Experimental training in molecular pharmacology education based on drug-target interactions.

INTRODUCTION: Drug development has been challenged by the dual drawbacks involving unpredictable disease outcomes and drug resistance, which has placed greater demands on pharmacology education. Molecular pharmacology, as a frontier crossover field of pharmacology, focuses on the research of new drugs and targets. However, due to the lack of a systematic experimental training system, molecular pharmacology has not made a corresponding contribution in promoting the training of innovative talent in pharmacology. We aim to establish an experimental training program suitable for molecular pharmacology to improve students' ability to engage in drug development in future. METHODS: Based on the feasibility of drug-target projects, a comprehensive training program containing molecular docking, target stability experiment, and fluorescent probe detection of protein expression in living cells and mice was conducted among 20 pharmacy graduate students. The experimental training was assessed by the experimental training report and the student recognition questionnaires. RESULTS: All 20 students mastered the experimental principles and operations required for the training program. The experimental reports proved that the students were in good command of the experimental principles, operations and applications. The results of the Likert questionnaire indicated that the training program promoted the understanding of the drug research process and increased motivation to learn. CONCLUSION: The designed experimental training program has a positive effect on the training of pharmacology talents, and can be implemented as a part of molecular pharmacology education.

Evaluation of 18F-FAPI-04 Imaging in Assessing the Therapeutic Response of Rheumatoid Arthritis.

PURPOSE: Fibroblast activating protein (FAP) is highly expressed in the synovial tissues of rheumatoid arthritis (RA) patients. The aim of this study was to determine the feasibility of PET imaging with an Al[18F] F-NOTA-labeled FAP inhibitor 04(18F-FAPI-04) for the evaluation of arthritic progression and therapeutic response in experimental arthritis. METHODS: Fibroblast-like synoviocytes (FLSs) were obtained from patients with RA or osteoarthritis (OA), and the relationship between 18F-FAPI-04 uptake and the inflammatory activity of RA FLSs was investigated. Collagen-induce arthritis (CIA) mice models were established and treated with methotrexate (MTX) or etanercept (ETC). Then, PET imaging was performed 24 h following 18F-FAPI-04 injection. The imaging results were compared by assessing macroscopic arthritis scores and histological staining. RESULTS: 18F-FAPI-04 uptake was obvious in RA FLSs that characterizing FAP activation. The higher the uptake of 18F-FAPI-04, the more severity of the inflammatory phenotype in RA FLS. Furthermore, the uptake of 18F-FAPI-04 in inflamed joints could be found even before the deformity of the parental joints could be observed by histological examination. Both MTX and ETC were effective in inhibiting the progression of arthritis in CIA mice was confirmed by macroscopic, histological, and radiographic pathology scores. Importantly, 18F-FAPI-04 uptake declined accordingly in CIA models following MTX and ETC treatment. CONCLUSIONS: These findings suggest that PET imaging of 18F-FAPI-04 can be used to monitor treatment response in RA, and is more sensitive in disease speculation than macroscopic arthritis scoring.

ATF6α contributes to rheumatoid arthritis by inducing inflammatory cytokine production and apoptosis resistance.

Objective: The contribution of activating transcription factor 6α (ATF6α) in rheumatoid arthritis (RA) pathogenesis, especially on fibroblast-like synoviocytes (FLSs), has been suggested by its sensitivity to inflammatory stimulus. However, the exact role and therapeutic potential of ATF6α in RA remains to be fully elucidated. Methods: ATF6α expression was determined in joint tissues and FLS, and gain-of-function and loss-of-function analyses were applied to evaluate the biological roles of ATF6α in RA FLSs. A murine collagen-induced arthritis (CIA) model, combining both gene deletion of ATF6α and treatment with the ATF6α inhibitor Ceapin-A7, was employed. Joint inflammation, tissue destruction, circulating levels of inflammatory cytokines were assessed in CIA mice. Transcriptome sequencing analysis (RNASeq), molecular biology, and biochemical approaches were performed to identify target genes of ATF6α. Results: ATF6α expression was significantly increased in synovium of RA patients and in synovium of mice subjected to CIA. ATF6α silencing or inhibition repressed RA FLSs viability and cytokine production but induced the apoptosis. CIA-model mice with ATF6α deficiency displayed decreased arthritic progression, leading to profound reductions in clinical and proinflammatory markers in the joints. Pharmacological treatment of mice with Ceapin-A7 reduced arthritis severity in CIA models. RNA-sequencing of wild-type and knockdown of ATF6α in RA FLSs revealed a transcriptional program that promotes inflammation and suppresses apoptosis, and subsequent experiments identified Baculoviral IAP Repeat Containing 3 (BIRC3) as the direct target for ATF6α. Conclusion: This study highlights the pathogenic role of ATF6α-BIRC3 axis in RA and identifies a novel pathway for new therapies against RA.

