OBJECTIVES: Osteoarthritis (OA) is one of the most common chronic degenerative diseases, with chondrocyte apoptosis and extracellular matrix (ECM) degradation as the major pathological changes. The mechanical stimulation can attenuate chondrocyte apoptosis and promote ECM synthesis, but the underlying molecular mechanisms remain unclear. This study aims to investigate the role of primary cilia (PC) in mediating the effects of mechanical stimulation on OA progression. METHODS: In vivo, conditional knockout mice lacking intraflagellar transport 88 (IFT88^flox/flox IFT88 knockout; i.e., primary cilia-deficient mice) were generated, with wild-type mice as controls. OA models were established via anterior cruciate ligament transection combined with destabilization of the medial meniscus, followed by treadmill exercise intervention. OA progression was evaluated by hematoxylin-eosin staining, safranin O-fast green staining, and immunohistochemistry; apoptosis was assessed by TUNEL staining; and limb function by rotarod testing. In vitro, primary articular chondrocytes were isolated from mice and transfected with lentiviral vectors to suppress IFT88 expression, thereby constructing a primary cilia-deficient cell model. Interleukin-1β (IL-1β) was used to induce an inflammatory environment, while cyclic tensile strain (CTS) was applied via a cell stretcher to mimic mechanical loading on chondrocytes. Immunofluorescence and Western blotting were used to detect the protein expression levels of type II collagen α1 chain (COL2A1), primary cilia, IFT88, and caspase-12; reverse transcription polymerase chain reaction was performed to assess COL2A1 mRNA levels; and flow cytometry was used to evaluate apoptosis. RESULTS: In vivo, treadmill exercise significantly reduced Osteoarthritis Research Society International (OARSI) scores and apoptotic cell rates, and improved balance ability in wild-type OA mice, whereas IFT88-deficient OA mice showed no significant improvement. In vitro, CTS inhibited IL-1β-induced ECM degradation and apoptosis in primary chondrocytes; however, this protective effect was abolished in cells with suppressed primary cilia expression. CONCLUSIONS Mechanical stimulation delays OA progression by mediating signal transduction through primary cilia, thereby inhibiting cartilage degeneration and chondrocyte apoptosis.
Animals ; Chondrocytes/cytology* ; Apoptosis/physiology* ; Mice ; Cilia/metabolism* ; Osteoarthritis/pathology* ; Extracellular Matrix/metabolism* ; Mice, Knockout ; Disease Progression ; Interleukin-1beta ; Male ; Cells, Cultured

OBJECTIVE: To observe the effect of acupuncture on chondrocyte autophagy in rats of knee osteoarthritis (KOA) and explore its underlying mechanisms. METHODS: Forty male SPF-grade SD rats were randomized into a blank group, a model group, a suspension group, an acupuncture group, and a combined therapy group, 8 rats in each one. Except the blank group, KOA model was prepared by the injection with papain. The suspension exercise therapy (10 min each time, three times daily), acupuncture (at "Yanglingquan" [GB34], "Zusanli" [ST36], and "Dubi" [ST35] on the right side, 30 min each intervention, once daily) and the combined therapy (the suspension exercise therapy combined with acupuncture) were delivered in the suspension group, the acupuncture group and the combined therapy group, respectively. The intervention of each group was performed continuously for 6 days, and 4 consecutive weeks, at the interval of 1 day. Before and after intervention, Lequesne MG score was assessed in the rats. After intervention, HE staining was adopted to observe the cartilaginous tissue morphology of the right knee joints, and Mankin score was evaluated; the serum levels of interleukin (IL)-1β, IL-6, and tumor neurosis factor-α (TNF-α) were measured using ELISA; the real-time PCR was provided to determine the mRNA expression of collagen protein type Ⅱ(COL2), collagen protein type Ⅹ (COL10), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and autophagy-regulated protein (Beclin-1) in the cartilaginous tissue of the right knee joint; Western blot was employed to detect the protein expression of PI3K, phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), mTOR, phosphorylated mTOR (p-mTOR) and Beclin-1 in the cartilaginous tissue of the right knee joint. RESULTS: Compared with the blank group, the rats in the model group showed the higher Lequesne MG score (P<0.01), thinner cartilage of the right knee, reduced chondrocytes and disordered arrangement, and higher Mankin score (P<0.01). Besides, in the model group, the serum levels of IL-1β, IL-6 and TNF-α were elevated (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 decreased (P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR increased (P<0.01) in the cartilaginous tissue of the right knee joint. Compared with the model group, in the suspension group, the acupuncture group and the combined therapy group, the Lequesne MG scores were reduced (P<0.01), the cartilage of the right knee was thickened, the arrangement of chondrocytes was improved, and the Mankin scores were lower (P<0.01). Besides, in these intervention groups, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05, P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. When compared with the suspension group and the acupuncture group, in the combined therapy group, the Lequesne MG score was reduced (P<0.01), and the Mankin score was reduced (P<0.05, P<0.01). Besides, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.05), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. CONCLUSION Acupuncture can promote cartilage regeneration of knee joint and autophagy in KOA rats, alleviate inflammation, so as to retard cartilage degeneration, which may be possibly associated with the PI3K/Akt/mTOR signaling pathway.
Animals ; Male ; Acupuncture Therapy ; Proto-Oncogene Proteins c-akt/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Osteoarthritis, Knee/physiopathology* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Signal Transduction ; Rats, Sprague-Dawley ; Chondrocytes/metabolism* ; Humans ; Autophagy ; Acupuncture Points

