragastrically with OVA. Mice in Ful group were treated with 200 mg/kg of Ful by subcutaneous injection once daily for 22 days. The mice in FA and NS groups were used as positive control and negative control, respectively, and were treated with normal saline solution by subcutaneous injection for 22 days. Just 48 hours after the last challenge, the mice in each group were sacrificed and specimens of jejunum were taken. The mRNA expressions of transforming growth factor β1 (TGF-β1), interleukin-6 (IL-6), interleukin-17A (IL-17A) and forkhead box P3 (Foxp3) in jejunum were detected by reverse transcription-PCR (RT-PCR). The protein expressions of TGF-β1, IL-6, and IL-17A in jejunum were detected by immunohistochemical method. The activation of neutrophils in jejunum was assayed by the levels of MPO.

RESULTThe expressions of TGF-β1, IL-6, IL-17A mRNA [(0.370 ± 0.013), (0.475 ± 0.015), (0.541 ± 0.013)] and related protein [(53,075.70 ± 20,727.06), (256,881.66 ± 36,561.79), (435,064.25 ± 69,911.48)] in jejunum were increased and the Foxp3 mRNA [(0.231 ± 0.014) vs. (0.365 ± 0.015)] expression was decreased in group FA. After the treatment with Ful, IL-6 and IL-17A mRNA [(0.196 ± 0.005), (0.204 ± 0.008)] and protein [(114,040.30 ± 20,295.25), (218,200.74 ± 30,077.69)] expressions were decreased and Foxp3 mRNA (0.578 ± 0.021) expression was increased, and no change of TGF-β1 expression was unchanged. There were no significant differences of the levels of MPO among the three groups.

CONCLUSIONInflammatory reaction which was characterized by the increase of IL-6 and IL-17A expressions was found in intestine of ovalbumin-sensitized BALB/c mice. Ful could decrease overexpression of IL-6 and IL-17A, and increase the expression of specific transcription factor Foxp3 of regulatory T cells significantly in intestine. It may be one of the mechanisms that Ful improved intestinal inflammation.

Animals ; Female ; Food Hypersensitivity ; metabolism ; Forkhead Transcription Factors ; metabolism ; Inflammation ; Interleukin-17 ; metabolism ; Interleukin-6 ; metabolism ; Intestines ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Ovalbumin ; adverse effects ; Saponins ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo evaluate the expression and possible modulation of heme oxygenase-1 (HO-1) in nasal polyps of patients with chronic rhinosinusitis with nasal polyps (CRSwNP).

METHODSNasal polyps and uncinate process tissues were collected from 25 CRSwNP patients and 19 healthy controls with nasal septal deviation. HO-1 expression was examined using qRT-PCR, immunohistochemistric staining and Western blot analysis. Moreover, additional uncinate process mucosal samples of 15 healthy controls with nasal septal deviation were harvested for nasal explant culture experiments. HO-1 expression was measured in cultured nasal explant in response to specific inflammatory and glucocorticoid stimulation. SPSS 20.0 software was used to analyze the data.

RESULTSThe mRNA and protein expression of HO-1 was significantly increased in polyp tissues, 1.220±0.397 in mRNA and 1.409±0.701 in protein, compared with healthy controls 0.464±0.318 in mRNA and 0.017±0.1147 in protein (U=22.00 in mRNA and U=1.00 in protein, both P< 0.05). The immunohistochemical results showed that HO-1 was mainly distributed in the epithelial layer, submucosal glands and inflammatory cells in nasal tissues. Nasal explant culture experiments demonstrated that HO-1 mRNA was upregulated by IL-17A. The HO-1 mRNA level before the stimulation was 1.000, and 17.264±4.275 after the stimulation of 1 ng/ml IL-17A (U=0, P<0.05), 19.128±4.605 after the stimulation of 10 ng/ml IL-17A (U=0, P<0.05), but was significantly suppressed after stimulation with glucocorticoids (dexamethasone, DEX). The mRNA level after the glucocorticoids stimulation was 0.370±0.101 (U=0, P<0.05) and 0.316±0.167 (U=0, P<0.05) respectively. Furthermore, the HO-1 mRNA was inhibited by TGF-β1, the mRNA level was 0.217±0.322 (U=0, P<0.05), 0.070±0.070 (U=0, P<0.05), respectively.

CONCLUSIONIncreased HO-1 expression may play a role in the pathogenesis of CRSwNP, which may be considered as the therapeutic target.

Blotting, Western ; Case-Control Studies ; Chronic Disease ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Heme Oxygenase-1 ; metabolism ; Humans ; Interleukin-17 ; pharmacology ; Nasal Polyps ; complications ; metabolism ; RNA, Messenger ; metabolism ; Rhinitis ; complications ; metabolism ; Sinusitis ; complications ; metabolism ; Tissue Culture Techniques ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of ginseng-derived compound K (C-K) on apoptosis, immunosuppressive activity, and pro-inflammatory cytokine production of myeloid-derived suppressor cells (MDSCs) from mice bearing colorectal cancer xenograft.

METHODSFlow-sorted bone marrow MDSCs from Balb/c mice bearing CT26 tumor xenograft were treated with either C-K or PBS for 96 h and examined for apoptosis with Annexin V/7-AAD, Cox-2 and Arg-1 expressions using qRT-PCR, and supernatant IL-1β, IL-6, and IL-17 levels with ELISA. C-K- or PBS-treated MDSCs were subcutaneously implanted along with CT26 tumor cells in WT Balb/c mice, and the tumor size and morphology were evaluated 21 days later.

RESULTSC-K treatment significantly increased the percentages of early and late apoptotic MDSCs in vitro (P<0.01 and P<0.05, respectively), decreased the expressions of immunosuppression-related genes Cox-2 (P<0.05) and Arg-1 (P<0.01), and suppressed the production of IL-1β (P<0.05), IL-6 (P<0.01), and IL-17 (P<0.05) by the MDSCs . Compared with PBS-pre-treated cells, C-K-pretreated MDSCs showed significantly attenuated activity in promoting CT26 tumor growth in mice (P<0.01).

CONCLUSIONC-K can suppress the immunosuppresive effect of MDSCs to inhibit tumor cell proliferation in mice, which suggests a new strategy of tumor therapy by targeting MDSCs.

Animals ; Apoptosis ; Cell Proliferation ; Colorectal Neoplasms ; pathology ; Disease Models, Animal ; Ginsenosides ; pharmacology ; Humans ; Immunosuppression ; Interleukin-17 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Myeloid Cells ; drug effects ; Neoplasm Transplantation

OBJECTIVETo generate engineering Th17 cells from mice CD4(+)CD25(-) naïve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Th17 cells.

METHODSRecombinant lentivirus carrying mouse RORγt (pXZ9-RORγt) and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4(+)CD25(-) naïve T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-β plus IL-6 and divided into five groups: pXZ9-RORγt (group A), pXZ9 + TGF-β + IL-6 (group B), pXZ9-RORγt + TGF-β + IL-6 (group C), pXZ9 (group D) and control (group E). Production efficiency of engineering Th17 cells was referred as the percentage of IL-17A producing cells. Cytokine production profiles of these cells were assayed by realtime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis (EAE).

RESULTS(1) High-title lentivirus was prepared and was succeeded to transduce CD4(+)CD25(-) naïve T cells. Forced expression of RORγt (group A) resulted in (40.25 ± 5.46)% CD4(+)CD25(-) naïve T cells converted into engineering Th17 cells and the convert efficiency increased to (60.59 ± 8.15)% in addition of TGF-β and IL-6 (group C), or decreased to (14.36 ± 5.27)% when presence of TGF-β and IL-6 only (group B). (2) IL-17A, IL-17F and IL-21 production of pXZ9-RORγt infected cells combined with TGF-β and IL-6 were most similar to natural Th17 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. (3) Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized cells and polarized of RORγt transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models.

CONCLUSIONHigh yield of engineering Th17 cells was prepared from CD4(+)CD25(-) naïve T cells by over expression RORγt plus TGF-β and IL-6 polarization. These engineering Th17 cells were similar to the natural Th17 cells in phenotypes and functional identification.

Animals ; Cells, Cultured ; Genetic Techniques ; Interleukin-17 ; pharmacology ; Interleukin-6 ; pharmacology ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; Th17 Cells ; cytology ; metabolism ; Transforming Growth Factor beta ; pharmacology

OBJECTIVE: To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect. METHODS: We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells. RESULTS: The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01). CONCLUSION The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.
Arthritis, Rheumatoid/pathology* ; Cells, Cultured ; Diterpenes/pharmacology* ; Epoxy Compounds/pharmacology* ; Fibroblasts/pathology* ; Humans ; Interleukin-17/metabolism* ; Interleukin-4/metabolism* ; Interleukin-6/metabolism* ; Janus Kinase 2/metabolism* ; Leukocytes, Mononuclear/metabolism* ; Phenanthrenes/pharmacology* ; RNA, Circular/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Synovial Membrane/pathology*

OBJECTIVETo observe the effect of Sanshui Baihu Decoction (SBD) containing serum on the proliferation of in vitro cultured fibroblast-like synoviocytes (FLS) derived from rheumatoid arthritis (RA) and osteoarthritis (OA) and its secretion of interleukin-6 (IL-6) and IL-17, and to explore the pharmacological mechanism of SBD.

