Oxymatrine ameliorates rheumatoid arthritis by regulation of Tfr/Tfh cell balance via the TLR9-MyD88-STAT3 signaling pathway.

BACKGROUND: Oxymatrine (OMT) is one of the authentic Chinese herbal medicines which has rich and complex active ingredients. however,the relevant potential targets of oxymatrine on rheumatoid arthritis and the mechanism remains unreported. The aim of this study was to determine the regulation of oxymatrine on rheumatoid arthritis using a collagen-induced arthritis (CIA) mice models and blood samples from RA patients. RESULTS: Our results indicated that Tfr cells in RA patients express low levels of Blimp-1 and CTLA-4. Oxymatrine treatment of CIA mice alleviated joint swelling, reduced the arthritis score, and improved joint damage. While flow cytometry results showed that oxymatrine treatment reduced Tfh cells and B cells, and increased Tfr cells in the spleen of CIA mice. In addition, oxymatrine treatment significantly down-regulated the expression of TLR9（Toll-like Receptors 9), IL-21, MyD88, STAT3, p-STAT3, and CXCR5 in the synovial tissues of CIA mice, and up-regulated the expression of Foxp3, Blimp-1, and CTLA-4. CONCLUSION: Oxymatrine can alleviate rheumatoid arthritis by regulating the TLR9-MyD88-STAT3 signaling pathway to maintain immune balance between Tfr-Tfh cells. © 2023 Society of Chemical Industry.

Case Report of a Female Patient with Adult-onset Still's Disease and Review of The Literature.

BACKGROUND: Adult-onset Still's disease (AOSD), which presents many non-specific symptoms, such as rash leukocytosis, spiking fever, and sore throat, is a rare auto inflammatory disease. Other clinical features that are frequently observed include lymphadenopathy, arthralgia, serositis, splenomegaly, and hepatomegaly. Laboratory tests show high levels of C-reactive protein, ferritin, and erythrocyte sedimentation rate reflecting the systemic inflammatory process in AOSD patients. CASE PRESENTATION: The patient was a middle-aged woman with a high fever (39.8 C), sore throat, rashes on limbs with pruritus, mainly at the joints (elbow, knee, and ankle), muscle aches, dizziness, infirmity, weakness, and poor appetite without arthralgia. The ferritin level was above 1500 (normal value: 14-233) ng/L. Antineutrophil, antinuclear antibodies, and rheumatoid factor were negative. Combining the symptoms such as fever, rash, stress-induced acute inflammation, arthritis, and ferritin levels, the patient was eventually diagnosed with adult Still's disease. She received methylprednisolone 40mg intravenously every 12 hours for one week. On the second week, the dose was reduced to 40mg in the morning and 20mg in the evening, and finally, the dose was reduced to 40mg oral intake in the morning and 8mg in the evening. After half a month of treatment, the patient's high fever and skin rashes subsided, and the other symptoms also gradually relieved. CONCLUSIONS: A case of a middle-aged woman diagnosed with adult Still's disease is reported, and the possible pathogenesis and treatment of the disease are discussed. This case highlights the importance of early diagnosis and timely treatment of adult Still's disease to prevent potentially fatal complications.

An imbalance between blood CD4+CXCR5+Foxp3+ Tfr cells and CD4+CXCR5+Tfh cells may contribute to the immunopathogenesis of rheumatoid arthritis.

BACKGROUND: Follicular helper T (Tfh) cells are a subgroup of activated CD4+ T cells which can assist the formation and maintenance of germinal centers. Follicular regulatory T (Tfr) cells are a new class of regulatory T cells which play a major role in suppressing cells in humoral immunity. In contrast to the role of Tfh cells, Tfr cells can inhibit the function of Tfh cells and B cells. Imbalance of blood Tfr/Tfh ratio resulted in the expansion of auto-reactive B cells and auto-antibody production (). However, the effect of Tfr cells and Tfh cells in the pathogenesis of RA (rheumatoid arthritis) is unclear. The purpose of this study was to investigate the function of Tfr cells and Tfh cells in the pathogenesis of RA. METHODS: We recruited 20 patients fulfilled the the American College of Rheumatology diagnosis criteria and 20 healthy controls (HCs). The number of CD4+CXCR5+Foxp3+ Tfr cells and CD4+CXCR5+ Tfh cells in 20 RA patients were measured by flow cytometry analysis. Furthermore, the correlations between the Tfr/Tfh ratio and the characteristic clinical parameters were assessed. The serum levels of IL-21(interleukin-21), CXCL13 (chemokine (C-X-C motif) ligand 13) and TGF-ß (Transforming growth factor-ß) were measured by ELISA. The formation of ectopic germinal center (GC) of synovial membrane was examined by H&E staining. The transcriptional levels of CXCR5 (C-X-C chemokine receptor type 5), CXCL13, ICOS (inducible co-stimulater) and TGF-ß mRNA were also analyzed. In addition, the expression of Bcl-6 (B-cell lymphoma 6), CXCR5, CXCL13 and ICOS in synovial membrane were examined by immunohistochemistry. RESULTS: RA patients had more Tfh cells in peripheral blood, conversely, the frequency of blood Tfr cells (p < 0.05) and the ratio of Tfr/Tfh were significantly decreased compared to healthy controls (p < 0.05, p < 0.01). Furthermore, the ratio of Tfr/Tfh was negatively correlated with values of ESR (r=-0.57, p < 0.05), RF (r=-0.5275, p < 0.001), CRP (r=-0.4486, p < 0.001), IgG (r=-0.4631, p < 0.05), DAS28 scores (r=-0.5645, p < 0.01), as well as the levels of IL-21(r=-0.7398, p < 0.01), CXCL13 (r=-0.4832, p < 0.05). However, the ratio of Tfr/Tfh was positively with the serum level of TGF-ß (r=0.5115, p < 0.05). Higher mRNA expression of CXCR5, CXCL13, ICOS and lower TGF-ß mRNA expression were observed in RA patients. The serum expression level of IL-21, CXCL13 was significantly increased and expression of TGF-ß was significantly decreased in RA patients. Furthermore, ectopic germinal center formation and higher expression of Bcl-6, CXCR5, ICOS, CXCL13 in the synovial membrane of the joints in RA patients were observed. CONCLUSIONS: The decreased blood CD4+CXCR5+Foxp3+ Tfr cells/CD4+CXCR5+ Tfh cells may be responsible for the immunopathogenesis of RA.

Pathologic characteristics of spinal tuberculosis: analysis of 181 cases.

OBJECTIVE: This study aimed to provide a basis for the diagnosis of spinal TB by analyzing its pathologic characteristics. METHODS: The data of 181 patients with spinal TB who underwent surgery from January 2013 to January 2019 at the General Hospital of Ningxia Medical University were retrospectively analyzed. The participants comprised 80 men and 101 women with an average age of 45.1 ± 16.5 (range: 14-78) years. Based on the assessment of tissue samples, five patients had cervical TB, 49 had thoracic TB, 86 had lumbar TB, 22 had thoracolumbar TB, and 19 had lumbosacral TB. Tuberculous granulation tissue, sclerotic bone, sequestrum, and intervertebral disc tissue were collected for hematoxylin and eosin staining. The proportion of patients with atypical and typical pathologic characteristics was identified and compared for statistical analysis. RESULTS: The typical pathologic characteristics included tubercles, granulomas, caseous necrosis, multinuclear giant cells, infiltration of acute inflammatory cells, sequestration, and fibroblastic proliferation. A total of 119 patients had caseous necrosis, 95 had multinuclear giant cells, 68 had granulomatous inflammation, and 21 had tubercles. Moreover, 46 (25.4%) patients had at least three pathologic characteristics and only 12 (6.6%) exhibited all the pathologic characteristics. Of the 35 (19.3%) patients with atypical pathologic characteristics, 17 had lymphocyte infiltration, 10 had fibroblastic proliferation, 2 had hyaline changes, 1 had local hemorrhage, 1 chronic inflammatory change, 2 had sequestration, 1 had dilated and congested vessels, and 1 had acute suppurative inflammation. CONCLUSIONS: The most common pathologic characteristics were caseous necrosis, multinuclear giant cells, granulomatous inflammation, and tubercles. Moreover, multiple pathologic characteristics were observed in patients with spinal TB and one type of these characteristics was dominant. However, atypical pathologic characteristics were also noted. Thus, both pathologic examination and clinical analysis must be performed to improve the diagnostic rate of spinal TB.