Objective To explore the effect of rehmanniae radix extract(RRE)on chondrocyte apoptosis in the rabbit model of knee osteoarthritis(KOA)by regulating the miR-485-5p/heat shock protein 90 beta family member 1(Hsp90b1)axis.Methods New Zealand rabbits were randomly assigned into control,KOA,low-dose RRE,medium-dose RRE,high-dose RRE,celecoxib,high-dose RRE+antagonist control,and high-dose RRE+miR-485-5p antagonist groups,with 12 rabbits in each group.Rabbits in other groups except the control group were modeled for KOA with the improved Hulth method.After modeling for 8 weeks,the rabbits were administrated with corresponding agents for 4 weeks.The changes in the activity rating of rabbits were recorded.ELISA was employed to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the serum.Safranine O-fast green staining was conducted to reveal the pathological changes in the cartilage tissue and Mankin scoring was performed.TUNEL was employed to detect chondrocyte apoptosis.Real-time fluorescence quantitative PCR was performed to determine the expression of miR-485-5p in the cartilage tissue.Western blot was employed to determine the protein levels of Hsp90b1,cleaved cysteinyl aspartate-specific proteinase-3(Caspase-3),and Bcl2-associated-X(Bax)in the cartilage tissue.The dual-luciferase reporter assay was employed to examine the relationship between miR-485-5p and Hsp90b1.Results Compared with the control group,the KOA group showed down-regulated expression of miR-485-5p,elevated levels of TNF-α and IL-6 in the serum,cartilage erosion and losses,and increases in activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.001).Compared with the KOA group,RRE at low,medium,and high doses,and celecoxib up-regulated the expression of miR-485-5p,lowered the levels of TNF-α and IL-6 in the serum,alleviated the pathological damage to the cartilage tissue,and decreased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).Compared with the high-dose RRE group and the high-dose RRE+antagonist control group,high-dose RRE+miR-485-5p antagonist down-regulated the expression of miR-485-5p,elevated the levels of TNF-α and IL-6 in the serum,exacerbated the pathological damage to the cartilage tissue,and increased the activity rating,Mankin score,chondrocyte apoptosis rate,and protein levels of Hsp90b1,cleaved Caspase-3,and Bax(all P<0.05).The results indicated that there was a targeted regulatory relationship between miR-485-5p and Hsp90b1.Conclusion RRE may inhibit the expression of Hsp90b1 by up-regulating miR-485-5p,thereby inhibiting chondrocyte apoptosis in the rabbit model of KOA.
Animals ; Rabbits ; Apoptosis/drug effects* ; Chondrocytes/pathology* ; Osteoarthritis, Knee/drug therapy* ; MicroRNAs/metabolism* ; Rehmannia/chemistry* ; Disease Models, Animal ; Tumor Necrosis Factor-alpha/blood* ; Plant Extracts/pharmacology* ; Interleukin-6/blood* ; HSP90 Heat-Shock Proteins/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology*