PET imaging to assess fibroblast activation protein inhibitor biodistribution: A training program adapted to pharmacology education.

In the process of pharmacology education, practical teaching is an important complement to theoretical teaching. These activities include the use of experimental animals to obtain certain pharmacological parameters or to help students understand certain classical concepts. However, the growing interest in laboratory animal welfare, the rapid development of pharmacology research and the challenges of cultivating innovative pharmacy talent create a need for innovative and flexible in vitro experiments for teaching purposes. Here, we report the application of positron emission tomography (PET) imaging of 18 F-labeled fibroblast activation protein inhibitor (18 F-FAPi) to practical pharmacology teaching, enabling dynamic visualization of the distribution and excretion process of FAPi in mice. Students can quantitatively analyze the distribution of FAPi in various tissues and organs without sacrificing the mice. Furthermore, the newly implemented method resulted in highly reproducible results and was generally appreciated by the students. Additionally, the application of PET imaging in pharmacokinetic teaching can not only greatly reduce the use of experimental animals but also need not sacrificing animals. Of note is that dynamic scanning data from this project can be used for online practical teaching during COVID-19 pandemic.

Preclinical evaluation and pilot clinical study of [18F]AlF-NOTA-FAPI-04 for PET imaging of rheumatoid arthritis.

PURPOSE: Fibroblast-like synoviocytes (FLSs) are key effector cells in the inflamed joints of patients with rheumatoid arthritis (RA). Previous studies have suggested that fibroblast activation protein (FAP) is highly expressed in RA-derived FLSs and is a specific marker of activated RA FLSs. In this study, we developed aluminum-[18F]-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid-conjugated FAP inhibitor 04 ([18F]AlF-NOTA-FAPI-04) to image RA-FLSs in vitro and arthritic joints in collagen-induced arthritis (CIA) mice and RA patients. METHODS: RA FLSs and NIH3T3 cells transfected with FAP were used to perform in vitro-binding studies. Biodistribution was conducted in normal DBA1 mice. Collagen-induced arthritis (CIA) models with different arthritis scores were subjected to [18F]AlF-NOTA-FAPI-04 and 18F-FDG PET imaging. Histological examinations were performed to evaluate FAP expression and Cy3 dye-labeled FAPI-04(Cy3-FAPI-04) uptake. Blocking studies with excess unlabeled FAPI-04 in CIA mice and NIH3T3 xenografts in immunocompromised mice were used to evaluate the binding specificity of [18F]AlF-NOTA-FAPI-04. Additionally, [18F]AlF-NOTA-FAPI-04 PET imaging was performed on two RA patients. RESULTS: The binding of [18F]AlF-NOTA-FAPI-04 increased significantly in RA FLSs and NIH3T3 cells overexpressing FAP compared to their parental controls (FAP-GFP-NIH3T3 vs. GFP-NIH3T3, 2.40 ± 0.078 vs. 0.297 ± 0.05% AD/105 cells; RA FLSs vs. OA FLSs, 1.54 ± 0.064 vs. 0.343 ± 0.056% AD/105 cells). Compared to 18F-FDG imaging, [18F]AlF-NOTA-FAPI-04 showed high uptake in inflamed joints in the early stage of arthritis, which was positively correlated with the arthritic scores (Pearson r=0.834, P<0.001). In addition, the binding of [18F]AlF-NOTA-FAPI-04 to cells with high FAP expression and the uptake of [18F]AlF-NOTA-FAPI-04 in arthritic joints both could be blocked by excessive unlabeled FAPI-04. Fluorescent staining showed that the intensity of Cy3-FAPI-04 binding to FAP increased accordingly as the expression of FAP protein increased in cells and tissue sections. Furthermore, the uptake of [18F]AlF-NOTA-FAPI-04 in FAP-GFP-NIH3T3 xenografts was significantly higher than that in GFP-NIH3T3 xenograft (35.44 ± 4.27 vs 7.92 ± 1.83% ID/mL). Finally, [18F]AlF-NOTA-FAPI-04 PET/CT imaging in RA patients revealed nonphysiologically high tracer uptake in the synovium of arthritic joints. CONCLUSION: [18F]AlF-NOTA-FAPI-04 is a promising radiotracer for imaging RA FLSs and could potentially complement the current noninvasive diagnostic parameters.

Euphorbia factor L3 ameliorates rheumatoid arthritis by suppressing the inflammatory response by targeting Rac family small GTPase 1.

Euphorbia factor L3 (EFL3) is extracted from Euphorbia lathyris and is known for its anti-inflammatory properties. This study focused on the potential anti-inflammatory and therapeutic effects of EFL3 on rheumatoid arthritis (RA) using fibroblast-like synoviocytes (FLSs) and arthritis animal models. Functional analysis showed that EFL3 could ameliorate the inflammatory phenotype of FLSs derived from RA patients, as evidenced by the decreases in cell viability, migration, invasion and cytokine production. Luciferase activity, Western blotting and immunofluorescence assays demonstrated that EFL3 inhibited the nuclear translocation of the p65 subunit and the subsequent activation of the nuclear factor kappa-Β (NF-κB) pathway. Furthermore, the therapeutic effects of EFL3 against arthritic progression were evidenced by decreases in joint swelling, arthritis scores, inflammatory factor production, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) and tumor necrosis factor-α (TNF-α) transgenic (TNF-tg) mouse models. Molecular analysis identified Rac family small GTPase 1 (Rac1) as the potential target that was required for EFL3-mediated suppression of the inflammatory RA FLS phenotype. In summary, this study uncovered the therapeutic potential of EFL3 in RA, which suggests its future clinical use.

ATF6α promotes prostate cancer progression by enhancing PLA2G4A-mediated arachidonic acid metabolism and protecting tumor cells against ferroptosis.

BACKGROUND: Despite the clinical success of androgen receptor (AR)-targeted therapies, prostate cancer (PCa) inevitably progresses to castration-resistant prostate cancer (CRPC). Transcription factor 6 α (ATF6α), an effector of the unfolded protein response (UPR) that modulates the cellular response to endoplasmic reticulum (ER) stress, has been linked to tumor development, metastasis, and relapse. However, the role of ATF6α in CRPC remains unclear. METHODS: The effect of ATF6α on the CRPC-like phenotype in PCa cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-Oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), 5-Bromo-2-deoxyUridine (BrdU) incorporation analysis, and cell death assay. Mechanistically, bioinformatic analysis was utilized to evaluate the potential of PLA2G4A as the target of ATF6α. Moreover, Western blot analysis, real-time polymerase chain reaction, chromatin immunoprecipitation, arachidonic acid (AA), and prostaglandin E2 (PGE2) assays were performed to identify the regulatory effect of ATF6α on PLA2G4A. RESULTS: In this study, we found that the increase of ATF6α expression in response to androgen deprivation generates PCa cells with a CRPC-like phenotype. PCa cells with high levels of ATF6α expression are resistant to ferroptosis, and genetic and pharmacological inhibition of ATF6α could, therefore, promote the ferroptotic death of tumor cells and delay PCa progression. Molecular analyses linked ATF6α regulation of ferroptosis to the PLA2G4A-mediated release of AA and the resulting increase in PGE2 production, the latter of which acts as an antiferroptotic factor. CONCLUSIONS: This study defines ATF6α as a novel antiferroptotic regulator that exacerbates PCa progression. In addition, our data establish ATF6α-PLA2G4A signaling as an important pathological pathway in PCa, and targeting this pathway may be a novel treatment strategy.

Azithromycin alleviates the severity of rheumatoid arthritis by targeting the unfolded protein response component of glucose-regulated protein 78 (GRP78).

BACKGROUND AND PURPOSE: Azithromycin is a macrolide antibiotic with anti-inflammatory properties. We aim to substantiate the treatment potential of azithromycin in rheumatoid arthritis. EXPERIMENTAL APPROACH: Gene expression profiles were collected by RNA sequencing and the effects of azithromycin were assessed by in vitro and in vivo assays on the effects of azithromycin-mediated blockade of glucose-regulated protein 78 (GRP78). Anti-inflammatory activity of azithromycin was measured in fibroblast-like synoviocytes from rheumatoid arthritis patients and in collagen-induced arthritis in DBA/1 mice. Characterization of the binding of azithromycin to GRP78 was performed using drug affinity responsive target stability, proteomics and cellular thermal shift assays. Azithromycin-mediated inhibition of GRP78 and its relationship to its anti-arthritic activity was assessed. KEY RESULTS: Azithromycin reduced proinflammatory factor production, cell migration, invasion and chemoattraction and enhanced apoptosis, reducing the deleterious inflammatory response of rheumatoid arthritis fibroblast-like synoviocytes in vitro. Azithromycin ameliorated the severity of collagen-induced arthritis lesions as efficiently as the TNFα inhibitor etanercept. Transcriptional analyses suggested that azithromycin treatment impairs signalling cascades associated with cholesterol and lipid biosynthesis. GRP78 was identified as a novel target of azithromycin. Azithromycin-mediated activation of the unfolded protein response via the inhibition of GRP78 activity is required not only for inducing the expression of C/EBP-homologous protein (ChOP) but also for the activating sterol-regulatory element binding protein (SREBP) and its targeted genes involved in cholesterol and lipid biosynthetic processes. Furthermore, deletion of GRP78 abolished the anti-arthritic activity of azithromycin. CONCLUSION AND IMPLICATIONS: These findings indicate that azithromycin can used to treat rheumatoid arthritis.

IL13Rα1 prevents a castration resistant phenotype of prostate cancer by targeting hexokinase 2 for ubiquitin-mediated degradation.

OBJECTIVE: Androgen deprivation therapy (ADT) is still the principal treatment option for prostate cancer (PCa). In addition to reactivation of androgen receptor signaling, the resistance of PCa to apoptosis during ADT also contributes to castration resistant PCa (CRPC). A previous study reported that gene transfer of IL-13Rα2 into PCa cells sensitized the cells to the IL-13R-targeted cytotoxin IL13Rα1, leading to apoptosis. Compared with IL-13Rα2, IL13Rα1 is more constitutively expressed in PCa cells, but its function in PCa remains to be established. METHODS: We determined the role and expression of IL13Rα1 in PCa cancer cells using western blotting, flow cytometry, and cell proliferation assays. Co-immunoprecipitation and mass spectrometry were used to identify the proteins that interacted with IL13Rα1, to elucidate its function. RESULTS: In this study, we showed that IL13Rα1 was selectively suppressed in androgen-deprived PCa cells and that its suppression tended to be associated with poor prognoses of PCa patients. IL13Rα1 overexpression promoted apoptosis and inhibited tumor growth under androgen-deprived or castrated conditions (P < 0.01). Mechanistically, IL13Rα1 recruited and facilitated ubiquitin protein ligase E3C-mediated ubiquitination and degradation of hexokinase 2 (HK2), resulting in glycolytic inhibition and eventually leading to PCa cell apoptosis. Furthermore, our data revealed that mutated ataxia-telangiectasia kinase phosphorylated and facilitated the selective ubiquitin proteasome-mediated degradation of HK2. Notably, IL13Rα1-overexpressing PCa cells were more susceptible to apoptosis and exhibited reduced tumor growth after exposure to the HK2 inhibitor, 2-deoxy-D-glucose (P < 0.01). CONCLUSIONS: Our data identified a tumor suppressor role for IL13Rα1 in preventing the resistance of PCa cells to apoptosis during androgen deprivation by inhibiting glycolysis. IL13Rα1-mediated signaling involving HK2 may therefore provide a novel treatment target and strategy for CRPC.

Isopsoralen ameliorates rheumatoid arthritis by targeting MIF.

BACKGROUND: Isopsoralen (IPRN), one of the active ingredients of Psoralea corylifolia Linn, has anti-inflammatory properties. We attempted to investigate the inhibitory effects of IPRN on rheumatoid arthritis (RA) and characterize its potential mechanism. METHODS: RA fibroblast-like synoviocytes (FLSs) and mice with collagen-induced arthritis (CIA) were used as in vitro and in vivo models to analyze the antiarthritic effect of IPRN. Histological analysis of the inflamed joints from mice with CIA was performed using microcomputed tomography (micro-CT) and hematoxylin-eosin (HE) staining. RNA sequencing (RNA-Seq), network pharmacology analysis, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to evaluate the targets of IPRN. RESULTS: IPRN ameliorated the inflammatory phenotype of RA FLSs by inhibiting their cytokine production, migration, invasion, and proangiogenic ability. IPRN also significantly reduced the severity of CIA in mice by decreasing paw thickness, arthritis score, bone damage, and serum inflammatory cytokine levels. A mechanistic study demonstrated that macrophage migration inhibitory factor (MIF), a key protein in the inflammatory process, was the specific target by which IPRN exerted its anti-inflammatory effects in RA FLSs. CONCLUSION: Our study demonstrates the antiarthritic effect of IPRN, which suggests the therapeutic potential of IPRN in RA.

IL13Rα1 protects against rheumatoid arthritis by combating the apoptotic resistance of fibroblast-like synoviocytes.

BACKGROUND: Endoplasmic reticulum (ER) stress is closely related with the pathological progression of rheumatoid arthritis (RA), and fibroblast-like synoviocytes (FLSs) are known as its resistance against ER stress-induced apoptosis. Studies on overcoming such resistance would provide a novel treatment strategy for RA in a clinical setting. METHODS: IL13Rα1 expression was assessed in the synovial tissue by RT-qPCR, immunohistology, and Western blot. Gain or loss of functional analysis was applied to evaluate the biological roles of IL13Rα1 in RA FLSs. Cell viability and apoptosis were assessed by MTS, Western blot, and flow cytometry. The therapeutic effects of IL13Rα1 on the severity of type II collagen-induced arthritis (CIA) in DBA-/1 mouse model were evaluated by scoring synovitis, hyperplasia, cartilage degradation, and bone destruction. RESULTS: IL13Rα1 expression was selectively downregulated when RA FLSs were stimulated by ER stress inducers. Functionally, IL13Rα1 overexpression could inhibit the viability, but induce the apoptosis of RA FLSs in the presence of ER stress inducers. Mechanistically, IL13Rα1 promotes cell apoptosis via transcriptionally activating trail expression. Besides, IL13Rα1 could interact and stabilize DR5 protein, thus forming a positive loop involving trail and DR5 to render RA FLSs more susceptible to apoptosis. Additionally, intraarticular injection of IL13Rα1 conferred therapeutic effects in CIA models and showed a limited degree of synovial proliferation and joint destruction. CONCLUSIONS: Together, our data establishes a regulatory role for IL13Rα1 to combat the apoptotic resistance of RA FLSs against ER stress. The inhibitory effects of IL13Rα1 on arthritis progression suggest the therapeutic potential in RA.

TXNDC5 protects synovial fibroblasts of rheumatoid arthritis from the detrimental effects of endoplasmic reticulum stress.

TXNDC5 is an endoplasmic reticulum (ER)-resident chaperone that protects the endothelium from secondary effects of ER stress. Previous studies by the current authors identified TXNDC5 as a key pathological factor in promoting the inflammatory phenotype of fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis (RA). However, its activity in RA FLSs under ER stress remains unclear. The current study found that TXNDC5 is responsive to ER stress in RA FLSs since its expression was induced by ER stress at both the endogenous and secretory level. A functional study indicated that silencing TXNDC5 reduced the viability of RA FLSs more markedly in the presence of ER stressors. In contrast, rhTXNDC5 attenuated a decrease in cell viability as a result of ER stress. Moreover, silencing TXNDC5 attenuated the induction of IL-6 and IL-8 from RA FLSs in response to ER stress. In addition, rhTXNDC5 induced a greater increase in VEGF production during ER stress. These findings confirm the pro-survival and pro-inflammation roles of TXNDC5 under ER stress in RA FLSs. TXNDC5 appears to act as a mediator linking ER stress and inflammation of RA.

A Network Pharmacology Approach for Uncovering the Osteogenic Mechanisms of Psoralea corylifolia Linn.

BACKGROUND AND AIM: Psoralea corylifolia Linn (PCL) is an herb that is commonly used for alleviating osteoporosis and vitiligo. Although accumulating evidence has demonstrated the antiosteoporotic effect of PCL, the identities of the osteogenic compounds in PCL and their functional targets remain elusive. To investigate the osteogenic ingredients in PCL and their functional mechanisms, network pharmacology analysis was performed on the targets of PCL and osteogenesis. METHODS: The active components of PCL were screened by literature review. The potential protein targets of the active PCL components were predicted with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Search Tool for Interactions of Chemicals (STITCH), SwissTargetPrediction, and PubChem. The target networks related to PCL and osteogenic differentiation were constructed by using Cytoscape. MC3T3-E1 cells were used to verify the targets. RESULTS: Twenty-three active components of PCL and 162 potential target proteins were identified. Further analysis reduced the number of potential target proteins to 71. Of the 23 components, bavachalcone, psoralen, bavachinin, neobavaisoflavone, methoxsalen, psoradin, bakuchiol, and angelicin may be the main active components of PCL that promote bone formation. PPARγ and aryl hydrocarbon receptor (AhR) were verified as targets of PCL in MC3T3-E1 cells, and the western blot and immunofluorescence staining results showed that compared to the control, PCL reduced the expression of these targets. CONCLUSIONS: The active components of PCL and the mechanisms by which they promoted osteogenic differentiation were successfully identified using network pharmacology.

CD109 regulates the inflammatory response and is required for the pathogenesis of rheumatoid arthritis.

OBJECTIVE: The aim of this study was to investigate the role of CD109 in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and to evaluate its potential as a therapeutic target. METHODS: CD109 expression was examined in synovial tissues and FLSs from RA patients and collagen-induced arthritis (CIA) model mice. CD109-deficient mice were developed to evaluate the severity of CIA. Small interfering RNAs and a neutralising antibody against CD109 (anti-CD109) were designed for functional or treatment studies in RA FLSs and CIA. RESULTS: CD109 was found to be abundantly expressed in the synovial tissues from RA patients and CIA mice. CD109 expression in RA FLSs was upregulated by inflammatory stimuli, such as interleukin-1ß and tumour necrosis factor-α. Silencing of CD109 or anti-CD109 treatment reduced proinflammatory factor production, cell migration, invasion, chemoattractive potential and osteoclast differentiation, thereby reducing the deleterious inflammatory response of RA FLSs in vitro. Mice lacking CD109 were protected against arthritis in the CIA model. Anti-CD109 treatment prevented the onset and ameliorated the severity of CIA lesions. CONCLUSION: Our study uncovers an antiarthritic role for CD109 and suggests that CD109 inhibition might serve as a promising novel therapeutic strategy for RA.

Bavachinin exhibits antitumor activity against non­small cell lung cancer by targeting PPARγ.

Bavachinin (BNN), one of the main active ingredients of Psoraleacorylifolia, can activate peroxisome proliferator­activated receptor Î³ (PPARγ). PPARγ has become a promising therapeutic target in cancer. The aim of the present study was to explore the antitumor effects of BNN in non­small cell lung cancer (NSCLC). Cell Counting Kit­8 and lactate dehydrogenase release assays were performed to measure cell toxicity. Western blotting and immunofluorescence were used to analyze the expression of apoptosis­related factors and PPARγ. The ability of PPARγ to bind to BNN was evaluated by drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). A reactive oxygen species (ROS) assay kit was used to detect the ROS level. The results revealed that the survival rates and cell viability of A549 cells were reduced by BNN in a dose­dependent manner. The present results also demonstrated that BNN dose­dependently changed the expression of Bcl­2, Bax, caspases­3/9 and PPARγ. In addition, through the cytotoxic and anti­proliferative effects, the apoptosis­related proteins' inhibitive properties of BNN were completely inhibited by the PPARγ antagonists T0070907 and GW9662. The DARTS and CETSA results confirmed the protein binding activity of PPARγ. Furthermore, it was demonstrated that the BNN­induced ROS generation was dependent on PPARγ activation. Taken together, the present study demonstrated that BNN induced the death of A549 cells by activating PPARγ, an effect mediated by the increased ROS level. These results highlighted the potential role of BNN as a chemotherapeutic agent against NSCLC.

Inhibition of hexokinases holds potential as treatment strategy for rheumatoid arthritis.

