Rutin is a potent senomorphic agent to target senescent cells and can improve chemotherapeutic efficacy.

Aging is a major risk factor for most chronic disorders, for which cellular senescence is one of the central hallmarks. Senescent cells develop the pro-inflammatory senescence-associated secretory phenotype (SASP), which significantly contributes to organismal aging and age-related disorders. Development of senotherapeutics, an emerging class of therapeutic agents to target senescent cells, allows to effectively delay aging and alleviate chronic pathologies. Here we report preliminary outputs from screening of a natural medicinal agent (NMA) library for senotherapeutic candidates and validated several agents with prominent potential as senomorphics. Rutin, a phytochemical constituent found in a number of plants, showed remarkable capacity in targeting senescent cells by dampening expression of the full spectrum SASP. Further analysis indicated that rutin restrains the acute stress-associated phenotype (ASAP) by specifically interfering with the interactions of ATM with HIF1α, a master regulator of cellular and systemic homeostasis activated during senescence, and of ATM with TRAF6, part of a key signaling axis supporting the ASAP development toward the SASP. Conditioned media produced by senescent stromal cells enhanced the malignant phenotypes of prostate cancer cells, including in vitro proliferation, migration, invasion, and more importantly, chemoresistance, while rutin remarkably downregulated these gain-of-functions. Although classic chemotherapy reduced tumor progression, the treatment outcome was substantially improved upon combination of a chemotherapeutic agent with rutin. Our study provides a proof of concept for rutin as an emerging natural senomorphic agent, and presents an effective therapeutic avenue for alleviating age-related pathologies including cancer.

PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy.

Senescent cells remain metabolically active, but their metabolic landscape and resulting implications remain underexplored. Here, we report upregulation of pyruvate dehydrogenase kinase 4 (PDK4) upon senescence, particularly in some stromal cell lines. Senescent cells display a PDK4-dependent increase in aerobic glycolysis and enhanced lactate production but maintain mitochondrial respiration and redox activity, thus adopting a special form of metabolic reprogramming. Medium from PDK4+ stromal cells promotes the malignancy of recipient cancer cells in vitro, whereas inhibition of PDK4 causes tumor regression in vivo. We find that lactate promotes reactive oxygen species production via NOX1 to drive the senescence-associated secretory phenotype, whereas PDK4 suppression reduces DNA damage severity and restrains the senescence-associated secretory phenotype. In preclinical trials, PDK4 inhibition alleviates physical dysfunction and prevents age-associated frailty. Together, our study confirms the hypercatabolic nature of senescent cells and reveals a metabolic link between cellular senescence, lactate production, and possibly, age-related pathologies, including but not limited to cancer.

Combinational study with network pharmacology, molecular docking and preliminary experiments on exploring common mechanisms underlying the effects of weijing decoction on various pulmonary diseases.

Objective: 'Homotherapy for heteropathy' is a theory by which different diseases with similar pathogenesis can be treated with one Chinese formula. We aimed to explore the key components and core targets of Weijing decoction (WJD) in treating various lung diseases, namely, pneumonia, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), pulmonary fibrosis, pulmonary tuberculosis and non-small cell lung cancer (NSCLC), via network pharmacology, molecular docking and some experiments. Significance: This is the first study on the mechanism of WJD in treating various lung diseases by 'homotherapy for heteropathy'. This study is helpful for the transformation of TCM formula and development of new drugs. Methods: Active components and therapeutic targets of WJD were obtained via TCMSP and UniProt databases. Targets of the six pulmonary diseases were harvested from the GeneCards TTD, DisGeNet, UniProt and OMIM databases. Drug-disease intersection targets, corresponding Venn diagrams, herb-component-target networks and protein-protein interaction networks were established. Furthermore, GO biological function and KEGG enrichment analysis were completed. Moreover, the binding activity between main compounds and core targets was measured through molecular docking. Finally, the xenograft NSCLC mouse model was established. Immune responses were evaluated by flow cytometry and mRNA expression levels of critical targets were measured by real-time PCR. Results: JUN, CASP3 and PTGS2 were the most critical targets in six pulmonary diseases. The active compounds beta-sitosterol, tricin and stigmasterol stably bound to many active sites on target proteins. WJD had extensive pharmacological regulation, involving pathways related to cancer, inflammation, infection, hypoxia, immunity and so on. Conclusions: Effects of WJD against various lung diseases involve lots of compounds, targets and pathways. These findings will facilitate further research as well as clinical application of WJD.

Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Network Pharmacology-Based Exploration on the Intervention of Qinghao Biejia Decoction on the Inflammation-Carcinoma Transformation Process of Chronic Liver Disease via MAPK and PI3k/AKT Pathway.

Chronic liver disease(CLD) is a slow-developing and long-term disease that can cause serious damage to the liver. Thus far, it has been associated with viral hepatitis, non-alcoholic fatty liver disease(NAFLD), alcoholic liver disease(ALD), hepatic fibrosis(HF), liver cirrhosis (LC), and liver cancer. Qinghao Biejia Decoction (QBD) is a classic ancient Chinese herbal prescription with strong immune-enhancing, anti-inflammatory, and anti-tumor effects. In this study, we used a network pharmacology approach to investigate the molecular mechanisms of QBD in the inflammation-carcinoma transformation process of chronic liver disease. Two key drug targets, MAPK1 and PIK3CA, were screened using network pharmacology and molecular docking techniques, revealing dihydroartemisinin, artesunate, 12-O-Nicotinoylisolineolone, caffeic acid, and diincarvilone A as active ingredients involved in QBD mechanisms. The main signaling pathways involved were the PI3K-AKT signaling pathway and MAPK signaling pathway. In summary, our results indicated that QBD affects the inflammatory transformation of chronic liver disease through MAPK1 and PIK3CA and signaling pathways MAPK and PI3K/AKT. These data provide research direction for investigating the mechanisms underlying the inflammation-carcinoma transformation process in QBD for chronic liver disease.

Qualitative and quantitative analysis of the major components in Qinghao Biejia decoction by UPLC-Orbitrap Fusion-MS/MS and UPLC-QQQ-MS/MS and evaluation of their antibacterial activities.

OBJECTIVE: In the present study, the chemical components of Qinghao Biejia decoction (QBD) were qualitatively and quantitatively analyzed using UPLC-Orbitrap Fusion-MS/MS and UPLC-QQQ-MS/MS techniques, followed by identification of each component's origin and evaluation of the antibacterial activity of QBD and its components. METHODS: High-resolution mass spectrometry was used to obtain information on the precise molecular weight, retention time, and fragmentation ion peaks of the compounds used to identify the components of QBD and establish a method for their quantification. In vitro assays including determination of the minimal inhibitory concentration and growth curves were used to assess the antibacterial activity of QBD and its components. RESULTS: A total of 39 components, including fatty acids, phenolic acids, amino acids, flavonoids, coumarins, terpenoids, and alkaloids, were identified by UPLC-Orbitrap Fusion-MS/MS. A high-performance analytical method was also established to quantify 12 components of QBD. The content of mangiferin was relatively high (estimated to be 814 µg/g). The results of the antibacterial assays indicated that mangiferin exhibits antibacterial effects against two strains causing respiratory tract infections. CONCLUSIONS: The present study suggests that mangiferin may serve as a natural compound which shows high antibacterial activity. The results can aid the discovery and analysis of the active antimicrobial components present in QBD and further provide a reference for quality assessment of multi-component herbal prescriptions.

Ultrastructural analysis of different skeletal cell types in mucopolysaccharidosis dogs at the onset of postnatal growth.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Epiphyseal cartilage canal architecture and extracellular matrix remodeling in mucopolysaccharidosis VII dogs at the onset of postnatal growth.

Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII.Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining.Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII.Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.

Altered IHH signaling contributes to reduced chondrocyte proliferation in the growth plate of MPS VII mice.