BACKGROUND: Knee osteoarthritis (OA) is still challenging to prevent or treat. Enhanced endoplasmic reticulum (ER) stress and increased pyroptosis in chondrocytes may be responsible for cartilage degeneration. This study aims to investigate the effect of ER stress on chondrocyte pyroptosis and the upstream regulatory mechanisms, which have rarely been reported. METHODS: The expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), microRNA-142-3p (miR-142-3p), and high mobility group box 1 (HMGB1) and the levels of ER stress, pyroptosis, and metabolic markers in normal and OA chondrocytes were investigated by western blotting, quantitative polymerase chain reaction, immunohistochemistry, fluorescence in situ hybridization, fluorescein amidite-tyrosine-valine-alanine-aspartic acid-fluoromethyl ketone (FAM-YVAD-FMK)/Hoechst 33342/propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and cell viability assessments. The effects of EZH2, miR-142-3p, and HMGB1 on ER stress and pyroptosis and the hierarchical regulatory relationship between them were analyzed by chromatin immunoprecipitation, luciferase reporters, gain/loss-of-function assays, and rescue assays in interleukin (IL)-1β-induced OA chondrocytes. The mechanistic contribution of EZH2, miR-142-3p, and HMGB1 to chondrocyte ER stress and pyroptosis and therapeutic prospects were validated radiologically, histologically, and immunohistochemically in surgically induced OA rats. RESULTS: Increased EZH2 and HMGB1, decreased miR-142-3p, enhanced ER stress, and activated pyroptosis in chondrocytes were associated with OA occurrence and progression. EZH2 and HMGB1 exacerbated and miR-142-3p alleviated ER stress and pyroptosis in OA chondrocytes. EZH2 transcriptionally silenced miR-142-3p via H3K27 trimethylation, and miR-142-3p posttranscriptionally silenced HMGB1 by targeting the 3'-UTR of the HMGB1 gene. Moreover, ER stress mediated the effects of EZH2, miR-142-3p, and HMGB1 on chondrocyte pyroptosis. In vivo experiments mechanistically validated the hierarchical regulatory relationship between EZH2, miR-142-3p, and HMGB1 and their effects on chondrocyte ER stress and pyroptosis. CONCLUSIONS A novel EZH2/miR-142-3p/HMGB1 axis mediates chondrocyte pyroptosis and cartilage degeneration by regulating ER stress in OA, contributing novel mechanistic insights into OA pathogenesis and providing potential targets for future therapeutic research.
Enhancer of Zeste Homolog 2 Protein/genetics* ; Osteoarthritis, Knee/pathology* ; Chondrocytes/metabolism* ; Pyroptosis/physiology* ; HMGB1 Protein/genetics* ; MicroRNAs/metabolism* ; Endoplasmic Reticulum Stress/genetics* ; Humans ; Animals ; Rats ; Male ; Rats, Sprague-Dawley ; Middle Aged

BACKGROUND: Osteoarthritis (OA) is a prevalent joint disorder that significantly impairs quality of life among elderly individuals because of chronic pain and physical disability. As the global burden of OA continues to rise, novel therapeutic strategies are urgently needed. Kaempferide (KA), a flavonoid derived from traditional Chinese herbal medicine, is known for its anti-inflammatory properties. However, the effect of KA on the progression of OA has not been well investigated. This study aimed to explore the therapeutic potential of KA in an OA model and investigate the underlying mechanisms via transcriptomic sequencing. METHODS: An in vitro OA model was established using SW1353 cells treated with interleukin-1 beta (IL-1β) and different concentrations of KA (30, 60, or 90 μmol/L) for 24 h. The anti-inflammatory effects of KA were assessed using quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting. In vivo , a papain-induced OA rat model was used to evaluate the therapeutic effects of KA through histological and behavioral analyses. Transcriptomic sequencing was performed to explore the differentially expressed genes (DEGs) and related signaling pathways. Statistical analysis was conducted using one-way analysis of variance. RESULTS: KA significantly increased cell viability in the OA chondrocyte model and downregulated the expression of inflammatory cytokines and cartilage degradation markers, with the greatest reduction observed at 90 μmol/L. In vivo , KA treatment mitigated cartilage degradation and improved gait behavior in OA rats. Transcriptomic analysis revealed substantial modulation of DEGs, implicating the hypoxia-inducible factor-1 (HIF-1) signaling pathway as a key mechanism. Further blocking and rescue experiments revealed that KA regulated key molecules within the HIF-1 pathway, specifically interferon-gamma (IFN-γ) and hypoxia-inducible factor 1-alpha (HIF-1α), confirming their critical roles in mediating the therapeutic effects of KA. CONCLUSION KA inhibited the progression of OA by targeting the HIF-1 signaling pathway, reducing inflammation, and cartilage degradation.
Animals ; Osteoarthritis/metabolism* ; Signal Transduction/drug effects* ; Rats ; Rats, Sprague-Dawley ; Humans ; Male ; Hypoxia-Inducible Factor 1/metabolism* ; Interleukin-1beta

This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*

A combination of the "disease-syndrome-symptom" approach was used to study the syndrome characterization and features of dampness-heat obstruction syndrome in papain-induced knee osteoarthritis(KOA) model rats during the disease process. Forty-eight male SD rats were randomly divided into sham and model groups. The KOA model was established by injecting a mixture of papain and L-cysteine into the joint cavity on days 1, 3, and 5. During the 8 weeks following model establishment, the rats were assessed weekly for the plantar mechanical pain threshold, knee joint diameter, local skin temperature of the knee joint, weight-bearing difference between the two hind feet, and the modified Lequesne MG score of the knee joint. Samples were collected at 1, 2, 4, 6, and 8 weeks after model establishment to observe the gross lesions in cartilage and synovium. Histopathological changes in joint tissues were examined using hematoxylin-eosin, Masson's trichrome, and Senna red O-solid green staining. ELISA and immunohistochemical analysis were performed to detect the levels of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, prostaglandin E2(PGE2), and the expression of aquaporins(AQP) 1 and 3 in serum and synovium. The results showed that the ink score of articular cartilage in the model group significantly increased from 4 to 8 weeks, the cartilage Mankin's score and the percentage of Masson-positive area in cartilage increased significantly from 1 to 8 weeks. The percentage of red-stained area for cartilage proteoglycans decreased significantly from 1 to 8 weeks. The synovitis score from 1 to 6 weeks and the percentage of blue-stained collagen fibers in the synovium from 1 to 8 weeks increased significantly, with statistically significant differences compared to the sham group. The mechanical pain threshold in the model group significantly decreased from 1 to 8 weeks, the knee joint diameter significantly increased from 1 to 6 weeks, and the local skin temperature of the knee joint, the weight-bearing difference between the two hind feet, and the modified Lequesne MG score from 1 to 5 weeks significantly increased, all with statistically significant differences compared to the sham group. The levels of IL-1β, IL-6, TNF-α, and PGE2 in serum and synovium of the model group significantly increased from 1 to 6 weeks. Serum TNF-α and PGE2, and synovial IL-1β, also significantly increased at 8 weeks. The levels of cartilage AQP1 and AQP3 significantly increased from 1 to 4 weeks, while synovial AQP1 and AQP3 increased significantly from 1 to 6 weeks, with all differences statistically significant compared to the sham group. In conclusion, papain-induced KOA rats exhibited pathological changes, including articular cartilage degeneration and synovial inflammation, within 1 week of induction. The KOA rats showed characteristics of dampness-heat obstruction syndrome, such as joint pain, swelling, elevated skin temperature, and decreased function, as well as increased inflammatory factors and AQP1、AQP3 in serum and joint tissues within 5 to 6 weeks of disease onset. These results provide an experimental model for studying the syndromes of KOA with dampness-heat obstruction syndrome.
Animals ; Male ; Rats, Sprague-Dawley ; Rats ; Osteoarthritis, Knee/physiopathology* ; Disease Models, Animal ; Humans ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Knee Joint/pathology*

This study aims to reveal the correlation between the pharmacokinetics(PK) and pharmacodynamics(PD) of multiple components in Epimedii Folium-Chuanxiong Rhizoma and clarify the pharmacodynamic material basis and mechanism of this herb pair in treating osteoarthritis. The Hulth method was used to establish the rat model of osteoarthritis and plasma was collected at various time points after drug administration. The plasma concentrations of multiple components were measured. Enzyme-linked immunosorbent assay(ELISA) was used to measure the plasma concentrations of matrix metalloproteinase(MMP)-3, MMP-13, interleukin-1β(IL-1β), nitric oxide(NO), and tumor necrosis factor-α(TNF-α) as pharmacodynamic indicators. Self-defined weighting coefficients were used to calculate the PK and PD data, and a Sigmoid E_(max) fitting model was used to evaluate the synergistic effect of the compatibility of Epimedii Folium-Chuanxiong Rhizoma. The PK-PD models for Epimedii Folium, Chuanxiong Rhizoma, and Epimedii Folium-Chuanxiong Rhizoma were E=(1.926×C~(2.652))/(0.136 6~(2.652)+C~(2.652)), E=(1.618×C~(345.2))/(0.118 4~(345.2)+C~(345.2)), and E=(2.305×C~(2.786))/(0.240 3~(2.786)+C~(2.786)), respectively. The E_(max) of Epimedii Folium-Chuanxiong Rhizoma was larger than those of the two herbal medicines alone. The EC_(50) of the herb pair was lower than the sum of Epimedii Folium and Chuanxiong Rhizoma alone. The concentrations of MMP-3, MMP-13, IL-1β, NO, and TNF-α were correlated with mass concentrations of multiple components in Epimedii Folium and Chuanxiong Rhizoma, and the compatibility was better than single use. Epimedii Folium, Chuanxiong Rhizoma, and Epimedii Folium-Chuanxiong Rhizoma may play a role in the treatment of osteoarthritis by inhibiting MMP-3, MMP-13, IL-1β, NO, and TNF-α.
Animals ; Rats ; Drugs, Chinese Herbal/pharmacology* ; Male ; Rats, Sprague-Dawley ; Osteoarthritis/metabolism* ; Epimedium/chemistry* ; Interleukin-1beta/blood* ; Tumor Necrosis Factor-alpha/blood* ; Disease Models, Animal ; Nitric Oxide/blood* ; Humans ; Rhizome/chemistry*

To investigate the mechanism of action of Syngnathus extract in treating knee osteoarthritis of rats, forty-eight male SD rats were randomly divided into the blank group, model group, positive drug group, as well as low-dose, medium-dose, and high-dose groups of Syngnathus extract. The rat model of knee osteoarthritis was constructed by intra-articular injection of sodium iodoacetate. After successful modeling, celecoxib(18 mg·kg~(-1)·d~(-1)) and Syngnathus extract(0.4, 0.8, and 1.6 g·kg~(-1)·d~(-1)) were given in different groups by gavage intervention for two weeks. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes of cartilage in knee joints, and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of inflammatory factors in serum. Real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to detect the levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR) pathway-related mRNA and protein expression. The results showed that, comparied with the blank group, the cartilage surface of the knee joints of rats in the model group was uneven, with disorganized levels and defective cartilage tissue. The serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) and the mRNA levels of PI3K, Akt, and mTOR in cartilage tissue, as well as the protein expression levels of phosphorylated PI3K(p-PI3K)/PI3K, phosphorylated Akt(p-Akt)/Akt, phosphorylated mTOR(p-mTOR)/mTOR, and P62 were significantly increased. Beclin1 protein expression was decreased. Comparied with the model group, the number of chondrocytes in the knee joint of rats in each group of Syngnathus extract increased, and the arrangement of chondrocytes was relatively neat. The cartilage layer was restored, and the serum levels of IL-1β, IL-6, and TNF-α, as well as the mRNA expression levels of PI3K, Akt, and mTOR in cartilage tissue were significantly reduced. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and P62 were significantly reduced in the rats in the middle-dose and high-dose groups of Syngnathus extract, and the Beclin1 protein expression was significantly increased. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, and P62 in rats in the low-dose group of Syngnathus extract were significantly reduced. In summary, Syngnathus extract may be used to treat knee osteoarthritis by inhibiting the expression of PI3K/Akt/mTOR signaling pathway, so as to alleviate the inflammatory response in the organism, enhance the autophagy activity of chondrocytes, and reduce the apoptosis of chondrocytes.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Male ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Humans

This study aims to systematically characterize the targeted effects of Quanduzhong Capsules on cartilage lesions in knee osteoarthritis by integrating spatial transcriptomics data mining and animal experiments validation, thereby elucidating the related molecular mechanisms. A knee osteoarthritis model was established using Sprague-Dawley(SD) rats, via a modified Hulth method. Hematoxylin and eosin(HE) staining was employed to detect knee osteoarthritis-associated pathological changes in knee cartilage. Candidate targets of Quanduzhong Capsules were collected from the HIT 2.0 database, followed by bioinformatics analysis of spatial transcriptomics datasets(GSE254844) from cartilage tissues in clinical knee osteoarthritis patients to identify spatially specific disease genes. Furthermore, a "formula candidate targets-spatially specific genes in cartilage lesions" interaction network was constructed to explore the effects and major mechanisms of Quanduzhong Capsules in distinct cartilage regions. Experimental validation was conducted through immunohistochemistry using animal-derived biospecimens. The results indicated that Quanduzhong Capsules effectively inhibited the degenerative changes in the cartilage of affected joints in rats, which was associated with the regulation of Quanduzhong Capsules on the thioredoxin-interacting protein(TXNIP)-NOD-like receptor family pyrin domain containing 3(NLRP3)-bone morphogenetic protein receptor type 2(BMPR2)-fibronectin 1(FN1)-matrix metallopeptidase 2(MMP2) signal axis in the articular cartilage surface and superficial zones, subsequently inhibiting cartilage matrix degradation leading to oxidative stress and inflammatory diffusion. In summary, this study clarifies the spatially specific targeted effects and protective mechanisms of Quanduzhong Capsules within pathological cartilage regions in knee osteoarthritis, providing theoretical and experimental support for the clinical application of this drug in the targeted therapy on the inflamed cartilage.
Animals ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Male ; Humans ; Capsules ; Female ; Disease Models, Animal