α-Solanine attenuates chondrocyte pyroptosis to improve osteoarthritis via suppressing NF-κB pathway.

α-Solanine has been shown to exhibit anti-inflammatory and anti-tumour properties; however, its efficacy in treating osteoarthritis (OA) remains ambiguous. The study aimed to evaluate the therapeutic effects of α-solanine on OA development in a mouse OA model. The OA mice were subjected to varying concentrations of α-solanine, and various assessments were implemented to assess OA progression. We found that α-solanine significantly reduced osteophyte formation, subchondral sclerosis and OARSI score. And it decreased proteoglycan loss and calcification in articular cartilage. Specifically, α-solanine inhibited extracellular matrix degradation by downregulating collagen 10, matrix metalloproteinase 3 and 13, and upregulating collagen 2. Importantly, α-solanine reversed chondrocyte pyroptosis phenotype in articular cartilage of OA mice by inhibiting the elevated expressions of Caspase-1, Gsdmd and IL-1ß, while also mitigating aberrant angiogenesis and sensory innervation in subchondral bone. Mechanistically, α-solanine notably hindered the early stages of OA progression by reducing I-κB phosphorylation and nuclear translocation of p65, thereby inactivating NF-κB signalling. Our findings demonstrate the capability of α-solanine to disrupt chondrocyte pyroptosis and sensory innervation, thereby improving osteoarthritic pathological progress by inhibiting NF-κB signalling. These results suggest that α-solanine could serve as a promising therapeutic agent for OA treatment.

Atrazine exposure promotes cardiomyocyte pyroptosis to exacerbate cardiotoxicity by activating NF-κB pathway.

Atrazine is a ubiquitous herbicide with persistent environmental presence and accumulation in the food chain, posing potential health hazards to organisms. Increasing evidence suggests that atrazine may have detrimental effects on various organ systems, including the nervous, digestive, and immune systems. However, the specific toxicity and underlying mechanism of atrazine-induced cardiac injury remain obscure. In this study, 4-week-old male C57BL/6 mice were administered atrazine via intragastric administration at doses of 50 and 200 mg/kg for 4 and 8 weeks, respectively. Our findings showed that atrazine exposure led to cardiac fibrosis, as evidenced by elevated heart index and histopathological scores, extensive myofiber damage, and interstitial collagen deposition. Moreover, atrazine induced cardiomyocyte apoptosis, macrophage infiltration, and excessive production of inflammatory factors. Importantly, atrazine upregulated the expressions of crucial pyroptosis proteins, including NLRP3, ASC, CASPASE1, and GSDMD, via the activation of NF-κB pathway, thus promoting cardiomyocyte pyroptosis. Collectively, our findings provide novel evidence demonstrating that atrazine may exacerbate myocardial fibrosis by inducing cardiomyocyte pyroptosis, highlighting its potential role in the development of cardiac fibrosis.

Cornus officinalis: a potential herb for treatment of osteoporosis.

Osteoporosis (OP) is a systemic metabolic skeletal disorder characterized by a decline in bone mass, bone mineral density, and deterioration of bone microstructure. It is prevalent among the elderly, particularly postmenopausal women, and poses a substantial burden to patients and society due to the high incidence of fragility fractures. Kidney-tonifying Traditional Chinese medicine (TCM) has long been utilized for OP prevention and treatment. In contrast to conventional approaches such as hormone replacement therapy, TCM offers distinct advantages such as minimal side effects, low toxicity, excellent tolerability, and suitability for long-term administration. Extensive experimental evidence supports the efficacy of kidney-tonifying TCM, exemplified by formulations based on the renowned herb Cornus officinalis and its bioactive constituents, including morroniside, sweroside, flavonol kaempferol, Cornuside I, in OP treatment. In this review, we provide a comprehensive elucidation of the underlying pathological principles governing OP, with particular emphasis on bone marrow mesenchymal stem cells, the homeostasis of osteogenic and osteoclastic, and the regulation of vascular and immune systems, all of which critically influence bone homeostasis. Furthermore, the therapeutic mechanisms of Cornus officinalis-based TCM formulations and Cornus officinalis-derived active constituents are discussed. In conclusion, this review aims to enhance understanding of the pharmacological mechanisms responsible for the anti-OP effects of kidney-tonifying TCM, specifically focusing on Cornus officinalis, and seeks to explore more efficacious and safer treatment strategies for OP.

Inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, offering a therapeutic target for osteoporosis.

BACKGROUND: Pyroptosis, an emerging inflammatory form of cell death, has been previously demonstrated to stimulate a massive inflammatory response, thus hindering the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Nevertheless, the impact of pyroptosis in thwarting osteogenic differentiation and exacerbating the advancement of osteoporosis (OP) remains enigmatic. METHODS: We evaluated the expression levels of pyroptosis-associated indicators, including NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), CASPASE-1, IL-1ß, and IL-18, in specimens obtained from femoral heads of OP patients, as well as in an ovariectomy-induced mouse model of OP. Subsequently, the precise roles of pyroptosis in osteogenic differentiation were investigated using bioinformatics analysis, alongside morphological and biochemical assessments. RESULTS: The pivotal pyroptotic proteins, including NLRP3, Caspase-1, IL-1ß, and IL-18, exhibited significant upregulation within the bone tissue samples of clinical OP cases, as well as in the femoral tissues of ovariectomy (OVX)-induced mouse OP model, displaying a negatively associated with compromised osteogenic capacity, as represented by lessened bone mass, suppressed expression of osteogenic proteins such as Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), Osterix (OSX), and Osteopontin (OPN), and increased lipid droplets. Moreover, bioinformatics analysis substantiated shared gene expression patterns between pyroptosis and OP pathology, encompassing NLRP3, Caspase-1, IL-1ß, IL-18, etc. Furthermore, our in vitro investigation using ST2 cells revealed that dexamethasone treatment prominently induced pyroptosis while impeding osteogenic differentiation. Notably, gene silencing of Caspase-1 effectively counteracted the inhibitory effects of dexamethasone on osteogenic differentiation, as manifested by increased ALP activity and enhanced expression of RUNX2, ALP, OSX, and OPN. CONCLUSION: Our findings unequivocally underscore that inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, providing a promising therapeutic target for managing OP.

Study on Deterioration Characteristics of a Composite Crossarm Mandrel in a 10 kV Distribution Network Based on Multi-Factor Aging.

This paper presents a study that conducted 5000 h of multi-factor aging tests on 10 kV composite crossarms, considering the natural environment in coastal areas and actual power line operations. Various aging conditions, such as voltage, rain, temperature, humidity, salt fog, ultraviolet light, and mechanical stress, were applied during the tests. The research initially analyzed the influence of multi-factor aging on the bending and tensile properties of the full-size composite crossarm. Subsequently, a detailed investigation was carried out to assess the impact of aging on the mechanical properties, electrical insulation properties, and microscopic characteristics of the composite crossarm core bar. Results indicated that the tensile strength and bending strength of the full-size composite crossarm mandrel experienced minimal changes after aging, remaining well within operational requirements. However, the silicone rubber outer sheath's hydrophobicity decreased, leading to the appearance of cracks and holes on the surface, which provided pathways for moisture and salt infiltration into the mandrel. As a consequence, the bending strength and shear strength of the mandrel material were reduced by 16.5% and 37.7%, respectively. Moreover, the electrical performance test demonstrated a slight change in the mandrel's leakage current, while the electrical breakdown strength decreased by 22.8%. Microscopic analysis using SEM, three-dimensional CT, and TGA revealed that a small amount of resin matrix decomposed and microcracks appeared on the surface. Additionally, the fiber-matrix interface experienced debonding and cracking, leading to an increased moisture absorption rate of the mandrel material.

AR/PCC herb pair inhibits osteoblast pyroptosis to alleviate diabetes-related osteoporosis by activating Nrf2/Keap1 pathway.

