Geniposide downregulates the VEGF/SphK1/S1P pathway and alleviates angiogenesis in rheumatoid arthritis in vivo and in vitro.

The VEGF/SphK1/S1P pathway is closely related to angiogenesis in rheumatoid arthritis (RA), but the precise underlying mechanisms are unclear at present. Here, we explored the involvement of the VEGF/SphK1/S1P cascade in RA models and determined the effects of GE intervention. Our results showed abnormal expression of proteins related to this pathway in RA synovial tissue. Treatment with GE effectively regulated the signal axis, inhibited angiogenesis, and alleviated RA symptoms. In vitro, TNF-ɑ enhanced the VEGF/SphK1/S1P pathway in a co-culture model of fibroblast-like synoviocytes (FLS) and vascular endothelial cells (VEC). GE induced downregulation of VEGF in FLS, restored the dynamic balance of pro-/antiangiogenic factors, and suppressed SphK1/S1P signaling in VEC, resulting in lower proliferation activity, migration ability, tube formation ability, and S1P secretion ability of VEC cells. Additionally, SphK1-specific small interfering RNA (siRNA) blocked the VEGF/SphK1/S1P cascade, which can effectively alleviate the stimulatory effect of FLS on VEC and further enhanced the therapeutic effect of GE. Taken together, our results demonstrate that GE suppresses the VEGF/SphK1/S1P pathway and alleviates the stimulation of VEC by FLS, thereby preventing angiogenesis and promoting therapeutic effects against RA.

Safety and efficacy of sequential treatments for postmenopausal osteoporosis: a network meta-analysis of randomised controlled trials.

Background: The sequential anti-osteoporotic treatment for women with postmenopausal osteoporosis (PMO) is important, but the order in which different types of drugs are used is confusing and controversial. Therefore, we performed a network meta-analysis to compare the efficacy and safety of available sequential treatments to explore the most efficacious strategy for long-term management of osteoporosis. Methods: In this network meta-analysis, we searched the PubMed, EMBASE, Web of Science, the Cochrane Library, and ClinicalTrials.gov from inception to September 19, 2023 to identify randomised controlled trials comparing sequential treatments for women with PMO. The identified trials were screened by reading the title and abstract, and only randomised clinical trials involving sequential anti-osteoporotic treatments and reported relevant outcomes for PMO were included. The main outcomes included vertebral fracture risk, the percentage change in bone mineral density (BMD) in different body parts, and all safety indicators in the stage after switching treatment. A frequentist network meta-analysis was performed using the multivariate random effects method and evaluated using the surface under the cumulative ranking curve (SUCRA). Certainty of evidence was assessed using the Confidence in the Network Meta-Analysis (CINeMA) framework. This study is registered with PROSPERO: CRD42022360236. Findings: A total of 19 trials comprising 18,416 participants were included in the study. Five different sequential treatments were investigated as the main interventions and compared to the corresponding control groups. The intervention groups in this study comprised the following treatment switch protocols: switching from an anabolic agent (AB) to an anti-resorptive agent (AR) (ABtAR), transitioning from one AR to another AR (ARtAAR), shifting from an AR to an AB (ARtAB), switching from an AB to a combined treatment of AB and AR (ABtC), and transitioning from an AR to a combined treatment (ARtC). A significant reduction in the incidence of vertebral fractures was observed in ARtC, ABtAR and ARtAB in the second stage, and ARtC had the lowest incidence with 81.5% SUCRA. ARtAAR and ABtAR were two effective strategies for preventing fractures and improving BMD in other body parts. Especially, ARtAAR could improve total hip BMD with the highest 96.1% SUCRA, and ABtAR could decrease the risk of total fractures with the highest 94.3% SUCRA. Almost no difference was observed in safety outcomes in other comparisons. Interpretation: Our findings suggested that the ARtAAR and ABtAR strategy are the effective and safe sequential treatment for preventing fracture and improving BMD for PMO. ARtC is more effective in preventing vertebral fractures. Funding: The National Natural Science Foundation of China (82170900, 81970762), the Hunan Administration of Traditional Chinese Medicine, and the Hunan Province High-level Health Talents "225" Project.

Adipose Tissue Plasticity: A Comprehensive Definition and Multidimensional Insight.

