Analysis of Enrichment Pathway, Hub Gene, and Protein-Protein Interaction Network in Rheumatoid Arthritis and Construction of Molecular Subtypes in Peripheral Blood.

Objective: To analyze the enrichment pathway, hub gene, and Protein-protein interaction (PPI) network of rheumatoid arthritis (RA) and construct peripheral blood subtypes based on integrated bioinformatics analysis. Methods: Suitable datasets were screened from the GEO database based on titles and abstracts, batch positive analysis was performed using R language, and KEGG enrichment analysis and GO enrichment analysis were performed. After screening the differential genes, the PPI network was constructed, and the hubba plug-in of Cytoscape software was used to obtain the top 10 hub genes(key regulatory genes). hub genes were used as the typing condition to identify the molecular subtypes of synovial tissue and peripheral blood of arthritis. Results: GSE12021 and GSE93272 have been chosen for analysis. GSE12021 presents the transcriptome analysis of human joint synovial tissue, comprising 12 samples from patients with rheumatoid arthritis and 9 samples from normal healthy individuals. On the other hand, GSE93272 includes human peripheral blood samples, comprising 232 samples from patients with rheumatoid arthritis and 43 samples from normal healthy individuals. The main results of GSE12021 KEGG enrichment were Parathyroid hormone synthesis, Relaxin signaling pathway, TNF signaling pathway, Rheumatoid arthritis, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, Toll-like receptor signaling pathway and so on. The main results of GSE12021 GO enrichment were regulation of feeding behavior, regulation of neuron death, positive regulation of cell-cell adhesion, and positive regulation of leukocyte activation. The top 10 hub genes were CD8A, JUN, CTLA4, CD19, LCK, FOS, CCL5, IL7R, CCR7 and CD247. Synovial tissue and peripheral blood subtypes of rheumatoid arthritis showed that the two classification methods maintained consistency. Conclusion: Identifying the Hub gene in peripheral blood helps screen molecular subtypes of rheumatoid arthritis.

Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

Celastrol inhibits prostaglandin E2-induced proliferation and osteogenic differentiation of fibroblasts isolated from ankylosing spondylitis hip tissues in vitro.

BACKGROUND: Heterotopic ossification on the enthesis, which develops after subsequent inflammation, is one of the most distinctive features in ankylosing spondylitis (AS). Prostaglandin E2 (PGE-2) serves as a key mediator of inflammation and bone remodeling in AS. Celastrol, a well-known Chinese medicinal herb isolated from Tripterygium wilfordii, is widely used in treating inflammatory diseases, including AS. It has been proven that it can inhibit lipopolysac-charide-induced expression of various inflammation mediators, such as PGE-2. However, the mechanism by which celastrol inhibits inflammation-induced bone forming in AS is unclear. OBJECTIVE: To investigate whether celastrol could inhibit isolated AS fibroblast osteogenesis induced by PGE-2. METHODS: Hip synovial tissues were obtained from six AS patients undergoing total hip replacement in our hospital. Fibroblasts were isolated, primarily cultured, and then treated with PGE-2 for osteogenic induction. Different doses of celastrol and indometacin were added to observe their effects on osteogenic differentiation. Cell proliferation, osteogenic markers, alizarin red staining as well as the activity of alkaline phosphatase were examined in our study. RESULTS: Celastrol significantly inhibits cell proliferation of isolated AS fibroblasts and in vitro osteogenic differentiation compared with control groups in a time- and dose-dependent manner. CONCLUSION: Our results demonstrated that celastrol could inhibit isolated AS fibroblast proliferation and in vitro osteogenic differentiation. The interaction of PI3K/AKT signaling and Wnt protein may be involved in the process. Further studies should be performed in vivo and animal models to identify the potential effect of celastrol on the bone metabolism of AS patients.

Downregulation of dickkopf-1 enhances the proliferation and osteogenic potential of fibroblasts isolated from ankylosing spondylitis patients via the Wnt/ß-catenin signaling pathway in vitro.

BACKGROUND: Heterotopic ossification of the entheses is one of the most distinctive features in ankylosing spondylitis (AS). Fibroblasts are potential target cells for heterotopic ossification. The Wnt/ß-catenin pathway and its inhibitor dickkopf-1 (DKK-1) regulate bone formation. DKK-1 expression in human AS tissues has not been documented. OBJECTIVE: The purpose of the current study was to investigate the expression of DKK-1 in AS tissues and to elucidate its role in fibroblasts proliferation and osteogenesis in AS. METHODS: DKK-1 expression was assessed by western blotting, real time-polymerase chain reaction (RT-PCR), and immunohistochemistry analysis of hip synovial tissues obtained from AS and control patients. Fibroblasts were isolated, cultured, and transfected with lentiviral vectors for overexpressing human DKK-1 or an shRNA for silencing DKK-1. MTS [(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium] and a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay were used to detect AS fibroblasts proliferation after transfection. The expression levels of ß-catenin, phosphorylated ß-catenin, c-Myc, cyclin D1, and the osteogenesis markers alkaline phosphatase (ALP), osteocalcin (OCN), and Runt-related transcription factor 2 (Runx2) were then examined by western blot analysis. Alizarin red staining (ARS) was also used to observe biomineralization activity. RESULTS: DKK-1 was downregulated in hip synovial tissues from AS patients compared to that observed in controls. AS fibroblasts exhibited excessive proliferation, a higher growth rate, and a decreased apoptotic rate. EdU assay demonstrated that DKK-1 suppressed the growth of AS fibroblasts. Downregulation of DKK-1 decreased the phosphorylation of ß-catenin and upregulated the expression of ß-catenin, c-Myc, cyclin D1, and osteogenesis markers. Overexpression of DKK-1 had the opposite effect, resulting in the inhibition of the Wnt/ß-catenin pathway. ARS showed an increase in biomineralization activity after the inhibition of DKK-1. CONCLUSIONS: AS fibroblasts are characterized by an imbalance between proliferation and apoptosis. DKK-1 may play a role in switching to new bone formation in AS progression.

Ligament, nerve, and blood vessel anatomy of the lateral zone of the lumbar intervertebral foramina.

PURPOSE: To provide an anatomical basis for intrusive treatment using an approach through the lateral zones of the lumbar intervertebral foramina (LIF), especially for acupotomology lysis, percutaneous transforaminal endoscopy, and lumbar nerve root block. METHODS: Blood vessels, ligaments, nerves, and adjacent structures of ten cadavers were exposed through the L1-2 to L5-S1 intervertebral foramina and examined. RESULTS: The lateral zones of the LIF were almost filled by ligaments, nerves, and blood vessels, which were separated into compartments by superior/inferior transforaminal ligaments and corporotransverse superior/inferior ligaments. Two zones relatively lacking in blood vessels and nerves (triangular working zones) were found beside the lamina of the vertebral arch and on the root of the transverse processus. Both the ascending lumbar vein and branches of the intervetebral vein were observed in 12 Kambin's triangles, and in only seven Kambin's triangles were without any veins. CONCLUSIONS: Nerves and blood vessels are fixed and protected by transforaminal ligaments and/or corporotransverse ligaments. It is necessary to distinguish the ligaments from nerves using transforaminal endoscopy so that the ligaments can be cut without damaging nerves. Care needs to be taken in intrusive operations because of the veins running through Kambin's triangle. We recommend injecting into the lamina of the vertebral arch and the midpoint between the adjacent roots of the transverse processus when administering nerve root block. Blind percutaneous incision and acupotomology lysis is dangerous in the lateral zones of the LIF, as they are filled with nerves and blood vessels.