Osteoporosis is a prevalent complication of diabetes, characterized by systemic metabolic impairment of bone mass and microarchitecture, particularly in the spine. Anemarrhenae Rhizoma/Phellodendri Chinensis Cortex (AR/PCC) herb pair has been extensively employed in Traditional Chinese Medicine to manage diabetes; however, its potential to ameliorate diabetic osteoporosis (DOP) has remained obscure. Herein, we explored the protective efficacy of AR/PCC herb pair against DOP using a streptozotocin (STZ)-induced rat diabetic model. Our data showed that AR/PCC could effectively reduce the elevated fasting blood glucose and reverse the osteoporotic phenotype of diabetic rats, resulting in significant improvements in vertebral trabecular area percentage, trabecular thickness and trabecular number, while reducing trabecular separation. Specifically, AR/PCC herb pair improved impaired osteogenesis, nerve ingrowth and angiogenesis. More importantly, it could mitigate the aberrant activation of osteoblast pyroptosis in the vertebral bodies of diabetic rats by reducing increased expressions of Nlrp3, Asc, Caspase1, Gsdmd and IL-1ß. Mechanistically, AR/PCC activated antioxidant pathway through the upregulation of the antioxidant response protein Nrf2, while concurrently decreasing its negative feedback regulator Keap1. Collectively, our in vivo findings demonstrate that AR/PCC can inhibit osteoblast pyroptosis and alleviate STZ-induced rat DOP, suggesting its potential as a therapeutic agent for mitigating DOP.

Study on Blending Modification of Bisphenol A Epoxy.

Epoxy-resin-based composites in the field of current electrical materials often work in high temperature, high humidity or salt spray conditions. In order to improve the operation reliability of the composite insulator cross arm in a high temperature, high humidity and high salt spray environment, and analyze the aging mechanism and performance characteristics of resin, in this paper, wet heat aging and salt spray aging experiments were carried out on the blended resin system composed of bisphenol A type epoxy resin (E-51), aliphatic epoxy modified bisphenol A epoxy resin (2021P/E-51) and dimeric acid modified bisphenol A epoxy resin (EPD-172/E-51). Among them, 10 wt% and 20 wt% of 2021P blend resin and 10 wt% of EPD blend resin have superior thermo-mechanical properties. Under humid and hot conditions, the dielectric loss of 10 wt% EPD blend system before and after aging is 39.9% and 49.5% lower than that of pure E51 resin system, respectively. Under the condition of salt spray, the dielectric loss of 20 wt% and 10 wt% EPD blends decreased by 73.1% and 74.6% after aging. The leakage current of 10 wt% 2021P blend resin and 10 wt% EPD blend resin decreased by 7% and 3.8% before aging, respectively. After aging, they decreased by 3.7% and 2.2%, respectively. The bending strength of 2021P blend resin before and after aging reached 29.3 MPa and 26.6 MPa, respectively. The above three blending resin systems exhibit good electrical properties and good mechanical properties, their ageing resistance performance is strong and they are suitable as the matrix resin of compound cross arm mandrel material.

Cadmium exposure promotes thyroid pyroptosis and endocrine dysfunction by inhibiting Nrf2/Keap1 signaling.

Cadmium (Cd) is a ubiquitous toxic metal and environmental pollutant. Increasing studies have shown that Cd exposure increases the incidence of various endocrine system diseases, including thyrotoxicity reflected by thyroid structural damage and endocrine toxicity. However, the observed outcomes are complex and conflicting, leading to the mechanism of Cd-induced thyrotoxicity remaining obscure. In this study, 4-week-old male C57BL/6 mice were given 2 or 7 mg/kg Cadmium Chloride (CdCl2) intragastrically for 4 and 8 weeks, and the Cd-mediated thyrotoxicity was evaluated by determining alterations in thyroid structure and endocrine function, and alterations of oxidant stress, apoptosis, and pyroptosis. Our data showed that Cd exposure could reduce body weight and induce thyrotoxicity by impairing thyroid follicular morphology and endocrine function, accompanied by elevated oxidative stress and apoptosis, macrophage infiltration, and inflammatory cytokine secretion. Importantly, Cd significantly promoted thyroid follicular cell pyroptosis by increasing Nlrp3, Asc, Caspase-1, Gsdmd, IL-1ß, and IL-18 expression. Mechanistical analysis suggested that Cd treatment could inhibit antioxidant pathway by downregulating antioxidant response protein, Nrf2, and upregulating its negative feedback regulator, Keap1. Collectively, our in vivo findings suggest that Cd exposure could facilitate thyroid follicular cell pyroptosis by inhibiting Nrf2/Keap1 signaling, thereby disrupting thyroid tissue structure and endocrine function, which offers novel insights into the Cd-mediated detrimental consequences on thyroid homeostasis.

Morroniside attenuates nucleus pulposus cell senescence to alleviate intervertebral disc degeneration via inhibiting ROS-Hippo-p53 pathway.

