Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in Transwell^TM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. Transwell^TM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.
Humans ; Arthritis, Rheumatoid/genetics* ; Interleukin-23 ; Macrophages ; RNA, Messenger ; RNA, Small Interfering/genetics* ; Smad7 Protein/genetics* ; Transforming Growth Factor beta1/genetics* ; Smad3 Protein/genetics* ; Gene Silencing

OBJECTIVE: To observe the effect of electroacupuncture (EA) on vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), proinflammatory factors and apoptosis in myocardial tissue in mice with acute myocardial ischemia (AMI), and to explore the mechanism of EA for AMI. METHODS: Fifty male C57BL/6 mice were randomly divided into a sham operation group, a model group, an EA group, an inhibitor group and an inhibitor+EA group, 10 mice in each group. Except for the sham operation group, the mice in the remaining groups were intervented with ligation at the left anterior descending (LAD) coronary artery to establish AMI model. The mice in the sham operation group were intervented without ligation after thoracotomy. The mice in the EA group were intervented with EA at "Shenmen" (HT 7) and "Tongli" (HT 5), disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity, 30 min each time, once a day, for 3 d. The mice in the inhibitor group were treated with intraperitoneal injection of SAR 131675 (12.5 mg•kg^-1•d^-1, once a day for 3 d). The mice in the inhibitor+EA group were injected intraperitoneally with SAR 131675 30 min before EA. The ECG before modeling, 30 min after modeling and 3 d after intervention was detected, and the ST segment displacement was recorded; after the intervention, the ELISA method was applied to measure the contents of serum creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) as well as tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) in myocardial tissue; the HE staining method was used to observe the morphological changes of myocardial tissue; the immunofluorescence double labeling method was applied to measure the number of co-expression positive cells of VEGF-C/VEGFR-3 in myocardial tissue; the TUNEL method was used to detect the level of cardiomyocyte apoptosis; the Western blot method was applied to measure the protein expressions of VEGF-C, VEGFR-3, b-lymphoma-2 (Bcl-2), activated caspase-3 (Cleaved Caspase-3) and activated poly adenosine diphosphate ribose polymerase-1 (Cleaved PARP-1). RESULTS: Compared with the sham operation group, in the model group the ST segment displacement was increased (P<0.01); the contents of CK-MB, AST, TNF-α and IL-23 were increased (P<0.01); the arrangement of myocardial fibers was disordered, and interstitial inflammatory cell infiltration was obvious; the number of co-expression positive cells of VEGF-C/VEGFR-3 was decreased (P<0.01); the number of cardiomyocyte apoptosis was increased (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were decreased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were increased (P<0.01). Compared with the model group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.01); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). There was no significant difference in all the indexes between the model group and the inhibitor group (P>0.05). Compared with the model group, the protein expression of VEGF-C was increased in the inhibitor+EA group (P<0.01). Compared with the inhibitor group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.05); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). Compared with the inhibitor+EA group, all the indexes in the EA group were improved except the protein expression of VEGF-C (P<0.01). CONCLUSION EA could relieve the inflammatory reaction and apoptosis in AMI mice, and its mechanism may be related to activating VEGF-C/VEGFR-3 pathway and promoting lymphangion genesis.
Mice ; Male ; Animals ; Electroacupuncture ; Vascular Endothelial Growth Factor Receptor-3 ; Caspase 3 ; Vascular Endothelial Growth Factor C ; Tumor Necrosis Factor-alpha/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Poly(ADP-ribose) Polymerase Inhibitors ; Mice, Inbred C57BL ; Myocardial Ischemia/metabolism* ; Apoptosis ; Interleukin-23 ; Proto-Oncogene Proteins c-bcl-2

OBJECTIVE: To observe the expression of helper T cells 17(Th17), interleukin 23 (IL-23) in peripheral blood in patients with acute myeloid leukemia (AML), to analyze the relationship between Th17, IL-23 in peripheral blood and immunophenotype. METHODS: 105 patients with AML in the hospital from January 2019 to January 2021 were prospectively selected as the research subjects, the expression of Th17 and IL-23 in peripheral blood of patients with AML was detected by flow cytometry; immunophenotype was detected and counted. The relationship between the expression of Th17, IL-23 in peripheral blood and immunophenotype of AML patients was analyzed. Draw ROC curve and analyze the predictive value of Th17 and IL-23 expression in peripheral blood to immunophenotype. RESULTS: The immunophenotype results of AML patients showed that myeloid antigen, lymphoid antigen and hematopoietic stem/progenitor cell marker antigen were positive expressed for various antigens in 105 AML patients, in myeloid antigens, CD13⁺ accounted for the highest proportion (93.33%), in lymphoid antigens, CD56⁺ accounted for the highest proportion (32.38%), and in hematopoietic stem/progenitor cell marker antigens, CD38⁺ accounted for the highest proportion (68.57%). The expression of Th17 in peripheral blood of AML patients with CD56⁺, CD7⁺, CD34⁺ and human leukocyte antigen DR⁺(HLA-DR⁺) were higher than that of AML patients with CD56^-, CD7^-, CD34^-, HLA-DR^-, the expression of IL-23 in peripheral blood of AML patients with CD56⁺, CD34⁺ and HLA-DR⁺ were higher than that of AML patients with CD56^-, CD34^-, HLA-DR^-, the differences were statistically significant (P<0.05); compared the expression of Th17 and IL-23 in peripheral blood between other antibody positive and negative patients, there was no statistical significant difference (P>0.05). Logistic regression analysis showed that the high expression of Th17 in patients with AML was related to the positive expression of CD56, CD7, CD34 and HLA-DR in the detection of immunophenotype, the high expression of IL-23 was related to the positive expression of CD56, CD34 and HLA-DR in the detection of immunophenotype. The ROC curve showed that the AUC of expression levels of Th17 and IL-23 in peripheral blood alone and in combination for predicting CD56⁺, CD34⁺, HLA-DR⁺ and Th17 in peripheral blood for predicting CD7⁺ were mostly 0.5-0.7, which had certain predictive value, but the predictive performance was low. CONCLUSION Myeloid antigen, lymphoid antigen and hematopoietic hematopoietic stem/progenitor cell marker antigen are positive expressed for various antigens in AML patients, the high expression of Th17 in peripheral blood of AML patients is related to the positive expression of CD56, CD7, CD34 and HLA-DR in detection of immunophenotyping, the high expression of IL-23 is related to the positive expression of CD56, CD34 and HLA-DR in the detection of immunophenotype.
Antigens, CD34 ; Flow Cytometry/methods* ; HLA-DR Antigens/analysis* ; Humans ; Immunophenotyping ; Interleukin-23 ; Interleukin-23 Subunit p19/blood* ; Leukemia, Myeloid, Acute/genetics* ; Th17 Cells

OBJECTIVES: The mechanism for traditional Chinese medicine in treating of recurrent spontaneous abortion is not clear. This study aims to explore the mechanism of baotaiyin in the treatment of recurrent abortion by regulating the immune inflammatory axis of interleukin (IL)-23/helper T cell (Th)17. METHODS: Spontaneous abortion model mice were randomly divided into a model group, 3 dose (low, medium, and high) groups of baotaiyin, with 10 mice in each group. After 14 days of medication, the levels of IL-17, IL-23, IL-10, and TGF-β in serum were detected with enzyme-linked immunosorbent assay. The proportion of Th17 and regulatory T cells (Treg) cells in spleen lymphocytes was tested with flow cytometry. The expressions of (retinoid-related orphan receptor γt, ROR-γt) and forkhead box P3 (FOXP3) mRNA in decidua tissues was detected with RT-PCR. Embryo absorption rate was counted. RESULTS: Compared with the model group, the absorption rate of embryo and Th17/Treg cell ratio in baotaiyin medium- and high-dose groups were decreased significantly (all P<0.05); the levels of IL-17 and IL-23 in serum were decreased (both P<0.05), while the levels of TGF-β and IL-10 in baotaiyin medium- and high-dose groups were increased (P<0.05, P<0.01, respectively); the expression of ROR-γt mRNA was decreased and the expression of FOXP3 mRNA was increased (all P<0.01) in decidua tissues of baotaiyin medium- and high-dose groups. CONCLUSIONS Baotaiyin inhibits the positive feedback cycle of IL-23/Th17 immune inflammatory axis, which regulates Th17/Treg cell balance, mediates the maternal and fetal immune tolerance, and prevents the recurrent abortion.
Mice ; Animals ; Female ; Humans ; Pregnancy ; Interleukin-23 ; Interleukin-17/genetics* ; Interleukin-10 ; Abortion, Habitual ; Transforming Growth Factor beta/genetics*