INTRODUCTION: Abnormal glycolytic metabolism contributes to joint inflammation and destruction in rheumatoid arthritis (RA). We examine the expression and function of hexokinases in RA and evaluate the potential of their specific inhibitor for clinical treatment. METHODS: Detection of HKs was assessed in synovial tissue by immunohistology and Western blot. SiRNA and a specific hexokinases inhibitor, lonidamine (LND), were used to evaluate the role of hexokinase-I/II (HK-I/II). Pro-inflammatory and glycolysis factors, cell viability, and apoptosis were assessed by ELISA, RT-qPCR, MTS, and flow cytometry. The clinical effects of LND on type II collagen-induced arthritis (CIA) in DBA-/1 mouse model was evaluated by scoring their clinical responses, synovitis, and cartilage destructions, and ELISA was employed to analyze the concentrations of antibody in the serum of CIA model. RESULTS: HK-I/II expression and their activities increased in the synovium of RA compared with osteoarthritis (OA). Silencing HK-I/II (siHK-I/II) or LND treatment decreased the production of pro-inflammatory factors, such as IL-6, IL-8, CXCL9, CXCL10, and CXCL11, and cell viability, but induced cell apoptosis of RASFs. The expression of TNF-α and IL-1ß of macrophage in response to LPS stimulation were depressed as well after treatment with siHK-I/II or LND. Furthermore, leucocyte infiltration co-cultured with RASFs was also suppressed after inhibiting the expression or activity of HK-I/II. These anti-inflammatory effects overlapped with their anti-glycolytic activities. Treatment with LND in mice with CIA decreased the production of antibodies against IgG1, IgG2a, and IgG2b and consequently attenuated joint inflammation and destruction. CONCLUSIONS: HK-I/II contribute to shape the inflammatory phenotype of RASFs and macrophages. LND may be a potential drug in treating patients with RA.

A protocol for isolation and identification and comparative characterization of primary osteoblasts from mouse and rat calvaria.

Calvaria from neonatal mouse and rat is ideal resource for osteoblasts but can be easily contaminated by other cells such as fibroblasts. Here, we established a protocol for isolation and purification of primary osteoblast by enzyme sequential digestion and differential adhesion. In addition, we compared the phenotypic and functional traits of osteoblasts from mouse and rat which are commonly employed in studies. The method applied equally to rat and mouse in osteoblasts isolation and was corroborated its feasibility and validity. The results also provided us evidences for other experiments such as choosing a certain time point to give intervention and do the relevant tests.

ERα and Wnt/ß­catenin signaling pathways are involved in angelicin­dependent promotion of osteogenesis.

Reports of the ameliorative effect of angelicin on sex hormone deficiency­induced osteoporosis have highlighted this compound as a candidate for the treatment of osteoporosis. However, the molecular mechanisms of action of angelicin on osteoblast differentiation have not been thoroughly researched. The aim of the present study was to evaluate the effect of angelicin on the proliferation, differentiation and mineralization of rat calvarial osteoblasts using a Cell Counting Kit­8, alkaline phosphatase activity and the expression of osteogenic genes and proteins. Treatment with angelicin promoted the proliferation, matrix mineralization and upregulation of osteogenic marker genes including collagen type I α 1 and bone γ­carboxyglutamate in fetal rat calvarial osteoblasts. Furthermore, angelicin promoted the expression of ß­catenin and runt related transcription factor 2, which serve a vital role in the Wnt/ß­catenin signaling pathway. Consistently, the osteogenic effect of angelicin was attenuated by the use of a Wnt inhibitor. Moreover, angelicin increased the expression of estrogen receptor α (ERα), which also serves a key role in osteoblast differentiation. Taken together, these results demonstrated that angelicin may promote osteoblast differentiation through activation of ERα and the Wnt/ß­catenin signaling pathway.

Isopsoralen Enhanced Osteogenesis by Targeting AhR/ERα.

Isopsoralen (IPRN), one of the main effective ingredients in Psoralea corylifolia Linn, has a variety of biological effects, including antiosteoporotic effects. In vivo studies show that IPRN can increase bone strength and trabecular bone microstructure in a sex hormone deficiency-induced osteoporosis model. However, the mechanism underlying this osteogenic potential has not been investigated in detail. In the present study, we investigated the molecular mechanism of IPRN-induced osteogenesis in MC3T3-E1 cells. Isopsoralen promoted osteoblast differentiation and mineralization, increased calcium nodule levels and alkaline phosphatase (ALP) activity and upregulated osteoblast markers, including ALP, runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 chain (COL1A1). Furthermore, IPRN limited the nucleocytoplasmic shuttling of aryl hydrocarbon receptor (AhR) by directly binding to AhR. The AhR target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was also inhibited in vitro and in vivo. This effect was inhibited by the AhR agonists indole-3-carbinol (I3C) and 3-methylcholanthrene (3MC). Moreover, IPRN also increased estrogen receptor alpha (ERα) expression in an AhR-dependent manner. Taken together, these results suggest that IPRN acts as an AhR antagonist and promotes osteoblast differentiation via the AhR/ERα axis.