Adipose tissue is composed of adipocytes, stromal vascular fraction, nerves, surrounding immune cells, and the extracellular matrix. Under various physiological or pathological conditions, adipose tissue shifts cellular composition, lipid storage, and organelle dynamics to respond to the stress; this remodeling is called "adipose tissue plasticity". Adipose tissue plasticity includes changes in the size, species, number, lipid storage capacity, and differentiation function of adipocytes, as well as alterations in the distribution and cellular composition of adipose tissue. This plasticity has a major role in growth, obesity, organismal protection, and internal environmental homeostasis. Moreover, certain thresholds exist for this plasticity with significant individualized differences. Here, we comprehensively elaborate on the specific connotation of adipose tissue plasticity and the relationship between this plasticity and the development of many diseases. Meanwhile, we summarize possible strategies for treating obesity in response to adipose tissue plasticity, intending to provide new insights into the dynamic changes in adipose tissue and contribute new ideas to relevant clinical problems.

Metabonomic analysis of abnormal sphingolipid metabolism in rheumatoid arthritis synovial fibroblasts in hypoxia microenvironment and intervention of geniposide.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by a joint hypoxia microenvironment. Our previous untargeted metabolomics study found that sphingolipid (SPL) metabolism was abnormal in the joint synovial fluid samples from adjuvant arthritis (AA) rats. Geniposide (GE), an iridoid glycoside component of the dried fruit of Gardenia jasminoides Ellis, is commonly used for RA treatment in many Asian countries. At present, the mechanism of GE in the treatment of RA, especially in the joint hypoxia microenvironment, is not entirely clear from the perspective of SPL metabolism. The purpose of this research was to explore the potential mechanism of abnormal SPL metabolism in RA joint hypoxia microenvironment and the intervention effect of GE, through the untargeted metabolic analysis based on the ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Arthritis index, foot swelling and histopathology were used to assess whether the AA rat model was successfully established. The SPLs extracts collected from AA rats' synovial tissue, serum and rheumatoid arthritis synovial fibroblasts (RASFs, MH7A cells, hypoxia/normoxia culture) were analyzed by metabolomics and lipdomics approach based on UPLC-Q-TOF/MS, to identify potential biomarkers associated with disorders of GE regulated RA sphingolipid metabolism. As a result, 11 sphingolipid metabolites related to RA were screened and identified. Except for galactosylceramide (d18:1/20:0), GE could recover the change levels of the above 10 sphingolipid biomarkers in varying degrees. Western blotting results showed that the changes in ceramide (Cer) level regulated by GE were related to the down-regulation of acid-sphingomyelinase (A-SMase) expression in synovial tissue of AA rats. To sum up, this research examined the mechanism of GE in the treatment of RA from the perspective of SPL metabolism and provided a new strategy for the screening of biomarkers for clinical diagnosis of RA.

Geniposide inhibits SphK1 membrane targeting to restore macrophage polarization balance in collagen-induced arthritis mice.

Imbalance of macrophage polarization plays a critical role in the progression of rheumatoid arthritis (RA). Geniposide (GE) has been shown to exert anti-inflammatory effects. However, the effect of GE on macrophage polarization remains unclear. Here, we investigated the regulation of GE on the imbalance of macrophage polarization in RA and how it functions. We established a mouse model of collagen-induced arthritis (CIA) and isolated bone marrow-derived macrophages (BMDMs). The results confirmed that pro-inflammatory M1 macrophages were dominant in CIA mice, but the polarization imbalance of macrophages was restored to a certain extent after GE treatment. Furthermore, the membrane targeting of sphingosine kinase 1 (SphK1) was increased in BMDMs of CIA mice, as manifested by increased membrane and cytoplasmic expression of p-SphK1 and high secretion level of sphingosine-1-phosphate (S1P). RAW264.7 cells were stimulated with lipopolysaccharide (LPS)-interferon (IFN)-γ or interleukin (IL)-4 to induce M1 or M2 phenotype, respectively, to revalidate the results obtained in BMDMs. The results again observed SphK1 membrane targeting in LPS-IFN-γ-stimulated RAW264.7 cells. Selective inhibition of SphK1 by PF543 or inhibition of the S1P receptors by FTY720 both restored the proportion of M1 and M2 macrophages in LPS-IFN-γ-stimulated RAW264.7 cells, confirming that SphK1 membrane targeting mediated a proportional imbalance in M1 and M2 macrophage polarization. In addition, GE inhibited SphK1 membrane targeting and kinase activity. Taken together, results confirmed that the inhibition of SphK1 membrane targeting by GE was responsible for restoring the polarization balance of macrophages in CIA mice.

Geniposide alleviates VEGF-induced angiogenesis by inhibiting VEGFR2/PKC/ERK1/2-mediated SphK1 translocation.

BACKGROUND: Rheumatoid arthritis (RA) is an angiogenesis-dependent disease caused by the imbalance of pro- and anti-angiogenic factors. More effective strategies to block synovial angiogenesis in RA should be studied. Geniposide (GE), a natural product isolated from the fruit of Gardenia jasminoides Ellis (GJ), is reported to have anti-inflammatory, anti-angiogenic and other pharmacological effects. However, the underlying mechanism through which GE affects synovial angiogenesis in RA remains unclear. PURPOSE: In this research, we aimed to elucidate the effect and potential mechanisms of GE on angiogenesis in RA. MATERIALS AND METHODS: Synovial angiogenesis in patients with RA and a rat model of adjuvant arthritis (AA) was detected by hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and western blottiing. The biological functions of vascular endothelial cells (VECs) and sphingosine kinase 1 (SphK1) translocation were checked by CCK-8, EdU, Transwell, tube formation, co-immunoprecipitation assays, and laser scanning confocal microscopy. The effect of the SphK1 gene on angiogenesis was assessed by transfection of SphK1-siRNA in cells and mices. The effect of GE on VEGF-induced angiogenesis was measured by Matrigel plug assay in a mouse model of AA. RESULTS: GE effectively inhibited synovial angiogenesis and alleviated the disease process. SphK1, as a new regulatory molecule, has a potentially important relationship in regulating VEGF/VEGFR2 and S1P/S1PR1 signals. SphK1 translocation was activated via the VEGFR2/PKC/ERK1/2 pathway and was closely linked to the biological function of VECs. GE significantly reduced SphK1 translocation, thereby ameliorating the abnormal biological function of VECs. Furthermore, after transfection of SphK1 siRNA in VECs and C57BL/6 mice, silencing SphK1 caused effectively attenuation of VEGF-induced VEC biological functions and angiogenesis. In vivo, the Matrigel plug experiment indicated that GE significantly inhibited pericyte coverage, basement membrane formation, vascular permeability, and fibrinogen deposition. CONCLUSIONS: Our findings suggest that GE inhibited VEGF-induced VEC biological functions and angiogenesis by reducing SphK1 translocation. Generally, studies have revealed that GE down-regulated VEGFR2/PKC/ERK1/2-mediated SphK1 translocation and inhibited S1P/S1PR1 signaling activation, thereby alleviating VEGF-stimulated angiogenesis. The above evidences indicated that angiogenesis inhibition may provide a new direction for RA treatment.

Anti-Inflammatory Effect of Geniposide on Regulating the Functions of Rheumatoid Arthritis Synovial Fibroblasts via Inhibiting Sphingosine-1-Phosphate Receptors1/3 Coupling Gαi/Gαs Conversion.

The activated Gα protein subunit (Gαs) and the inhibitory Gα protein subunit (Gαi) are involved in the signal transduction of G protein coupled receptors (GPCRs). Moreover, the conversion of Gαi/Gαs can couple with sphingosine-1-phosphate receptors (S1PRs) and have a critical role in rheumatoid arthritis (RA). Through binding to S1PRs, sphingosine-1-phosphate (S1P) leads to activation of the pro-inflammatory signaling in rheumatoid arthritis synovial fibroblasts (RASFs). Geniposide (GE) can alleviate RASFs dysfunctions to against RA. However, its underlying mechanism of action in RA has not been elucidated so far. This study aimed to investigate whether GE could regulate the biological functions of MH7A cells by inhibiting S1PR1/3 coupling Gαi/Gαs conversion. We use RASFs cell line, namely MH7A cells, which were obtained from the patient with RA and considered to be the main effector cells in RA. The cells were stimulated with S1P (5 µmol/L) and then were treated with or without different inhibitors: Gαi inhibitor pertussis toxin (0.1 µg/mL), S1PR1/3 inhibitor VPC 23019 (5 µmol/L), Gαs activator cholera toxin (1 µg/mL) and GE (25, 50, and 100 µmol/L) for 24 h. The results showed that GE may inhibit the abnormal proliferation, migration and invasion by inhibiting the S1P-S1PR1/3 signaling pathway and activating Gαs or inhibiting Gαi protein in MH7A cells. Additionally, GE could inhibit the release of inflammatory factors and suppress the expression of cAMP, which is the key factor of the conversion of Gαi and Gαs. GE could also restore the dynamic balance of Gαi and Gαs by suppressing S1PR1/3 and inhibiting Gαi/Gαs conversion, in a manner, we demonstrated that GE inhibited the activation of Gα downstream ERK protein as well. Taken together, our results indicated that down-regulation of S1PR1/3-Gαi/Gαs conversion may play a critical role in the effects of GE on RA and GE could be an effective therapeutic agent for RA.