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of interleukin (IL) -23/IL-17 axis and Toll-like receptor 4 (TLR4) in the infarcted tissue in rats with myocardial infarction (MI), and to explore the mechanism of EA on alleviating MI injury. METHODS: Forty male SD rats were randomly divided into a sham-operation group, a sham-operation plus EA group, a model group and an EA group, 10 rats in each group. The MI models were established by ligation of left anterior descending coronary artery in the model group and EA group, while only threading was performed in the sham-operation group and sham-operation plus EA group. The rats in the sham-operation plus EA group and EA group were treated with EA at "Neiguan" (PC 6), disperse-dense wave, 2 Hz/100 Hz, 2 mA, once a day, 20 min each time, for 3 days. After the intervention, the ejection fraction (EF) was measured by echocardiography to evaluate the cardiac function; the infarct area was measured by TTC staining; the HE staining was used to observe the morphological changes of myocardial tissue; the levels of IL-23 and IL-17 in infarcted tissue were detected by ELISA; the protein expression of TLR4 in infarcted tissue was detected by Western blot. RESULTS: Compared with the sham-operation group, the EF was decreased ( CONCLUSION EA may alleviate the excessive inflammatory response after MI by inhibiting the expression of IL-23/IL-17 axis in MI rats, and TLR4 may be involved during the process.
Animals ; Electroacupuncture ; Interleukin-17/genetics* ; Interleukin-23/genetics* ; Male ; Myocardial Infarction/therapy* ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4/genetics*

OBJECTIVETo investigate the levels of cytokines related to T-helper (Th) 17 cells in serum and signal transducers in the psoriatic lesions of patients with psoriasis vulgaris of blood-heat syndrome (BHS) and blood-stasis syndrome (BSS).

METHODSSixty patients with psoriasis vulgaris were divided into the BHS and BSS groups according to the syndrome differentiation of Chinese medicine (CM). Ten healthy subjects were considered as the control group. Cytokine levels of interleukin (IL)-17, IL-23 and IL-6 in serum were determined by enzyme-linked immunosorbent assay. Expression levels of signal transducer and activator of transcription 3 (STAT3), p38-mitogen-activated protein kinase (MAPK) and STAT6 in the psoriatic lesions were determined using immunohistochemistry (IHC), Western blot, and real-time quantitative reverse transcription polymerase chain reaction, respectively.

RESULTSProduction of IL-17, IL-23 and IL-6 in the BHS group and BSS group were significantly increased compared with those in the control group (P<0.05). Levels of IL-17 and IL-23 in the BHS group were higher than those in the BSS group (P<0.05). Compared with the control group, IHC positive expressions and protein expressions of STAT3 and p38-MAPK, and the STAT3 mRNA expressions in the BHS and BSS groups were significantly higher (P<0.05 or P<0.01). The protein expression of STAT3 in the BHS group was significantly higher than that in the BSS group (P<0.05).

CONCLUSIONSCytokines in serum and signal transducers in the psoriatic lesions alter with various CM syndromes of psoriasis. The results provide scientific basis for the treatment based on syndrome differentiation of CM in treating psoriasis vulgaris.

Adult ; Female ; Gene Expression Regulation ; Humans ; Immunohistochemistry ; Interleukin-17 ; blood ; Interleukin-23 ; blood ; Interleukin-6 ; blood ; Male ; Psoriasis ; blood ; enzymology ; genetics ; immunology ; RNA, Messenger ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; STAT6 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Syndrome ; Th17 Cells ; immunology ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-beta production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases.
Adjuvants, Immunologic/pharmacology/*therapeutic use ; Animals ; Caco-2 Cells ; Cell Proliferation ; Colitis, Ulcerative/*drug therapy/metabolism ; Colon/pathology ; Humans ; Interleukin-23/genetics/metabolism ; Intestinal Mucosa/drug effects/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Oligopeptides/pharmacology/*therapeutic use ; Permeability ; Receptors, Formyl Peptide/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/metabolism

To determine the impact of IL-23 knockdown by RNA interference on the development and severity of ovalbumin (OVA)-induced asthmatic inflammation, and the potential mechanisms in mice, the IL-23-specific RNAi-expressing pSRZsi-IL-23p19 plasmid was constructed and inhaled into OVA-sensitized mice before each challenge, as compared with that of control mice treated with alum or budesonide. Inhalation of the pSRZsi-IL-23p19, significantly reduced the levels of OVA-challenge induced IL-23 in the lung tissues by nearly 75%, determined by RT-PCR. In addition, knockdown of IL-23 expression dramatically reduced the numbers of eosinophils and neutrophils in BALF and mitigated inflammation in the lungs of asthmatic mice. Furthermore, knockdown of IL-23 expression significantly decreased the levels of serum IgE, IL-23, IL-17, and IL-4, but not IFNgamma, and its anti-inflammatory effects were similar to or better than that of treatment with budesonide in asthmatic mice. Our data support the notion that IL-23 and associated Th17 responses contribute to the pathogenic process of bronchial asthma. Knockdown of IL-23 by RNAi effectively inhibits asthmatic inflammation, which is associated with mitigating the production of IL-17 and IL-4 in asthmatic mice.
Animals ; Asthma/chemically induced/genetics/metabolism/*prevention & control ; Bronchoalveolar Lavage Fluid/cytology ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; Female ; Inflammation/metabolism ; Interleukin-23/*genetics ; Leukocyte Count ; Mice ; Mice, Inbred BALB C ; Neutrophils ; Ovalbumin/pharmacology ; Plasmids/genetics ; *RNA Interference ; RNA, Small Interfering/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Th17 Cells/immunology

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

OBJECTIVETo detect the expression levels of transcription factors and associated cytokines of Th17 and Treg cells in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer, and explore the possible pathological mechanism of these cells involved in the development of gastric cancer.

METHODSThe mRNA levels of RORgammat, FoxP3 in PBMC were determined by quantitative real-time PCR (QRT-PCR) from 57 patients with gastric cancer, 31 patients with benign gastric illness and 40 healthy people. The concentration of IL-17, IL-23, TGF-beta, IL-10 in plasma were detected by enzyme linked immunosorbent assay (ELISA).

RESULTSCompared with healthy volunteers, patients with gastric cancer showed higher levels of RORgammat and FoxP3 in PBMC (P < 0.05). The ratio of FoxP3/RORgammat in gastric cancer group was higher than that in the volunteer group and benign gastric illness group (P < 0.05). The ratio of FoxP3/RORgammat was higher in advanced disease than early disease (P < 0.05). The expressions of IL-17, IL-23, TGF-beta and IL-10 were higher in patients with gastric cancer than that in healthy volunteers (P < 0.05). In addition, The expression of TGF-beta and IL-10 were significantly increased in the advanced disease group than that in the early group (P < 0.05), but IL-17 and IL-23 was not significantly changed between the two groups (P > 0.05).

CONCLUSIONThere are higher levels of Th17 and Treg cells in gastric cancer patients, and it also shows a persistent predominant tendency of Treg cells and a reduced tendency of Th17 cells in advanced disease. Detecting the expression of Th17/Treg transcription factor and related cytokines would contribute to the diagnosis and prediction of the disease development and prognosis.

Adult ; Aged ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Gastritis ; blood ; metabolism ; pathology ; Humans ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; Interleukin-23 ; blood ; Male ; Middle Aged ; Neoplasm Staging ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; blood ; metabolism ; pathology ; T-Lymphocytes, Regulatory ; metabolism ; Th17 Cells ; metabolism ; Transforming Growth Factor beta ; blood