Bone elongation is driven by chondrocyte proliferation and hypertrophy in the growth plate. Both processes are modulated by multiple signaling pathways including the Indian Hedgehog (IHH) signaling pathway. Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders characterized by accumulation of glycosaminoglycans (GAGs) in multiple tissues and organs, leading to a range of clinical symptoms including bone shortening through mechanisms that are not fully understood. Using MPS VII mice, we previously observed a reduction in the number of proliferating and hypertrophic chondrocytes and a reduced gene expression of Ihh in the tibial growth plate. We further demonstrate here that IHH secretion by MPS VII chondrocytes was reduced both in vitro and in vivo. While normal chondrocytes showed no response to exogenous IHH, proliferation of MPS VII chondrocytes was stimulated in response to exogenous IHH in vitro. This was accompanied by an elevated gene expression of patched receptor (Ptch1). The results from this study suggested that reduced proliferation in MPS VII growth plate may be partially due to dysfunction of the IHH signaling pathway.

Failures of Endochondral Ossification in the Mucopolysaccharidoses.

PURPOSE OF REVIEW: The mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders characterized by abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. MPS patients frequently exhibit failures of endochondral ossification during postnatal growth leading to skeletal deformity and short stature. In this review, we outline the current understanding of the cellular and molecular mechanisms underlying failures of endochondral ossification in MPS and discuss associated treatment challenges and opportunities. RECENT FINDINGS: Studies in MPS patients and animal models have demonstrated that skeletal cells and tissues exhibit significantly elevated GAG storage from early in postnatal life and that this is associated with impaired cartilage-to-bone conversion in primary and secondary ossification centers, and growth plate dysfunction. Recent studies have begun to elucidate the underlying cellular and molecular mechanisms, including impaired chondrocyte proliferation and hypertrophy, diminished growth factor signaling, disrupted cell cycle progression, impaired autophagy, and increased cell stress and apoptosis. Current treatments such as hematopoietic stem cell transplantation and enzyme replacement therapy fail to normalize endochondral ossification in MPS. Emerging treatments including gene therapy and small molecule-based approaches hold significant promise in this regard. Failures of endochondral ossification contribute to skeletal deformity and short stature in MPS patients, increasing mortality and reducing quality of life. Early intervention is crucial for effective treatment, and there is a critical need for new approaches that normalize endochondral ossification by directly targeting affected cells and signaling pathways.

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-ß, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

Cell cycle progression is disrupted in murine MPS VII growth plate leading to reduced chondrocyte proliferation and transition to hypertrophy.

Endochondral bone growth is abnormal in 6 of the 11 types of mucopolysaccharidoses (MPS) disorders; resulting in short stature, reduced size of the thoracic cavity and compromised manual dexterity. Current therapies for MPS have had a limited effect on bone growth and to improve these therapies or develop adjunct approaches requires an understanding of the underlying basis of abnormal bone growth in MPS. The MPS VII mouse model replicates the reduction in long bone and vertebral length observed in human MPS. Using this model we have shown that the growth plate is elongated but contains fewer chondrocytes in the proliferative and hypertrophic zones. Endochondral bone growth is in part regulated by entry and exit from the cell cycle by growth plate chondrocytes. More MPS VII chondrocytes were positive for Ki67, a marker for active phases of the cell cycle, suggesting that more MPS VII chondrocytes were in the cell cycle. The number of cells positive for phosphorylated histone H3 was significantly reduced in MPS VII chondrocytes, suggesting fewer MPS VII chondrocytes progressed to mitotic division. While MPS VII HZ chondrocytes continued to express cyclin D1 and more cells were positive for E2F1 and phos pRb than normal, fewer MPS VII HZ chondrocytes were positive for p57kip2 a marker of terminal differentiation, suggesting fewer MPS VII chondrocytes were able to exit the cell cycle. In addition, multiple markers typical of PZ to HZ transition were not downregulated in MPS VII, in particular Sox9, Pthrpr and Wnt5a. These findings are consistent with MPS VII growth plates elongating at a slower rate than normal due to a delay in progression through the cell cycle, in particular the transition between G1 and S phases, leading to both reduced cell division and transition to the hypertrophic phenotype.

Delayed development of ossification centers in the tibia of prenatal and early postnatal MPS VII mice.

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII.