Dynamic YAP expression in the non-parenchymal liver cell compartment controls heterologous cell communication.

INTRODUCTION: The Hippo pathway and its transcriptional effectors yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are targets for cancer therapy. It is important to determine if the activation of one factor compensates for the inhibition of the other. Moreover, it is unknown if YAP/TAZ-directed perturbation affects cell-cell communication of non-malignant liver cells. MATERIALS AND METHODS: To investigate liver-specific phenotypes caused by YAP and TAZ inactivation, we generated mice with hepatocyte (HC) and biliary epithelial cell (BEC)-specific deletions for both factors (YAPKO, TAZKO and double knock-out (DKO)). Immunohistochemistry, single-cell sequencing, and proteomics were used to analyze liver tissues and serum. RESULTS: The loss of BECs, liver fibrosis, and necrosis characterized livers from YAPKO and DKO mice. This phenotype was weakened in DKO tissues compared to specimens from YAPKO animals. After depletion of YAP in HCs and BECs, YAP expression was induced in non-parenchymal cells (NPCs) in a cholestasis-independent manner. YAP positivity was detected in subgroups of Kupffer cells (KCs) and endothelial cells (ECs). The secretion of pro-inflammatory chemokines and cytokines such as C-X-C motif chemokine ligand 11 (CXCL11), fms-related receptor tyrosine kinase 3 ligand (FLT3L), and soluble intercellular adhesion molecule-1 (ICAM1) was increased in the serum of YAPKO animals. YAP activation in NPCs could contribute to inflammation via TEA domain transcription factor (TEAD)-dependent transcriptional regulation of secreted factors. CONCLUSION: YAP inactivation in HCs and BECs causes liver damage, and concomitant TAZ deletion does not enhance but reduces this phenotype. Additionally, we present a new mechanism by which YAP contributes to cell-cell communication originating from NPCs.

Cinnamaldehyde Inhibits Inflammation of Human Synoviocyte Cells Through Regulation of Jak/Stat Pathway and Ameliorates Collagen-Induced Arthritis in Rats.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1ß-induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6-induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-κB pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1ß-induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug. SIGNIFICANCE STATEMENT: In this study, we found that cinnamaldehyde (Cin) suppressed proinflammatory cytokines secretion in rheumatology arthritis synoviocyte cells by Janus kinase/signal transducer and activator of transcription pathway. The in vivo results showed that Cin ameliorated collagen-induced arthritis in rats. These findings indicate that Cin is a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug.

Pyroptosis: A spoiler of peaceful coexistence between cells in degenerative bone and joint diseases.

BACKGROUND: As people age, degenerative bone and joint diseases (DBJDs) become more prevalent. When middle-aged and elderly people are diagnosed with one or more disorders such as osteoporosis (OP), osteoarthritis (OA), and intervertebral disc degeneration (IVDD), it often signals the onset of prolonged pain and reduced functionality. Chronic inflammation has been identified as the underlying cause of various degenerative diseases, including DBJDs. Recently, excessive activation of pyroptosis, a form of programed cell death (PCD) mediated by inflammasomes, has emerged as a primary driver of harmful chronic inflammation. Consequently, pyroptosis has become a potential target for preventing and treating DBJDs. AIM OF REVIEW: This review explored the physiological and pathological roles of the pyroptosis pathway in bone and joint development and its relation to DBJDs. Meanwhile, it elaborated the molecular mechanisms of pyroptosis within individual cell types in the bone marrow and joints, as well as the interplay among different cell types in the context of DBJDs. Furthermore, this review presented the latest compelling evidence supporting the idea of regulating the pyroptosis pathway for DBJDs treatment, and discussed the potential, limitations, and challenges of various therapeutic strategies involving pyroptosis regulation. KEY SCIENTIFIC CONCEPTS OF REVIEW: In summary, an interesting identity for the unregulated pyroptosis pathway in the context of DBJDs was proposed in this review, which was undertaken as a spoiler of peaceful coexistence between cells in a degenerative environment. Over the extended course of DBJDs, pyroptosis pathway perpetuated its activity through crosstalk among pyroptosis cascades in different cell types, thus exacerbating the inflammatory environment throughout the entire bone marrow and joint degeneration environment. Correspondingly, pyroptosis regulation therapy emerged as a promising option for clinical treatment of DBJDs.

Carbon Dot Nanozyme Ameliorating Ischemia-Reperfusion-Induced Muscle Injury by Antioxidation and Downregulating iNOS/COX-2 Pathway.

Skeletal muscle ischemia-reperfusion (IR) injury is a prevalent type of muscle injury caused by events, such as trauma, arterial embolism, and primary thrombosis. The development of an IR injury is associated with oxidative stress and an excessive inflammatory response. Nanozymes, which have exceptional free radical scavenging activities, have gained significant attention for treating oxidative stress. This study demonstrates that carbon dot (C-dot) nanozymes possess superoxide dismutase (SOD)-like activity and can act as free radical scavengers. The carbon dot nanozymes are presented to mitigate inflammation by downregulating the iNOS/COX-2 pathway and scavenging reactive oxygen-nitrogen species to reduce oxidative stress, thereby suppressing inflammation. In the IR injury of skeletal muscle mice, we demonstrate that C-dots can effectively reduce inflammatory cytokines and tissue edema in skeletal muscle following IR injury in the limb. These findings suggest that C-dots have potential as a therapeutic approach for IR injury of skeletal muscle with negligible systemic toxicity. This offers a promising strategy for clinical intervention.

Apoptotic body-inspired nanotherapeutics efficiently attenuate osteoarthritis by targeting BRD4-regulated synovial macrophage polarization.

Bromodomain-containing protein 4 (BRD4) is the most well-studied BET protein that is important for the innate immune response. We recently revealed that targeting BRD4 triggers apoptosis in tumor-associated macrophages, but its role in synovial macrophages and joint inflammation is largely unknown. Herein, we demonstrated that BRD4 was highly expressed in the iNOS-positive M1 macrophages in the human and mouse osteoarthritis (OA) synovium, and conditional knockout of BRD4 in the myeloid lineage using Lyz2-cre; BRD4flox/flox mice significantly abolished anterior cruciate ligament transection (ACLT)-induced M1 macrophage accumulation and synovial inflammation. Accordingly, we successfully constructed apoptotic body-inspired phosphatidylserine-containing nanoliposomes (PSLs) loaded with the BRD4 inhibitor JQ1 to regulate inflammatory macrophages. JQ1-loaded PSLs (JQ1@PSLs) exhibited a higher cellular uptake by macrophages than fibroblast-like synoviocytes (FLSs) in vitro and in vivo, as well as the reduction in proinflammatory M1 macrophage polarization. Intra-articular injections of JQ1@PSLs showed prolonged retention within the joint, and remarkably reduced synovial inflammation and joint pain via suppressing M1 polarization accompanied by reduced TRPA1 expression by targeted inhibition of BRD4 in the macrophages, thus attenuating cartilage degradation during OA development. The results show that BRD4-inhibiting JQ1@PSLs can targeted-modulate macrophage polarization, which opens a new avenue for efficient OA therapy via a "Trojan horse".

Prediction of folding patterns for intrinsic disordered protein.

The conformation flexibility of natural protein causes both complexity and difficulty to understand the relationship between structure and function. The prediction of intrinsically disordered protein primarily is focusing on to disclose the regions with structural flexibility involving relevant biological functions and various diseases. The order of amino acids in protein sequence determines possible conformations, folding flexibility and biological function. Although many methods provided the information of intrinsically disordered protein (IDP), but the results are mainly limited to determine the locations of regions without knowledge of possible folding conformations. Here, the developed protein folding fingerprint adopted the protein folding variation matrix (PFVM) to reveal all possible folding patterns for the intrinsically disordered protein along its sequence. The PFVM integrally exhibited the intrinsically disordered protein with disordering regions, degree of disorder as well as folding pattern. The advantage of PFVM will not only provide rich information for IDP, but also may promote the study of protein folding problem.

Genetically Engineered Biomimetic Nanoparticles for Targeted Delivery of mRNA to Treat Rheumatoid Arthritis.

