DYZY01 alleviates pulmonary hypertension via inhibiting endothelial cell pyroptosis and rescuing endothelial dysfunction.

Pulmonary hypertension (PH) is a malignant pulmonary vascular disease with a poor prognosis. Although the development of targeted drugs for this disease has made some breakthroughs in recent decades, PH remains incurable. Therefore, innovative clinical treatment methods and drugs for PH are still urgently needed. DYZY01 is a new drug whose main ingredient is high-purity cannabidiol, a non-psychoactive constituent of cannabinoids that was demonstrated to have anti-inflammatory and anti-pyroptosis properties. Several recent studies have found cannabidiol could improve experimental PH, whereas the mechanistic effect of it warrants further investigation. Thus, this study aimed to investigate whether DYZY01 can treat PH by inhibiting inflammation and pyroptosis and to reveal its underlying mechanism. We established hypoxia and monocrotaline (MCT)-induced PH rat models in vivo and treated them with either DYZY01 (10,50 mg/kg/d) or Riociguat (10 mg/kg/d) by oral administration. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), and extent of vascular remodeling were measured. Meanwhile, the effect of DYZY01 on human pulmonary arterial endothelial cells (HPAECs) was assessed in vitro. The results indicated that DYZY01 significantly reduced mPAP and RVHI in PH rats and reversed the extent of pulmonary vascular remodeling. This improvement may have been achieved by reducing endothelial cell pyroptosis via inhibiting the NF-κB/NLRP3/Caspase-1 pathway. Furthermore, DYZY01 could improve endothelial vascular function, possibly by regulating the secretion of vasodilator factors and inhibiting the proliferation and migration of pulmonary endothelial cells.

Unveiling the metabolic landscape of pulmonary hypertension: insights from metabolomics.

Pulmonary hypertension (PH) is regarded as cardiovascular disease with an extremely poor prognosis, primarily due to irreversible vascular remodeling. Despite decades of research progress, the absence of definitive curative therapies remains a critical challenge, leading to high mortality rates. Recent studies have shown that serious metabolic disorders generally exist in PH animal models and patients of PH, which may be the cause or results of the disease. It is imperative for future research to identify critical biomarkers of metabolic dysfunction in PH pathophysiology and to uncover metabolic targets that could enhance diagnostic and therapeutic strategies. Metabolomics offers a powerful tool for the comprehensive qualitative and quantitative analysis of metabolites within specific organisms or cells. On the basis of the findings of the metabolomics research on PH, this review summarizes the latest research progress on metabolic pathways involved in processes such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in the context of PH.

Iron metabolism disorder regulated by BMP signaling in hypoxic pulmonary hypertension.

BACKGROUNDS AND AIMS: Unexplained iron deficiency is associated with poorer survival in patients with pulmonary hypertension (PH). Bone morphogenetic protein (BMP) signaling and BMP protein type II receptor (BMPR2) expression are important in the pathogenesis of PH. BMP6 in hepatocytes is a central transcriptional regulator of the iron hormone hepcidin that controls systemic iron balance. This study aimed to investigate the effects of BMP signaling on iron metabolism and its implication in hypoxia-induced PH. METHODS AND RESULTS: PH was induced in Sprague-Dawley Rats under hypoxia for 4 weeks. Compared with the control group, right ventricular systolic pressure and right ventricle hypertrophy index were both markedly increased, and serum iron level was significantly decreased with iron metabolic disorder in the hypoxia group. In cultured human pulmonary artery endothelial cells (HPAECs), hypoxia increased oxidative stress and apoptosis, which were reversed by supplementation with Fe agent. Meanwhile, iron chelator deferoxamine triggered oxidative stress and apoptosis in HPAECs, and treatment with antioxidant alleviated iron-deficiency-induced apoptosis by reducing reactive oxygen species production. Expression of hepcidin, BMP6 and hypoxia-inducible factor (HIF)-1α were significantly upregulated, while expression of BMPR2 was downregulated in hepatocytes in the hypoxia group, both in vivo and in vitro. Expression of hepcidin and HIF-1α were significantly increased by BMP6, while pretreatment with siRNA-BMPR2 augmented the enhanced expression of hepcidin and HIF-1α induced by BMP6. CONCLUSIONS: Iron deficiency promoted oxidative stress and apoptosis in HPAECs in hypoxia-induced PH, and enhanced expression of hepcidin regulated by BMP6/BMPR2 signaling may contribute to iron metabolic disorder.