METHODSThe FLS obtained from cultured RA and OA patients' synovial tissue were cultured and passaged in vitro in a routine way. The cultured medium was changed to DMEM with 20% SBD containing serum and cultured for 72 h after cultured for 3 to 6 generations. The proliferation rate of FLS was detected by MTT assay. Levels of IL-6 and IL-17 in the supernatant were detected by ELISA. Leflunomide and saline containing serum were used as positive and negative control respectively.

RESULTSSBD containing serum significantly inhibited the proliferation of RA-FLS and OA-FLS, and decreased the secretion of IL-17 in RA-FLS. Its inhibition efficiency of SBD was equivalent to that of Leflunomide. No obvious inhibition on the secretion of IL-6 in RA-FLS was observed. It had no significant effect on the secretion of IL-17 and IL-6 in OA-FLS.

CONCLUSIONSBD could inhibit the proliferation of FLS and the secretion of IL-17 in RA-FLS, which might be one of its pharmacological mechanisms for treating RA.

Animals ; Arthritis, Rheumatoid ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Fibroblasts ; drug effects ; secretion ; Humans ; Interleukin-17 ; metabolism ; Interleukin-6 ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; drug effects

OBJECTIVETo explore the molecular mechanism of exocrine immune inflammatory injury of Sjögren's Syndrome and the intervention of Banxia Qinlian Decoction (BQD).

METHODSTotally 18 female NOD mice were randomly divided into the model group, the positive drug group, and the BQD group, 6 in each group. Six female BALB/c mice were recruited as a blank control group. Mice in the blank control group and the model group were gavaged with deionized water at the daily dose of 0.1 mL/10 g body weight. Tripterygium Tablet was administered by gastrogavage to mice in the positive group at the daily dose of 10 mg/kg. BQD was administered by gastrogavage to mice in the BQD group at the daily dose of 60 g crude drugs/kg. After 12 weeks of medication, mice were sacrificed. Their eyeballs were excised and blood collected. Tissues of bilateral parotids and submandibular glands were kept. mRNA transcriptional levels of IL-17, IL-6, type 3 muscarinic acetylcholine receptors (M3R), aquaporin protein-5 (AQP5) were detected by RT-PCR. Expression levels of M3R and AQP5 protein were detected by Western blot. Protein expression levels of IL-17 and IL-6 were detected by ELISA.

RESULTSCompared with the normal group, mRNA transcriptional levels and protein expression levels of IL-17, IL-6, M3R, and AQP5 were significantly up-regulated in the model group (P < 0.01). Compared with the model group, mRNA transcriptional levels and protein expression levels of IL-17, IL-6, M3R, and AQP5 were significantly down-regulated in the positive drug group and the BQD group with statistical difference (P < 0.01, P < 0.05). Compared with the BQD group, mRNA-transcriptional levels of IL-17, IL-6, and M3R, as well as M3R and AQP5 protein expression levels were significantly down-regulated in the positive drug group (all P < 0.01).

CONCLUSIONThe molecular mechanism of BQD in inhibiting SS exocrine neurotoxic injury might be possibly related to regulating Th17/IL-17 immune inflammatory way.

Animals ; Aquaporin 5 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Interleukin-17 ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Sjogren's Syndrome ; drug therapy ; immunology ; Submandibular Gland ; Th17 Cells ; Up-Regulation

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.
Adaptor Proteins, Vesicular Transport/genetics/metabolism ; Arthritis, Rheumatoid/*metabolism ; Blotting, Western ; Cells, Cultured ; Fibroblasts/drug effects/*metabolism ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/*metabolism ; Interleukin-12/pharmacology ; Interleukin-16/pharmacology ; Interleukin-17/pharmacology ; Interleukin-23/pharmacology ; Lipopolysaccharides/pharmacology ; Myeloid Differentiation Factor 88/genetics/*metabolism ; Poly I-C/pharmacology ; Polymerase Chain Reaction ; RNA, Small Interfering/genetics/physiology ; Synovial Membrane/*cytology ; Toll-Like Receptor 4/genetics/metabolism ; Tumor Necrosis Factor-alpha/pharmacology