In addition to inhibiting persistent inflammation, phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is known as an important therapeutic target for alleviating rheumatoid arthritis (RA) symptoms. Modulation of PTEN gene expression in synovial tissue using messenger RNA (mRNA) is a promising approach to combat RA. However, mRNA therapeutics are often hampered by unsatisfactory stability and inefficient localization in synovial tissue. In this study, a genetically engineered biomimetic membrane-coated mRNA (MR@P-mPTEN) carrier that effectively delivers mRNA-PTEN (mPTEN) directly to the RA joint is presented. By overexpressing tumor necrosis factor (TNF-α) receptors on macrophage biomimetic membranes via plasmid transfection, decoys that reduce inflammatory pathway activation are prepared for TNF-α. The resulting construct, MR@P-mPTEN, shows good stability and RA targeting based on in vivo fluorescence imaging. It is also found that MR@P-mPTEN competitively binds TNF-α and activates the PTEN pathway in vitro and in vivo, thereby inhibiting synovitis and joint damage. Clinical micro-computed tomography and histological analyses confirm the treatment effects. These results suggest that the genetically engineered biomimetic therapeutic platform MR@P-mPTEN both inhibits pro-inflammatory cytokines and upregulates PTEN protein expression to alleviate RA damage, providing a new a new combination strategy for RA treatment.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

Objective: The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Methods: The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. Results: The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. Conclusion: The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. The translational potential of this article: Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Astragaloside IV as a novel CXCR4 antagonist alleviates osteoarthritis in the knee of monosodium iodoacetate-induced rats.

BACKGROUND AND PURPOSE: C-X-C chemokine receptor type 4 (CXCR4) inhibition protects cartilage in osteoarthritis (OA) animal models. Therefore, CXCR4 has becoming a novel target for OA drug development. Since dietary and herbal supplements have been widely used for joint health, we hypothesized that some supplements exhibit protective effects on OA cartilage through inhibiting CXCR4 signaling. METHODS: The single-cell RNA sequencing data of OA patients (GSE152805) was re-analyzed by Scanpy 1.9.0. The docking screening of CXCR4 antagonists was conducted by Autodock Vina 1.2.0. The CXCR4 antagonistic activity was evaluated by calcium response in THP-1 cells. Signaling pathway study was conducted by bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. The anti-OA activity was evaluated in monosodium iodoacetate (MIA)-induced rats. RESULTS: Astragaloside IV (ASN IV), the predominate phytochemical in Astragalus membranaceus, has been identified as a novel CXCR4 antagonist. ASN IV reduced CXCL12-induced ADAMTS4,5 overexpression in chondrocytes through blocking Akt signaling pathway. Furthermore, ASN IV administration significantly repaired the damaged cartilage and subchondral bone in MIA-induced rats. CONCLUSION: The blockade of CXCR4 signaling by ASN IV could explain anti-OA activities of Astragalus membranaceus by protection of cartilage degradation in OA patients. Since ASN IV as an antiviral has been approved by China National Medical Products Administration for testing in people, repurposing of ASN IV as a joint protective agent might be a promising strategy for OA drug development.

Shang-Ke-Huang-Shui and coptisine alleviate osteoarthritis in the knee of monosodium iodoacetate-induced rats through inhibiting CXCR4 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Shang-Ke-Huang-Shui (SKHS) is a classic traditional Chinese medicine formula originally from the southern China city of Foshan. It has been widely used in the treatment of osteoarthritis (OA) but underlying molecular mechanisms remain unclear. AIM OF STUDY: Recently, activation of C-X-C chemokine receptor type 4 (CXCR4) signaling has been reported to induce cartilage degradation in OA patients; therefore, inhibition of CXCR4 signaling has becoming a promising approach for OA treatment. The aim of this study was to validate the cartilage protective effect of SKHS and test whether the anti-OA effects of SKHS depend on its inhibition on CXCR4 signaling. Additionally, CXCR4 antagonist in SKHS should be identified and its anti-OA activity should also be tested in vitro and in vivo. METHODS: The anti-OA effects of SKHS and the newly identified CXCR4 antagonist was evaluated by monosodium iodoacetate (MIA)-induced rats. The articular cartilage surface was examined by hematoxylin and eosin (H&E) staining and Safranin O-Fast Green (S-F) staining whereas the subchondral bone was examined by micro-CT. CXCR4 antagonist screenings were conducted by molecular docking and calcium response assay. The CXCR4 antagonist was characterized by UPLC/MS/MS. The bulk RNA-Seq was conducted to identify CXCR4-mediated signaling pathway. The expression of ADAMTS4,5 was tested by qPCR and Western blot. RESULTS: SKHS protected rats from MIA-induced cartilage degradation and subchondral bone damage. SKHS also inhibited CXCL12-indcued ADAMTS4,5 overexpression in chondrocytes through inhibiting Akt pathway. Coptisine has been identified as the most potent CXCR4 antagonist in SKHS. Coptisine reduced CXCL12-induced ADAMTS4,5 overexpression in chondrocytes. Furthermore, in MIA-induced OA model, the repaired cartilage and subchondral bone were observed in the coptisine-treated rats. CONCLUSION: We first report here that the traditional Chinese medicine formula SKHS and its predominate phytochemical coptisine significantly alleviated cartilage degradation as well as subchondral bone damage through inhibiting CXCR4-mediated ADAMTS4,5 overexpression. Together, our work has provided an important insight of the molecular mechanism of SKHS and coptisine for their treatment of OA.

FGF receptor-mediated gene delivery using ligands coupled to PEI-ß-CyD.

A novel vector with high gene delivery efficiency and special cell-targeting ability was developed using a good strategy that utilized low-molecular-weight polyethylenimine (PEI; molecular weight: 600 KDa [PEI600]) crosslinked to ß-cyclodextrin (ß-CyD) via a facile synthetic route. Fibroblast growth factor receptors (FGFRs) are highly expressed in a variety of human cancer cells and are potential targets for cancer therapy. In this paper, CY11 peptides, which have been proven to combine especially with FGFRs on cell membranes were coupled to PEI-ß-CyD using N-succinimidyl-3-(2-pyridyldithio) propionate as a linker. The ratios of PEI600, ß-CyD, and peptide were calculated based on proton integral values obtained from the (1)H-NMR spectra of the resulting products. Electron microscope observations showed that CY11-PEI-ß-CyD can efficiently condense plasmid DNA (pDNA) into nanoparticles of about 200 nm, and MTT assays suggested the decreased toxicity of the polymer. Experiments on gene delivery efficiency in vitro showed that CY11-PEI-ß-CyD/pDNA polyplexes had significantly greater transgene activities than PEI-ß-CyD/pDNA in the COS-7 and HepG2 cells, which positively expressed FGFR, whereas no such effect was observed in the PC-3 cells, which negatively expressed FGFR. Our current research indicated that the synthesized nonviral vector shows improved gene delivery efficiency and targeting specificity in FGFR-positive cells.

Effects of tetracyclines on bones: an ambiguous question needs to be clarified.

Tetracyclines have been widely used in bone histomorphometry to label new bone formation and apposition rate. However, most studies of tetracyclines have also shown their strong inhibitory action on osteoclasts and their effects on osteoblast activities as well. To even obtain the in-depth understanding on this issue, we have reviewed related studies in "Pubmed" by searching the keywords "tetracyclines and osteoclast", "tetracyclines and osteoblast", which retrieved 118 and 162 related documents, respectively. Among these papers, some described the application of tetracyclines as fluorescent marker in bone histomorphometry, while others discussed their role in protection of bone metabolism partly through inhibiting osteoclastogenesis or bone resorption and through enhancing osteogenesis. Based on the above mentioned, it seems that tetracyclines used as bone labeling markers may affect the results of bone histomorphometry to some extent. To even confirm the effect of tetracyclines on bones cells (osteoblast, osteoclast) and in vivo bone remodeling, related research work has been performed in our research team which indicated quite different results in vivo and in vitro. Therefore, the influence of tetracyclines on bones may differ in terms of different conditions which need to be further elucidated.

Peptide-based inhibitors hold great promise as the broad-spectrum agents against coronavirus.

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), and the recent SARS-CoV-2 are lethal coronaviruses (CoVs) that have caused dreadful epidemic or pandemic in a large region or globally. Infections of human respiratory systems and other important organs by these pathogenic viruses often results in high rates of morbidity and mortality. Efficient anti-viral drugs are needed. Herein, we firstly take SARS-CoV-2 as an example to present the molecular mechanism of CoV infection cycle, including the receptor binding, viral entry, intracellular replication, virion assembly, and release. Then according to their mode of action, we provide a summary of anti-viral peptides that have been reported in peer-reviewed publications. Even though CoVs can rapidly evolve to gain resistance to the conventional small molecule drugs, peptide-based inhibitors targeting various steps of CoV lifecycle remain a promising approach. Peptides can be continuously modified to improve their antiviral efficacy and spectrum along with the emergence of new viral variants.