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.

Inhibition of sphingosine 1-phosphate (S1P) receptor 1/2/3 ameliorates biological dysfunction in rheumatoid arthritis fibroblast-like synoviocyte MH7A cells through Gαi/Gαs rebalancing.

Sphingosine 1-phosphate (S1P) exerts its various physiological and pathological effects by interacting with G protein-coupled receptors. In addition, S1P can induce biological dysfunction in fibroblast-like synoviocytes (FLSs) in the development of rheumatoid arthritis (RA). However, the mechanism underlying this S1P-induced dysfunction remains unclear. An imbalance between Gαi and Gαs can affect the level of cAMP, an important regulator of numerous cell functions. Therefore, we studied the effects of S1P receptor (S1PR) 1-, 2-, and 3-associated Gαi/Gαs imbalance on the biological function of rheumatoid arthritis fibroblast-like synoviocyte (MH7A cells). The results showed that blocking S1PR1/3 and Gαi, and activating Gαs, inhibited the proliferation, migration, invasion, and proinflammatory cytokine release of MH7A cells in a S1P-induced inflammation model, whereas suppressing S1PR2 only affected the invasion and the release of proinflammatory cytokines of these cells. Analysis of the expression of S1PR1/2/3 and Gαi/Gαs further showed that S1PR1/2/3 could regulate the Gαi/Gαs balance. Furthermore, our data suggested that the level of cAMP was also affected. Combined, our results showed that impaired S1PR1/2/3 signalling can affect MH7A cells biological function via Gαi/Gαs-cAMP signalling, which can provide a new idea for the treatment of RA.

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.

[Pharmacokinetics of Achyranthes bidentata on adjuvant arthritis rats by microdialysis and UHPLC-MS/MS].

To investigate the " drug-guide" effect of Achyranthes bidentata saponins( ABS) and geniposide( GE) in the treatment on adjuvant arthritis( AA) rats. A UHPLC-MS/MS method for the quantitative determination of GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa in rat blood and joint dialysate was established. After single or combined administration with ABS and GE was given to AA rat model,a microdialysis sampling method for rat joint cavity and jugular vein blood vessels was established to collect microdialysis samples. Waters Acquity HSS C_(18) column was used to separate the above four components,with mobile phase as acetonitrile-0. 1% formic acid water as mobile phase for gradient elution. ESI source was adopted for mass spectra in a negative ion scanning mode. Multiple reaction monitoring( MRM) mode was applied to detect the above four components. The methodological results showed that GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa demonstrated a good linear relationship within the concentration ranges of 2-4 000,16-4 096,14-3 584,23-5 888 µg·L-1 respectively. The precision,accuracy,stability and matrix effect of these four ingredients reached the requirements of quantitative analysis of biological samples. The pharmacokinetic results demonstrated that the combined administration of ABS and GE( 60 mg·kg~(-1)+60 mg·kg~(-1)) can increase the degree of GE in joint cavity distribution,and the AUCjoint/AUCplasmwere twice of that of single administration of GE( 60 mg·kg~(-1)),which indicated that ABS might played a vital role in GE's distribution to joint cavity. Moreover,there was no significant difference between the distribution trend of total three ABS and GE in rats. The pharmacodynamics results showed that the combined administration of ABS and GE has stronger effects on paw swelling,arthritis index and synovial pathomorphology of AA rats than single administration of GE,which suggested that ABS might improve GE's anti-inflammatory effect in AA rats. Based on the above results,ABS has a targeting effect in increasing GE's concentration in joint cavity,with a synergy in efficacy.

Novel anti-inflammatory target of geniposide: Inhibiting Itgß1/Ras-Erk1/2 signal pathway via the miRNA-124a in rheumatoid arthritis synovial fibroblasts.

Geniposide (GE) is an active component isolated from the fruit of Gardenia jasminoides Ellis that has anti-inflammatory and other pharmacological effects; however, the underlying mechanism of GE action has not been elucidated in rheumatoid arthritis (RA). Previous studies have shown that GE plays a therapeutic role in RA via regulation of the integrin beta 1 (Itgß1)-mediated Ras-Erk1/2 signalling pathway. However, the specific mechanism of GE action on Itgß1 has not been clarified. Recent evidence indicates that microRNAs (miRNAs) are involved in the development of RA. In this study, we developed a miRNA-124a-based synoviocyte repair strategy. We demonstrated that miRNA-124a can directly inhibit the expression of the Itgß1 gene and decrease TNF-α-stimulated cell proliferation in vitro. MH7A cells were obtained from the patient with RA and treated with GE in the presence of TNF-α (10 ng/mL). Additionally, we demonstrated that the expression of miRNA-124a can be regulated by GE. GE upregulated the expression of miRNA-124a and decreased the expression of Itgß1 at the mRNA and protein levels. The results of the present study are the first to suggest that GE inhibits TNF-α-stimulated cell proliferation and blocks the activation of the Ras-Erk1/2 pathway via the upregulation of miRNA-124a expression. Our study elucidates the role of miRNA-124a as a protected miRNA in RA and may provide a novel strategy for the diagnosis and treatment of RA in the future.

Immunosuppressive Effect of Geniposide on Mitogen-Activated Protein Kinase Signalling Pathway and Their Cross-Talk in Fibroblast-Like Synoviocytes of Adjuvant Arthritis Rats.

Geniposide (GE), an iridoid glycoside compound derived from Gardenia jasminoides Ellis fruit, is known to have anti-inflammatory and immunoregulatory activities. The aim of this study was to investigate the protective mechanism of GE in the regulation of the mitogen-activated protein kinase (MAPK) signalling pathway and the cross-talk among the MAPK signalling pathway in fibroblast-like synoviocytes (FLS) of adjuvant arthritis (AA) rats. AA was induced by injecting with Freund's complete adjuvant. Male SD rats and FLS were subjected to treatment with GE (30, 60 and 120 mg/kg) in vivo from day 14 to 21 after immunization and GE (25, 50 and 100 µg/mL) in vitro, respectively. The proliferation of FLS was assessed by MTT. IL-4, IL-17, IFN-γ, and TGF-ß1 were determined by ELISA. Key proteins in the MAPK signalling pathway were detected by Western blot. GE significantly reduced the proliferation of FLS, along with decreased IFN-γ and IL-17 and increased IL-4 and TGF-ß1. In addition, GE decreased the expression of p-JNK, p-ERK1/2 and p-p38 in FLS of AA rats. Furthermore, disrupting one MAPK pathway inhibited the activation of other MAPK pathways, suggesting cross-talk among MAPK signalling. In vivo study, it was also observed that GE attenuated histopathologic changes in the synovial tissue of AA rats. Collectively, the mechanisms by which GE exerts anti-inflammatory and immunoregulatory effects may be related to the synergistic effect of JNK, ERK1/2 and p38. Targeting MAPK signalling may be a new therapeutic strategy in inflammatory/autoimmune diseases.

Vacuum metal deposition: visualisation of gold agglomerates using TEM imaging.

Vacuum metal deposition (VMD) is a well-established technique that can be used for the development of latent fingermarks on a range of polymer surfaces, including polyethylene (PE) bags exposed to harsh environmental conditions. The technique has also proved to be effective on difficult semi-porous surfaces such as the polymer banknotes in circulation in Australia and in an increasing number of other countries. VMD is a two-stage technique. In the first stage, a small amount of gold is deposited under high vacuum onto the exhibit. This is then followed by the deposition, onto the gold layer, of a much thicker layer of zinc. Normal VMD development is characterised by zinc depositing all over the surface except on the fingermark ridges themselves. A phenomenon of reverse development (zinc on the ridges but not on the surface) has been reported by many authors. Recent studies indicated that this phenomenon might occur on low-density polyethylene (LDPE) when the amount of deposited gold is above an optimum quantity. The results suggested that the size of the gold agglomerates formed on the surface of the polymer plays a critical role. This preliminary study was aimed at visualising, by transmission electron microscopy (TEM), the formation of gold agglomerates on polymer surfaces to gain an appreciation of how the density and size of these agglomerates changes with an increasing amount of evaporated gold.