Pharmacokinetic study of tanshinol and ligustrazine in rat plasma after intravenous administration of tanshinol and Danshen Chuanxiongqin Injection.

To investigate the effect of ligustrazine on the pharmacokinetic profile of tanshinol after intravenous administration in rats, a sensitive liquid chromatography tandem mass spectrometry method was developed and validated for quantitative determination of tanshinol and ligustrazine in rat plasma. After prepared by protein precipitation, the analytes were separated on a Waters Acquity HSS T3 column (100 × 2.1 mm, 1.8µm) and eluted by 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 ml/min. The precursor-product ion transitions were m/z 197.0 → 135.0 for tanshinol, m/z 417.1 → 255.1 for liquiritin (internal standard) in negative ion mode and m/z 137.1 → 55.0 for ligustrazine in positive ion mode. To avoid the interference of tanshinol metabolite transformation, the stability of analytes in samples collected after administration was assessed. The validated method was successfully applied to a pharmacokinetic study after intravenous administration of single tanshinol and Danshen Chuanxiongqin Injection. After Danshen Chuanxiongqin injection administration, the values of elimination half-time, area under the concentration-time curve and Co were 0.36 ± 0.13 h, 1.29 ± 0.37 µg/ml h and 10.51 ± 2.58 µg/ml for male rats, respectively. In the single tanshinol group, the corresponding values were 0.56 ± 0.24 h, 1.85 ± 0.44 µg/ml h and 14.11 ± 2.26 µg/ml for male rats-30-40% higher than those for the Danshen Chuanxiongqin Injection group. There was a significant different between male and female rats. This study provided information on the influence of ligustrazine on the pharmacokinetic characteristics of tanshinol after intravenous administration of Danshen Chuanxiongqin Injection in rats, which will be helpful for its clinical application.

Anti­inflammatory effects of oxymatrine on rheumatoid arthritis in rats via regulating the imbalance between Treg and Th17 cells.

Oxymatrine (OMT), a monosomic alkaloid extracted from the Chinese herb, Sophora flavescens Ait, has long been used as a traditional Chinese medicine for the treatment of inflammatory diseases. The aim of the present study was to investigate the potential anti­inflammatory effect of OMT, and its modulation on imbalance between regulatory T (Treg) cells and T helper (Th) 17 cells in rats with collagen­induced arthritis (CIA). Sprague­Dawley rats were immunized with type II collagen and following a second collagen immunization, the rats were treated with OMT or dexamethasone (DXM) intraperitoneally once a day for 43 days. Paw swelling, arthritic score and joint histopathology were evaluated. The Treg/Th17­mediated autoreactive response was assessed by determining serum levels of inflammatory response cytokines, including tumor necrosis factor (TNF)­α and interleukin (IL)­17, using an enzyme­linked immunosorbent assay. The mRNA levels of forkhead box P3 (FOXP3) and retinoic acid­related orphan receptor (ROR)γt in spleen cells stimulated with type II collagen were determined using reverse transcription­quantitative polymerase chain reaction analysis. In addition, the protein expression levels of FOXP3 and RORγt were measured using western blot analysis. The results showed that OMT treatment significantly reduced the severity of CIA, markedly abrogating paw swelling, arthritic scores and synovial hyperplasia, and the increased loss in body weight. OMT significantly reduced the production of TNF­α and IL­17A, upregulated FOXP3 and downregulated RORγt in rats with CIA. In conclusion, the present study demonstrated that OMT exhibited a protective effect on rheumatoid arthritis (RA) through the inhibition of inflammation and regulation of Treg/Th17 in the CIA rats, suggesting that OMT may be used as an immune suppressive and cartilage protective medicine in human RA.

The Expression of NOX4 in Smooth Muscles of Small Airway Correlates with the Disease Severity of COPD.

Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD), and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases (NOXs) produced reactive oxygen species (ROS) play a crucial role in COPD pathogenesis. In the present study, the expression of NOX4 and its correlation with the ASM hypertrophy/hyperplasia, clinical pulmonary functions, and the expression of transforming growth factor ß (TGF-ß) in the ASM of COPD small airways were investigated by semiquantitative morphological and/or immunohistochemistry staining methods. The results showed that an elevated expression of NOX4 and TGF-ß, along with an increased volume of ASM mass, was found in the ASM of small airways in COPD patients. The abundance of NOX4 protein in the ASM was increased with disease severity and inversely correlated with the pulmonary functions in COPD patients. In addition, the expression of NOX4 and ASM marker α-SMA was colocalized, and the increased NOX4 expression was found to accompany an upregulated expression of TGF-ß in the ASM of small airways of COPD lung. These results indicate that NOX4 may be a key regulator in ASM remodeling of small airway, in part through a mechanism interacting with TGF-ß signaling in the pathogenesis of COPD, which warrants further investigation.

SIRT1 in B[a]P-induced lung tumorigenesis.

Benzo[a]pyrene (B[a]P) is a carcinogen in cigarette smoke. We found that B[a]P induced SIRT1 in human bronchial epithelial BEAS-2B cell. SIRT1 was overexpressed in the lung of B[a]P-exposed mice and in human lung cancer biopsies. SIRT1 up-regulated TNF-α and ß-catenin and down-regulated the membrane fraction of E-cadherin. In addition, SIRT1 promoted invasion, migration and tumorigenesis of BEAS-2B cells in nude mice upon B[a]P exposure. Thus, SIRT1 is involved in B[a]P-induced transformation associated with activation of the TNF-α/ß-catenin axis and is as a potential therapeutic target for lung cancer.

Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.

Functional expression of TLR9 in esophageal cancer.

The Toll-like receptor 9 (TLR9) plays a crucial role in both innate and adaptive immune responses against infection and danger signals. Stimulation of TLR9 has been linked to invasion in various cancer cells in vitro. The present study evaluated the expression of TLR9 in human esophageal cancer (EC) cells and normal and malignant esophageal squamous epithelium, and examined the association between TLR9 expression, clinicopathological variables, and EC patient outcome. We further characterized the direct effects of TLR9 agonist CpG oligonucleotides (CpG ODN) and inhibitor chloroquine (CQ), on the proliferation and invasion of EC cells in vitro. RT-PCR, western blot, flow cytometry and immunohistochemical analysis were used to determine the expression of TLR9 in EC cell line TE10, and 90 cases of esophageal squamous cell carcinoma, including 30 cases of adjacent esophageal epithelium. The TLR9 expression was compared with tumor size, location, grade, stage and proliferation. We found basal expression of TLR9 in TE10 cells. Esophageal carcinomas exhibited TLR9 expression that was positively associated with tumor size, location and TNM stage (P<0.05). CpG ODN significantly enhanced the invasion of TE10 cells, which could be abrogated by a TLR9 inhibitor CQ. CpG ODN led to activation of NF­κB and enhanced expression of matrix metalloproteinase (MMP)-2, MMP-7 and cyclooxygenase-2 (COX-2) mRNA. Expression of TLR9 in EC suggests a role of TLR9 related to cell proliferation and differentiation. Our findings indicate that TLR9 may represent a novel therapeutic target in this disease.

Hyperglycemia increased brain ischemia injury through extracellular signal-regulated protein Kinase.

This study was to examine the alterations in the phosphorylation of mitogen-activated protein kinase (MAPK) family in transient brain ischemia under a hyperglycemia and to highlight the molecular mechanisms by which hyperglycemia exacerbates brain damage resulting from stroke. Extracellular signal-regulated protein kinase (ERK) expression was studied in rats subjected to global brain ischemia with pre-ischemic normoglycemic (CIN) and hyperglycemic (CIH) conditions. In another group, the hyperglycemic ischemic rats were pretreated with ERK inhibitor U0126 (U0126). Increased phospho-ERK1/2 immunoreactive neurons in the cingulate cortex and hippocampal CA3 were detected in CIN after ischemia and reperfusion. The numbers of phospho-ERK1/2-positive neurons were further increased significantly in CIH compared to the CIN. Pretreatment with U0126 in CIH rats significantly decreased ERK1/2 immunoreactive cells. Western blot analyses confirmed that phospho-ERK1/2 increased significantly after 30 min ischemia and reperfusion compared to non-ischemic controls in both the CIN and CIH groups. The increase of phospho-ERK1/2 was more prominent in the CIH than in the CIN group after 3 and 6h of reperfusion. Treatment with U0126 significantly reduced phospho-ERK1/2 in the CIH group. The findings presented here suggest that ERK1/2 may play a role in mediating neuronal cells death under hyperglycemic condition.