Intervertebral disc (IVD) degeneration (IVDD) which is highly prevalent within the elderly population, is a leading cause of chronic low back pain and disability. Nucleus pulposus (NP) cell senescence plays an indispensable role in the pathogenesis of IVDD. Morroniside is a major iridoid glycoside and one of the quality control metrics of Cornus officinalis Siebold & Zucc (CO). An increasing body of evidence suggests that morroniside and CO-containing formulae share many similar biological effects, including anti-inflammatory, anti-oxidative, and anti-apoptotic properties. In a previous study, we reported that Liuwei Dihuang Decoction, a CO-containing formula, is effective for treating IVDD by targeting p53 expression; however, the therapeutic role of morroniside on IVDD remains obscure. In this study, we assessed the pharmacological effects of morroniside on NP cell senescence and IVDD pathogenesis using a lumbar spine instability surgery-induced mouse IVDD model and an in vitro H2O2-induced NP cell senescence model. Our results demonstrated that morroniside administration could significantly ameliorate mouse IVDD progression, concomitant with substantial improvement in extracellular matrix metabolism and histological grading score. Importantly, in vivo and in vitro experiments revealed that morroniside could significantly reduce the increase in SA-ß-gal activities and the expression of p53 and p21, which are the most widely used indicators of senescence. Mechanistically, morroniside suppressed ROS-induced aberrant activation of Hippo signaling by inhibiting Mst1/2 and Lats1/2 phosphorylation and reversing Yap/Taz reduction, whereas blockade of Hippo signaling by Yap/Taz inhibitor-1 or Yap/Taz siRNAs could antagonize the anti-senescence effect of morroniside on H2O2-induced NP cell senescence model by increasing p53 expression and activity. Moreover, the inhibition of Hippo signaling in the IVD tissues by morroniside was further verified in mouse IVDD model. Taken together, our findings suggest that morroniside protects against NP cell senescence to alleviate IVDD progression by inhibiting the ROS-Hippo-p53 pathway, providing a potential novel therapeutic approach for IVDD.

α-Chaconine Facilitates Chondrocyte Pyroptosis and Nerve Ingrowth to Aggravate Osteoarthritis Progression by Activating NF-κB Signaling.

Background: With the rapid growth of the elderly population, the incidence of osteoarthritis (OA) increases annually, which has attracted extensive attention in public health. The roles of dietary intake in controlling joint disorders are perhaps one of the most frequently posed questions by OA patients, while the information about the interaction between dietary intake and OA based on scientific research is limited. α-Chaconine is the richest glycoalkaloid in eggplants such as potatoes. Previous evidence suggests that α-Chaconine is a toxic compound to nervous and digestive systems with potentially severe and fatal consequences for humans and farm animals, but its effect on OA development remains obscure. Objective: To determine whether α-Chaconine deteriorates OA progression through sensory innervation and chondrocyte pyroptosis via regulating nuclear factor-κB (NF-κB) signaling, providing evidence for a possible linkage between α-Chaconine and OA progression. Methods: We established a mouse OA model by destabilization of medial meniscus (DMM) surgery and then intra-articular injection of 20 or 100 µM α-Chaconine into the OA mice for 8 and 12 weeks. The severity of OA progression was evaluated by histological staining and radiographic analyses. The expressions of matrix metabolic indicators, Col2, Mmp3, and Mmp13, as well as pyroptosis-related proteins, Nlrp3, Caspase-1, Gsdmd, IL-1ß, IL-18, were determined by immunohistochemistry. And the changes in sensory nerve ingrowth and activity of NF-κB signaling were determined by immunofluorescence. Results: We found that α-Chaconine could exacerbate mouse OA progression, resulting in subchondral sclerosis, osteophyte formation, and higher OARSI scores. Specifically, α-Chaconine could augment cartilage matrix degradation and induce chondrocyte pyroptosis and nerve ingrowth. Mechanistical analysis revealed that α-Chaconine stimulated NF-κB signaling by promoting I-κB α phosphorylation and p65 nuclear translocation. Conclusion: Collectively, our findings raise the possibility that α-Chaconine intake can boost chondrocyte pyroptosis and nerve ingrowth to potentiate OA progression by activating NF-κB signaling.

Pyroptosis and Intervertebral Disc Degeneration: Mechanistic Insights and Therapeutic Implications.

Low back pain (LBP) is a common problem worldwide, resulting in great patient suffering and great challenges for the social health system. Intervertebral disc (IVD) degeneration (IVDD) is widely acknowledged as one of the key causes of LBP. Accumulating evidence suggests that aberrant pyroptosis of IVD cells is involved in the pathogenesis of IVDD progression, however, the comprehensive roles of pyroptosis in IVDD have not been fully established, leaving attempts to treat IVDD with anti-pyroptosis approaches questionable. In this review, we summarize the characteristics of pyroptosis and emphasize the effects of IVD cell pyroptosis on the pathological progression of IVDD, including secretion of cytokines, nucleus pulposus cell apoptosis and autophagy, accelerated extracellular matrix degradation, annulus fibrosus rupture, cartilage endplate calcification, vascularization, sensory and sympathetic fiber neoinnervation, and infiltrating lymphatic vessels. Finally, we discuss several interventions used to treat IVDD by targeting pyroptosis. This review provides novel insights into the crucial role of IVD cell pyroptosis in IVDD pathogenesis, and could be informative for developing novel therapeutic approaches for IVDD and LBP.

Corrective Osteotomy with Volar and Dorsal Fixation for Malunion of Intra-Articular Fracture of the Distal Radius: A Retrospective Study.

OBJECTIVES: Although corrective osteotomy with volar or dorsal plate fixation can treat malunion of distal radius fractures, each has its own disadvantages. Little is currently known on whether dorsal fixation combined with volar fixation may further improve recovery. This study aimed to evaluate the clinical value of corrective osteotomy combined with volar and dorsal plate fixation in patients with malunion of intra-articular fractures of the distal radius. METHODS: Seventeen patients with malunion of intra-articular fractures of the distal radius treated with corrective osteotomy with volar and dorsal plate fixation from 1 January 2016 to 31 November 2018 were retrospectively analyzed. The enrolled patients included seven males and 10 females with an average age of 54.9 years (range: 36-70 years). The radiographic parameters, including the radial length, the radial inclination angle, the ulnar variance, and the volar tilt, as well as clinical outcomes, including wrist and forearm range of motion (ROM), grip strength, the Mayo Modified Wrist Score (MMWS), and the disabilities of the Arm, Shoulder, and Hand (DASH) score, were examined at 3 months and 18 months after operation and compared with the preoperative state. The paired t-test was used for statistical analysis. RESULTS: After corrective osteotomy combined with volar and dorsal plate fixation, all included patients were followed up for 18 months, and there was no surgical site infection. Patients reported postoperative pain due to the irritation of extensor tendon (two cases) and wrist arthritis (two cases). The radial length increased from 1.34 ± 2.34 mm to 9.25 ± 2.65 mm and 9.03 ± 2.47 mm at 3 months and 18 months postoperatively (t = 8.257, 7.954, all p < 0.05). The radial inclination angle increased from 6.45° ± 0.76° to 19.35° ± 3.43° and 19.03° ± 3.63° at 3 and 18 months (t = 12.517, 12.122, all p < 0.05). The ulnar variance decreased from 5.11 ± 0.23 mm to 1.32 ± 0.31 mm and 1.54 ± 0.62 mm at 3 and 18 months (t = 4.214, 4.895, all p < 0.05). The volar tilt was corrected from 4.47° ± 3.46° to 15.51° ± 2.72° and 14.12° ± 2.41°, respectively (t = 11.247, 10.432, all p < 0.05). Moreover, wrist ROM increased from 42.53° ± 8.99° to 98.70° ± 7.61° and 101.24° ± 7.66° (t = 41.433, 46.627, all p < 0.05), while forearm ROM was increased from 94.82° ± 6.54° to 134.47° ± 5.06° and 137.24° ± 5.52°, respectively (t = 31.507, 32.584, all p < 0.05). Similarly, grip strength, MMWS, and DASH were also remarkably improved. There were no significant differences in the wrist and forearm ROM, grip strength, MMWS, and DASH scores between follow-up at 3 and 18 months (all p > 0.05). CONCLUSIONS: Corrective osteotomy with volar and dorsal fixation can improve recovery of volar tilt, relieve wrist pain, restore wrist and forearm function, and increase grip strength of patients with malunion of intra-articular fractures of the distal radius.

Emerging Roles of Platelets in Allergic Asthma.

Allergic asthma is a complex chronic inflammatory disease of the airways, driven by Th2 immune responses and characterized by eosinophilic pulmonary inflammation, airway hyperresponsiveness, excessive mucus production, and airway remodeling. Overwhelming evidence from studies in animal models and allergic asthmatic patients suggests that platelets are aberrantly activated and recruited to the lungs. It has been established that platelets can interact with other immune cells and secrete various biochemical mediators to promote allergic sensitization and airway inflammatory response, and platelet deficiency may alleviate the pathological features and symptoms of allergic asthma. However, the comprehensive roles of platelets in allergic asthma have not been fully clarified, leaving attempts to treat allergic asthma with antiplatelet agents questionable. In this review, we summarize the role of platelet activation and pulmonary accumulation in allergic asthma; emphasis is placed on the different interactions between platelets with crucial immune cell types and the contribution of platelet-derived mediators in this context. Furthermore, clinical antiplatelet approaches to treat allergic asthma are discussed. This review provides a clearer understanding of the roles of platelets in the pathogenesis of allergic asthma and could be informative in the development of novel strategies for the treatment of allergic asthma.

Network Pharmacology and Experimental Validation to Reveal the Pharmacological Mechanisms of Liuwei Dihuang Decoction Against Intervertebral Disc Degeneration.

PURPOSE: To explore the pharmacological mechanisms of Liuwei Dihuang Decoction (LWDHD) against intervertebral disc (IVD) degeneration (IVDD) via network pharmacology analysis combined with experimental validation. METHODS: First, active ingredients and related targets of LWDHD, as well as related genes of IVDD, were collected from public databases. The protein-protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to predict the core targets and pathways of LWDHD against IVDD. Secondly, the IVDD model of mice treated with LWDHD was selected to validate the major targets predicted by network pharmacology. RESULTS: By searching the intersection of the active ingredient targets and IVDD targets, a total of 110 targets matched the related targets of 30 active ingredients in LWDHD and IVDD were retrieved. PPI network analysis indicated that 17 targets, including Caspase-3, IL-1ß, P53, etc., were hub targets. GO and KEGG enrichment analyses showed that the apoptosis pathway was enriched by multiple targets and served as the target for in vivo experimental study validation. The results of animal experiments revealed that LWDHD administration not only restored the decrease in disc height and abnormal degradation of matrix metabolism in IVDD mice but also reversed the high expression of Bax, Caspase-3, IL-1ß, P53, and low expression of Bcl-2, thereby inhibiting the apoptosis of IVD tissue and ameliorating the progression of IVDD. CONCLUSION: Using a comprehensive network pharmacology approach, our findings predicted the active ingredients and potential targets of LWDHD intervention for IVDD, and some major target proteins involved in the predictive signaling pathway were validated experimentally, which gave us a new understanding of the pharmacological mechanism of LWDHD in treating IVDD at the comprehensive level.

Aberrant spinal mechanical loading stress triggers intervertebral disc degeneration by inducing pyroptosis and nerve ingrowth.

Aberrant mechanical factor is one of the etiologies of the intervertebral disc (IVD) degeneration (IVDD). However, the exact molecular mechanism of spinal mechanical loading stress-induced IVDD has yet to be elucidated due to a lack of an ideal and stable IVDD animal model. The present study aimed to establish a stable IVDD mouse model and evaluated the effect of aberrant spinal mechanical loading on the pathogenesis of IVDD. Eight-week-old male mice were treated with lumbar spine instability (LSI) surgery to induce IVDD. The progression of IVDD was evaluated by µCT and Safranin O/Fast green staining analysis. The metabolism of extracellular matrix, ingrowth of sensory nerves, pyroptosis in IVDs tissues were determined by immunohistological or real-time PCR analysis. The apoptosis of IVD cells was tested by TUNEL assay. IVDD modeling was successfully produced by LSI surgery, with substantial reductions in IVD height, BS/TV, Tb.N. and lower IVD score. LSI administration led to the histologic change of disc degeneration, disruption of the matrix metabolism, promotion of apoptosis of IVD cells and invasion of sensory nerves into annulus fibrosus, as well as induction of pyroptosis. Moreover, LSI surgery activated Wnt signaling in IVD tissues. Mechanical instability caused by LSI surgery accelerates the disc matrix degradation, nerve invasion, pyroptosis, and eventually lead to IVDD, which provided an alternative mouse IVDD model.

Morroniside attenuates apoptosis and pyroptosis of chondrocytes and ameliorates osteoarthritic development by inhibiting NF-κB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Corni Fructus (CF), the red fruit of Cornus officinalis Siebold & Zucc, has been used both as food and medicinal herb in traditional Chinese medicine (TCM). Our previous studies showed that Yougui pills and Bushenhuoxue formula, both TCM prescriptions containing Corni Fructus (CF), have protective effects on osteoarthritis (OA). However, the underlying detailed components in both TCM prescriptions that play therapeutic roles have not been fully defined. Morroniside is a major iridoid glycoside and one of the quality control metrics of CF, but the effects of morroniside on OA remain largely elusive. AIM OF THE STUDY: The study aims to assess the therapeutic effects of morroniside on cartilage degeneration using a mouse model of OA. MATERIAL AND METHODS: 8-week-old male C57BL/6J mice were randomly divided into 4 groups: Sham, destabilization of the medial meniscus (DMM)-treated with vehicle, DMM-treated with low dose morroniside and DMM-treated with high dose morroniside. Histological staining, immunostaining, and TUNEL staining were conducted to detect changes in tissue morphology, expression of key molecules in chondrocytes, and chondrocyte apoptosis, respectively. Osteophyte formation, meniscus calcification, and subchondral sclerosis were quantitated using micro-CT. The expression of chondrocyte markers was also analyzed by Western blot in primary chondrocytes derived from mice treated with morroniside. RESULTS: Morroniside attenuated the progression of OA in mice, resulting in substantially reduced osteophyte formation and subchondral sclerosis and lower OARSI scores. Specifically, morroniside significantly promoted cartilage matrix synthesis by increasing collagen type II expression and suppressing chondrocyte pyroptosis. Morroniside administration led to inhibition of matrix metalloproteinase-13 (MMP13), Caspase-1 and nod-like receptor protein-3 (NLRP3) expression in DMM mice and IL-1ß-stimulated chondrocytes. In addition, morroniside attenuated the progression of OA by enhancing chondrocyte proliferation and inhibiting chondrocyte apoptosis. Morroniside also attenuated the progression of OA by inhibiting nuclear factor-κB (NF-κB) signaling. CONCLUSION: Morroniside was protective against cartilage matrix degradation and reduced DMM-induced chondrocyte pyroptosis and apoptosis by the inhibition of NF-κB signaling.

Integration of Network Pharmacology and Experimental Validation to Explore the Pharmacological Mechanisms of Zhuanggu Busui Formula Against Osteoporosis.

Osteoporosis (OP) is a common skeletal disease, characterized by decreased bone formation and increased bone resorption. As a novel Chinese medicine formula, Zhuanggu Busui formula (ZGBSF) has been proved to be an effective prescription for treating OP in clinic, however, the pharmacological mechanisms underlying the beneficial effects remain obscure. In this study, we explored the pharmacological mechanisms of ZGBSF against OP via network pharmacology analysis coupled with in vivo experimental validation. The results of the network pharmacology analysis showed that a total of 86 active ingredients and 164 targets of ZGBSF associated with OP were retrieved from the corresponding databases, forming an ingredient-target-disease network. The protein-protein interaction (PPI) network manifested that 22 core targets, including Caspase-3, BCL2L1, TP53, Akt1, etc, were hub targets. Moreover, functional enrichment analyses revealed that PI3K-Akt and apoptosis signalings were significantly enriched by multiple targets and served as the targets for in vivo experimental study validation. The results of animal experiments revealed that ZGBSF not only reversed the high expression of Caspase-3, Bax, Prap, and low expression of Bcl-2 in osteoblasts of the OP mouse model but also contributed to the phosphorylation of Akt1 and expression of PI3K, thereby promoting osteogenesis and ameliorating the progression of OP. In conclusion, this study systematically and intuitively illustrated that the possible pharmacological mechanisms of ZGBSF against OP through multiple ingredients, targets, and signalings, and especially the inhibition of the apoptosis and the activation of PI3K-Akt signaling.

Comparative intra-articular gene transfer of seven adeno-associated virus serotypes reveals that AAV2 mediates the most efficient transduction to mouse arthritic chondrocytes.

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.

Biomechanical, histologic, and molecular characteristics of graft-tunnel healing in a murine modified ACL reconstruction model.

PURPOSE: The purpose of our study was to introduce and validate a metal-free, reproducible and reliable mouse model of anterior cruciate ligament (ACL) reconstruction (ACLR) surgery as an effective tool for a better understanding of molecular mechanisms of graft-tunnel healing after ACLR. METHODS: A total of 150 C57BL/6 mice were randomly allocated into five Groups: Group 1 (mice with intact ACL), Group 2-4 (mice underwent modified ACLR surgery and sacrificed 1-, 2-, and 4-weeks after surgery), and Group 5 (mice underwent unmodified ACLR surgery and sacrificed 4 weeks after surgery). Micro-computed tomography (CT), biomechanical histological as well as immunohistochemical (IHC) analyses were performed to characterize the modified ACLR. RESULTS: Micro-CT analysis demonstrated there is a non-significant increase in BV/TV and BMD of the bone tunnel during the tendon-to-bone healing following ACLR. Biomechanical tests showed that the mean load-to-failure forces of Group 3 and 4 are equal to 31.7% and 46.0% of that in Group 1, while the stiffness was 33.1% and 57.2% of that of Group 1, respectively. And no obvious difference in biomechanical parameters was found between Group 4 and 5. Histological analysis demonstrated that formation of fibrovascular tissue in the tibial tunnel and aperture in Groups 4 and 5 and direct junction appeared between tendon graft and tunnel both in Groups 4 and 5. IHC results showed that there are gradually enhanced expression of Patched1, Smoothened and Gli2 concomitant with decreased Gli3 protein in the tendon-bone interface during the tendon-bone healing process. CONCLUSION: We introduced a metal-free, reproducible and reliable mouse model of ACLR compared to the unmodified ACLR procedure, and characterized the expression pattern of key molecules in Ihh signaling during the graft healing process. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: In the present study we introduced and validated, for the first time, a metal-free, reproducible and reliable ACLR mouse model, which could be used to investigate the detailed molecular mechanisms of graft-tunnel healing after ACLR. We also explored new strategies to promote the healing of tendon-to-bone integration.

Loganin ameliorates cartilage degeneration and osteoarthritis development in an osteoarthritis mouse model through inhibition of NF-κB activity and pyroptosis in chondrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Corni Fructus (CF), the red fruit of Cornus officinalis Siebold & Zucc, has been used both as food and medicinal herb in traditional Chinese medicine (TCM). Loganin is a major iridoid glycoside and one of the quality control indexes of CF. In TCM clinical practice, prescription containing CF is commonly used to treat osteoarthritis (OA), but the underlying mechanisms of loganin are not yet utterly understood. AIM OF THE STUDY: The aims of the present study are to confirm the therapeutic effects of loganin in an OA mouse model and to determine the mechanisms involved in the OA protective effects. MATERIALS AND METHODS: The destabilization of the medial meniscus (DMM) procedure was performed on the right knee of 8-week-old C57BL/6 male mice. 30 or 100 µg/ml of loganin was then injected into articular space twice a week for 8 and 12-week. Safranin O/Fast green staining, H&E staining, micro-CT analysis were performed to analyze structural and morphological changes. The protein expression of collagen type II (Col2), metalloproteinase-3 (Mmp3), matrix metalloproteinase 13 (Mmp13) collagen type X (Col10), cryopyrin and caspase-1 were detected by immunochemistry staining. Immuno-fluorescence assay was performed to assess changes in expression of CD31, endomucin, p65 and p-I-κB. RESULTS: Results of histomorphometry showed that loganin delays the progression of OA in the DMM model. In cartilage, loganin decreased the OARSI score, increasing hyaline cartilage (HC) thickness and decreasing calcified cartilage (CC) thickness. Moreover, loganin inhibited osteophyte formation, reduced the bone volume fraction (BV/TV), lowered trabecular thickness (Tb.Th) and increased trabecular separation (Tb.Sp) in subchondral bone. Mechanistically, loganin increased the expressions of Col2, decreases the expression of Mmp3, Mmp13, Col10, cryopyrin and caspase-1 in cartilage. In parallel, loganin inhibited the expression of CD31 and endomucin in subchondral bone. Furthermore, loganin suppressed nuclear translocation of p65 protein, and decreased the amount of p-I-κB in chondrocytes. CONCLUSIONS: In summary, these results uncovered that loganin inhibits NF-κB signaling and attenuates cartilage matrix catabolism and pyroptosis of chondrocytes in articular cartilage. Loganin may serve as a potential therapeutic agent for OA treatment.