New use for old drug: Local delivery of puerarin facilitates critical-size defect repair in rats by promoting angiogenesis and osteogenesis.

Objectives: Large bone defect repair is a challenging clinical problem due to limited self-repair ability. A well-designed bone filling product should possess the ability to induce tissue in-growth and facilitate neovascularization and new bone formation. Puerarin has been used in clinics for a long time, and recently it was found to be able to promote osteogenesis. This study aimed to investigate a puerarin-based drug/delivery combination implant for promoting large bone defect repair. Methods: Puerarin was incorporated into the poly (lactic-co-glycolic acid)/ß-calcium phosphate (PLGA/TCP, PT) to form a porous PLGA/TCP/Puerarin (PTP) composite scaffold by low-temperature rapid prototyping technology. Its structural and degradation were analyzed in vitro. Then we employed a rat calvarial critical size defect model to assess the potency of the PTP scaffold. MC3T3-E1 cells and EA. hy 926 â€‹cells were used to investigate the underlying mechanism. Results: PTP scaffold inherited all advantages of PT scaffold in structural, mechanical, and biodegradation, meanwhile puerarin stably and continuously released from PTP scaffold and lasted for 5 months in vitro. At 8 weeks after implantation, the PTP scaffold triggered new bone formation in the macro-pores of the scaffold and inside the scaffold accompanied by the degrading materials. The underlying mechanism revealed that the PTP scaffold induced vascular infiltration and recruit repair cells through stimulating vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) expressions to promote angiogenesis and osteogenesis. Conclusion: Puerarin-enriched porous PTP scaffold was a promising local delivery system with sustained release of puerarin for facilitating defect repair through getting synergistic angiogenic and osteogenic effects. The Translational Potential of this Article: The PTP scaffold presents a potential drug/device combination medical implant for large bone defect repair, which also provides a new and innovative application for the "old drug" puerarin.

Interlayer Structure Engineering of MXene-Based Capacitor-Type Electrode for Hybrid Micro-Supercapacitor toward Battery-Level Energy Density.

Micro-supercapacitors are notorious for their low energy densities compared to micro-batteries. While MXenes have been identified as promising capacitor-type electrode materials for alternative zinc-ion hybrid micro-supercapacitors (ZHMSCs) with higher energy density, their tightly spaced layered structure renders multivalent zinc-ions with large radii intercalation inefficient. Herein, through insertion of 1D core-shell conductive BC@PPy nanofibers between MXene nanosheets, an interlayer structure engineering technique for MXene/BC@PPy capacitor-type electrodes towards ZHMSCs is presented. Owing to simultaneously achieving two objectives: (i) widening the interlayer space and (ii) providing conductive connections between the loose MXene layers, enabled by the conductive BC@PPy nanospacer, the approach effectively enhances both ion and electron transport within the layered MXene structure, significantly increasing the areal capacitance of the MXene/BC@PPy film electrode to 388 mF cm-2 , which is a 10-fold improvement from the pure MXene film electrode. Pairing with CNTs/MnO2 battery-type electrodes, the obtained ZHMSCs exhibit an areal energy density up to 145.4 µWh cm-2 with an outstanding 95.8% capacity retention after 25000 cycles, which is the highest among recently reported MXene-based MSCs and approaches the level of micro-batteries. The interlayer structure engineering demonstrated in the MXene-based capacitor-type electrode provides a rational means to achieve battery-levelenergy density in the ZHMSCs.

A Three-Dimensional Co-Culture Model for Rheumatoid Arthritis Pannus Tissue.

Three-dimensional (3D) co-culture models have closer physiological cell composition and behavior than traditional 2D culture. They exhibit pharmacological effects like in vivo responses, and therefore serve as a high-throughput drug screening model to evaluate drug efficacy and safety in vitro. In this study, we created a 3D co-culture environment to mimic pathological characteristics of rheumatoid arthritis (RA) pannus tissue. 3D scaffold was constructed by bioprinting technology with synovial fibroblasts (MH7A), vascular endothelial cells (EA.hy 926) and gelatin/alginate hydrogels. Cell viability was observed during 7-day culture and the proliferation rate of co-culture cells showed a stable increase stage. Cell-cell interactions were evaluated in the 3D printed scaffold and we found that spheroid size increased with time. TNF-α stimulated MH7A and EA.hy 926 in 3D pannus model showed higher vascular endothelial growth factor (VEGF) and angiopoietin (ANG) protein expression over time. For drug validation, methotrexate (MTX) was used to examine inhibition effects of angiogenesis in 3D pannus co-culture model. In conclusion, this 3D co-culture pannus model with biological characteristics may help the development of anti-RA drug research.

Treatment of collagen-induced arthritis rat model by using Notch signalling inhibitor.

BACKGROUND: The Notch signalling pathway has been reported to play a key role in rheumatoid arthritis (RA) development. Thus, inhibition of the activation of this signalling pathway may be a promising approach to the treatment of RA. In this study, the Notch signalling inhibitor LY411575, which can inhibit both Notch1 and Notch3, was used for the treatment of collagen-induced arthritis (CIA) rats. METHODS: Wistar rats were immunised with bovine type II collagen (CII) to establish rats CIA model. The inhibitory effects of LY411575 on Notch1 intracellular domain (N1ICD) and Notch3 intracellular domain (N3ICD) protein was verified by western blot (WB) in vitro. CIA rats were treated with different doses of LY411575 for 15 and 28 days, respectively. Methotrexate and sodium carboxymethyl cellulose (CMC-Na) were used as positive and negative (vehicle) control respectively. Destruction of the rat ankle joint and the bone loss on the periarticular side were evaluated by micro-computed tomography (Micro-CT). In addition, destruction of the ankle articular cartilage and the osteoclast numbers were determined by histology. Expression of N1ICD and N3ICD in the ankle joint was detected by immunohistochemistry. RESULTS: LY411575 could significantly inhibit the expression of N1ICD and N3ICD in vitro. Micro-CT test showed that the ankle joint destruction significantly improved after treatment with LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively). The bone quality in the LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively) groups were improved compared with the vehicle group. Histological analysis showed that LY411575 (5 â€‹mg/kg and 10 â€‹mg/kg, respectively) treatment reduced the severity of ankle joint inflammation in CIA rats (including ankle joint destruction, pannus formation, and cartilage damage) and reduced the expression of N1ICD and N3ICD in CIA rats ankle joints significantly. CONCLUSION: The inhibitor of Notch signalling LY411575 is an effective treatment for CIA. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our study provides new evidence to support the potential clinical application of Notch signalling pathway inhibitor LY411575 as a drug candidate for the treatment of RA.

Repurposing of Pirfenidone (Anti-Pulmonary Fibrosis Drug) for Treatment of Rheumatoid Arthritis.

Clinical studies have shown that pirfenidone (PFD) effectively relieves joint pain in rheumatoid arthritis (RA) patients. However, the detailed mechanisms underlying the anti-RA effects of PFD have not been investigated. This study was undertaken to investigate the repurposing of PFD for the treatment of RA, and explore its anti-rheumatic mechanisms. A collagen-induced arthritis (CIA) rat model was used to observe joint pathological changes following PFD treatment. Based on bioinformatics to predict the mechanism of PFD anti-RA, using EA. hy926 and TNF-α-induced MH7A cells to establish in vitro model to explore its biological mechanism from the perspectives of synovial inflammation and angiogenesis. PFD significantly relieved pathological changes, including joint swelling, synovial hyperplasia, inflammatory cell infiltration and joint destruction. PFD was also associated with reduced expression of MMP-3 and VEGF in articular chondrocytes and synovial cells of CIA rats (p < 0.05). Using bioinformatic methods, we predicted that PFD inhibits cell inflammation and migration by interfering with the JAK2/STAT3 and Akt pathways. These results were verified using in vitro models. In particular, PFD effectively reduced the expression of pro-inflammatory, chondrogenic, and angiogenic cytokines, such as IL-1ß, IL-6, IL-8, MMP-1/3/2/9 and VEGF (p < 0.05), in TNF-α-induced MH7A cells. In addition, PFD significantly reduced the production of MMP-2/9 and VEGF in EA. hy926 cells, thereby weakening migration and inhibiting angiogenesis (p < 0.05). These findings suggest that PFD may alleviate the pathological process in CIA rats, by inhibiting inflammation and angiogenesis through multiple pathways, and serve as a potential therapeutic drug for RA.

Biphasic Effect of Pirfenidone on Angiogenesis.

Pirfenidone (PFD), a synthetic arsenic compound, has been found to inhibit angiogenesis at high concentrations. However, the biphasic effects of different PFD concentrations on angiogenesis have not yet been elucidated, and the present study used an in vitro model to explore the mechanisms underlying this biphasic response. The effect of PFD on the initial angiogenesis of vascular endothelial cells was investigated through a Matrigel tube formation assay, and the impact of PFD on endothelial cell migration was evaluated through scratch and transwell migration experiments. Moreover, the expression of key migration cytokines, matrix metalloproteinase (MMP)-2 and MMP-9, was examined. Finally, the biphasic mechanism of PFD on angiogenesis was explored through cell signaling and apoptosis analyses. The results showed that 10-100 µM PFD has a significant and dose-dependent inhibitory effect on tube formation and migration, while 10 nM-1 µM PFD significantly promoted tube formation and migration, with 100 nM PFD having the strongest effect. Additionally, we found that a high concentration of PFD could significantly inhibit MMP-2 and MMP-9 expression, while low concentrations of PFD significantly promoted their expression. Finally, we found that high concentrations of PFD inhibited EA.hy926 cell tube formation by promoting apoptosis, while low concentrations of PFD promoted tube formation by increasing MMP-2 and MMP-9 protein expression predominantly via the EGFR/p-p38 pathway. Overall, PFD elicits a biphasic effect on angiogenesis through different mechanisms, could be used as a new potential drug for the treatment of vascular diseases.

The effect and mechanism of QufengZhitong capsule for the treatment of osteoarthritis in a rat model.

OBJECTIVE: The purpose of this study was to evaluate the therapeutic effects and mechanism of Qufeng Zhitong (QFZT)capsule for the treatment of osteoarthritis (OA) in a rat model. METHODS: 8-10-week-old male Sprague-Dawley rats were randomly divided into the sham group (vehicle-treated), OA group (vehicle-treated), high-dose, middle-dose, low-dose of QFZT capsule-treated groups. OA was induced by transecting the medial collateral ligament and the medial meniscus in the right limb. The Sprague-Dawley rats were treated daily for 12 weeks with different concentrations of QFZT capsule: low (QFZT-L, 128 â€‹mg/kg), medium (QFZT-M, 256.5 â€‹mg/kg), and high (QFZT-H, 513 â€‹mg/kg) by gavage administration for a period of 4 and 12 weeks respectively. Vehicle-treated rats served as controls and administered 0.5% Carboxymethyl Cellulose Sodium (CMC-Na) by gavage on the same schedule. Weekly measurement of dynamic weight-bearing capacity, grip strength, joint swelling was were performed to monitor the progression of disease for 3 weeks. After euthanasia, the knee joints were articular cartilage changes. Pro-inflammatory gene expression in synovial joints was examined to assess the bone and cartilage changes. Gene expression of pro-inflammatory cytokines in synovial joints was measured to determine the therapeutic effect of QFZT. RESULTS: 2 weeks after the treatment, the grip strength and weight-bearing capacity were significantly increased in the QFZT-M and QFZT-H groups, compared with the OA group. The joint widths were decreased significantly in the QFZT-L and QFZT- H groups, compared with the OA group as well. The mRNA level in the articular cartilage of knee joint of IL-1ß in the QFZT-L group and IL-6 in the QFZT-H group was significantly suppressed at week 4, compared with the OA group. The radiology score was significantly decreased in the QFZT-H group compared with the OA group 12 weeks after treatment. Furthermore, the rats on QFZT treatment decreased the progression of OA, which was characterised by decreased cartilage degradation. However, the bone changes were no different in OA group and QFZT groups. CONCLUSION: In a rat model of OA, QFZT capsule shows the tendency to reduce the destruction of cartilage, joint swelling and bone erosion which provides new evidence for the therapeutic potential of QFZT capsule in the treatment of OA in clinics. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The QFZT capsule can improve the symptoms of the OA in rodent animal rats by attenuating pain and retarding cartilage damage. This study indicated that the QFZT capsule has the potential clinical application of in OA therapy.