Cellular and molecular responses in progressive pseudorheumatoid dysplasia articular cartilage associated with compound heterozygous WISP3 gene mutation.

Progressive pseudorheumatoid dysplasia (PPD) is characterized by continuous degeneration and loss of articular cartilage, which has been attributed to mutations in the gene encoding WISP3. We collected a PPD family and analyzed their WISP3 genes mutation. Articular chondrocytes (ACs) were purified from the femurs of a PPD patient after hip replacement surgery. Cell growth, proliferation, and viability were examined. Gene expression profiling and analyses of matrix metalloproteinases (MMP)-1, -3, and -13 proteins were carried out using cDNA differential microarrays, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. We found that two probands carried a deletion (840delT) mutation in maternal allele, which leads to truncated WISP3 protein missing 43 residues in C terminus; and a 1000T>C substitution in paternal allele, which was also passed on to four other members in the PPD kindred. PPD ACs were heterogeneous in size with an enhanced rate of cell proliferation and viability compared with the normal ACs. MMP-1, -3, and -13 mRNA expressions were dereased in PPD ACs. MMP-1, -3, and -13 protein levels, however, were increased in cell lysates from PPD ACs, but markedly decreased in the supernatants from cultured ACs. WISP3 mRNA expression in PPD ACs was also decreased. Our results show, for the first time, a compound heterozygous mutation of WISP3 and a series of cellular and molecular changes disturbing the endochondral ossification in this PPD patient.

Dental caries and risk indicators for patients with leprosy in China.

BACKGROUND: In leprosy, oral health is often neglected and poorly understood. This study aimed to evaluate the prevalence and risk indicators of dental caries in patients with leprosy in China. METHODS: This cross-sectional, multicentre study included 613 patients with leprosy and 602 control subjects. Based on the established standards of the World Health Organization, we investigated dental caries in cluster samplings from six so-called 'leprosy villages' in three Chinese provinces. Clinical oral examinations were performed and data were reported as decayed (D), missing (M) and filled (F) teeth (DMFT scores). RESULTS: The average DMFT scores were 10.39 in patients with leprosy (D = 4.43; M = 5.94; and F = 0.02) and 4.39 in control individuals (D = 2.29; M = 2.02; F = 0.08). The DMFT scores were statistically significantly different in patients with different ages, educational backgrounds and daily brushing frequency (P < 0.05). High DMFT scores were related to age, low educational levels and poor toothbrushing habits. CONCLUSIONS: The results indicate that patients with leprosy have a high prevalence of severe dental caries. Effective therapy and oral health education should be enhanced for this group of patients.

Insulin receptor substrate 1 regulates the cellular differentiation and the matrix metallopeptidase expression of preosteoblastic cells.

Insulin receptor substrate 1 (IRS1) is an essential molecule for the intracellular signaling of IGF1 and insulin, which are potent anabolic regulators of bone metabolism. Osteoblastic IRS1 is essential for maintaining bone turnover; however, the mechanism underlying this regulation remains unclear. To clarify the role of IRS1 in bone metabolism, we employed RNA interference to inhibit IRS1 gene expression and observed the effects of silencing this gene on the proliferation and differentiation of and the expression of matrix metallopeptidase (MMP) and tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) in MC3T3-E1 cells. Our results showed that IRS1 short hairpin RNAs can effectively suppress the expression of IRS1, and inhibit the phosphorylation of AKT in IRS1 pathway; reduce the expression of MMP2, MMP3, MMP13, and MMP14, decrease the expression of TNFRSF11B and RANKL (also known as tumor necrosis factor (ligand) superfamily, member 11), however increase the RANKL/TNFRSF11B ratio; decrease cell survival, proliferation, and mineralization, and impair the differentiation of MC3T3-E1 cells. The downregulation of IRS1 had no effect on the expression of MMP1. Our findings suggest that IRS1 not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover.