Knee Osteoarthritis Rats Research Articles

Polydatin protects against articular cartilage degeneration by regulating autophagy mediated by the AMPK/mTOR signaling pathway.

Knee osteoarthritis (KOA) is one of the leading causes of disability. Polydatin has a potential effect on KOA treatment but the therapeutic mechanism is not clear. This study aims to investigate the therapeutic action of polydatin in KOA and its mechanism in activating autophagy via the AMP-activated protein kinase (AMPK)/mTOR signaling pathway. After a KOA rat model was established by anterior cruciate ligament transection surgery, model rats were treated with polydatin 40 mg/kg for 30 days. Subsequently, cartilage tissues were collected, and hematoxylin-eosin (HE), Safranin-O, and TUNEL staining, and western blotting were performed to evaluate the pathological damage and autophagy-related protein expression. Then, human chondrocyte C28/I2 cells were stimulated with lipopolysaccharide (LPS), and the effects of polydatin on C28/I2 cell viability, apoptosis, and autophagy-related protein expression were detected by MTT, Flow Cytometry, and western blot. In addition, an AMPK inhibitor (Dorsomorphin 2HCl) was used to probe the cell proliferation and apoptosis of polydatin-administered C28/I2 cells. Polydatin ameliorated the pathological damage in rat cartilage tissues and inhibited cell apoptosis in KOA rats. Meanwhile, in C28/I2 cells, polydatin promoted viability and reduced apoptosis. In addition, the protein expression of collagen II, LC3II/LC3I, Beclin-1, and p-AMPK/AMPK were upregulated, and p62 and p-mTOR/mTOR were downregulated by polydatin treatment. Interestingly, relative results showed that the protective effect of polydatin in LPS-stimulated-C28/I2 cells was blocked by the AMPK/mTOR inhibitor, dorsomorphin 2HCl. Our research showed that polydatin reduced apoptosis and activated autophagy both in vivo and in vitro by the AMPK/mTOR signaling pathway to protect against KOA, which provided the basis for further investigation into the potential therapeutic impact of polydatin on KOA.

The active ingredient of Evodia rutaecarpa reduces inflammation in knee osteoarthritis rats through blocking calcium influx and NF-κB pathway.

Chronic inflammation significantly contributes to the progression of osteoarthritis (OA), and an anti-inflammatory small molecule derived from medicinal herbs could be a potential drug candidate for OA. Herein, we investigated the function and mechanism of Evodiamine (EAE), the active ingredient from Evodia rutaecarpa, in chondrocytes and macrophages in vitro and in vivo. The cytotoxicity of EAE was determined using an MTT assay. And the anti-inflammatory and anti-extracellular matrix (ECM) degradation effects of EAE were investigated using qRT-PCR, western blot (WB), immunofluorescence (IF). Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), Fluo-4AM, IF and AutoDock were used to elucidate the molecular mechanisms and signalling pathways of the reducing-inflammatory properties of EAE on chondrocytes in vitro. Moreover, the effect of EAE on macrophage polarization was detected by IF and flow cytometry (FC). Ultimately, we explored the in vivo therapeutic efficacy of EAE in an anterior cruciate ligament transection (ACLT)-induced OA model. The finding demonstrated that EAE blocked the phosphorylation of IKBα and Ca2+ influx, thereby curbing inflammation and ECM degradation. Additionally, EAE can prevent the polarization towards the M1 phenotype. Thus, our findings suggest that EAE has great potential as a therapeutic drug for the treatment of OA.

Effects of acupuncture on serotonin, histamine, substance P, and tryptase levels at sensitized points in model rats with knee osteoarthritis

ObjectiveTo elucidate the differences in manual acupuncture effectiveness at sensitized points by investigating the mechanisms of local skin action at different sensitization points in rats with knee osteoarthritis (KOA). MethodsForty Sprague–Dawley rats were equally divided into control, model (1 mg of monoiodoacetate into the right knee joint cavity), sham operation, manual acupuncture at right Tianjing acupoint (MAR-SJ 10), and left SJ 10 groups. Safranine-O and fast green staining were used to assess the modeling. The morphological and functional changes in mast cells (MCs) were assessed during acupoint sensitization using toluidine blue and immunofluorescence staining. The levels of serotonin, histamine, substance P (SP), and tryptase at skin acupoints and serum levels of IL-β, IL-6, and TNF-α were detected using ELISA. ResultsAfter 14 days of treatment, the number of MCs and their degranulation rates were statistically higher in the model group than in the control group (both P < .001). After applying acupuncture, the levels of 5-HT, HA, and SP at skin acupoints were lower than those in the model group (all P < .05), and tryptase level was higher (both P < .05). Tryptase level was higher on the skin at the MAL-SJ 10 acupoint than that on the MAR-SJ 10 acupoint (P = .004). Compared with the model group, the serum levels of IL-1β, IL-6, and TNF-α in the MAR-SJ 10 and MAL-SJ 10 groups were lower (all P < .05). ConclusionAcupuncture at KOA-sensitized acupoints mitigates joint injury in KOA rats and may bidirectionally regulate local MCs of these acupoints. This finding not only enhances the reference value of sensitizing points in clinical diagnosis and treatment, but also contributes to the understanding of the biological mechanisms underlying acupuncture intervention at sensitizing points.

Amentoflavone alleviated cartilage injury and inflammatory response of knee osteoarthritis through PTGS2.

The role of amentoflavone on cartilage injury in knee osteoarthritis (KOA) rats and the underlying mechanismwere explored. KOA rat and IL-1β-stimulated chondrocyte models were constructed. MTT, colony formation, and ELISA were performed to determine the cytotoxicity, cell proliferation, and inflammatory factors. The role of PTGS2 in IL-1β-stimulated chondrocytes was also confirmed through transfecting PTGS2 overexpression and silencing plasmids. Further, we analyzed how amentoflavone regulated PTGS2 to improve IL-1β-stimulated chondrocytes in vitro. Additionally, we analyzed the expression of PTGS2 after amentoflavone treatment. In vivo, HE and Safranin-O staining were carried out, and the inflammatory response was detected by ELISA and HE staining. In addition, we also analyzed the regulatory effect of amentoflavone on PTGS2 and explored the mechanism effect of PTGS2 in vitro and in vivo. The results indicated that PTGS2 was the downstream molecule of amentoflavone, which was highly expressed in IL-1β-stimulated chondrocytes and KOA rats, and amentoflavone decreased PTGS2 expression. We also confirmed the potential role of amentoflavone on KOA, which was also characterized by the repair of cartilage injury, reduction of inflammatory infiltration, and improvement of functional disability. Consistent with in vivo results, in vitro experiments gave the same conclusions. Amentoflavone reduced PTGS2 expression in IL-1β-stimulated chondrocytes and inhibited inflammation of chondrocytes via PTGS2. Collectively, the results confirmed that this drug was the potential targeted drug for KOA, whose repair effect on cartilage injury was partly related to PTGS2.

The impact of sericin on inflammation, oxidative stress, and lipid metabolism in female rats with experimental knee osteoarthritis.

This study investigated the effects of sericin on inflammation, oxidative stress, and lipid metabolism in female rats with experimental knee osteoarthritis (KOA), focusing on evaluating its effectiveness via the sterol regulatory protein (SREBP)-1C and SREBP-2 pathways. The rats were randomly assigned to three experimental groups: the C group (control), the KOA group (KOA control), and the sericin group (KOA + sericin). The KOA model was created by injecting monosodium iodoacetate (MIA) into the knee joint. Sericin was administered intra-articularly to rats on days 1, 7, 14, and 21 (0.8g/kg/mL, 50 µL). After 21days, the rats were sacrificed, and serum samples were analyzed using an ELISA to measure tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-10, SREBP-1c, SREBP-2, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), cholesterol, triglyceride, and total oxidant-antioxidant status (TOS-TAS) levels. The KOA group exhibited higher serum TNF-α, IL-1β, TOS, SREBP-1C, ACC, FAS, triglyceride, SREBP-2, and cholesterol levels than the C group (P < 0.05). However, the levels of these cytokines, except cholesterol, were significantly lower in the sericin group than in the KOA group. The KOA group exhibited significantly lower serum TAS and IL-10 levels than the C group (P < 0.05). In the sericin group, there was a statistically significant increase (P < 0.05). Sericin shows promising potential for reducing inflammation, oxidative stress, and lipid metabolism in experimental models of KOA in rats. However, further clinical research is necessary to validate the potential of sericin as a therapeutic agent for treating KOA. Key Points • Sericin can reduce knee osteoarthritis (KOA) symptoms in an experimental rat model. • In particular, in the serum of an experimental KOA rat model, sericin specifically reduces the levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), and increases the levels of anti-inflammatory cytokines, such as IL-10. • Sericin reduced lipid metabolism via the sterol regulatory protein (SREBP)-1C and SREBP-2 pathways and oxidative stress in the serum of the experimental KOA rat model. • The intra-articular administration of sericin has been shown to significantly reduce lipid metabolism, oxidative stress, and inflammation, as supported by biochemical analysis. These findings suggest its promising potential as an alternative treatment option for KOA.

Curcumin regulates autophagy through SIRT3-SOD2-ROS signaling pathway to improve quadriceps femoris muscle atrophy in KOA rat model

Knee osteoarthritis (KOA) usually leads to quadriceps femoris atrophy, which in turn can further aggravate the progression of KOA. Curcumin (CUR) has anti-inflammatory and antioxidant effects and has been shown to be a protective agent for skeletal muscle. CUR has been shown to have a protective effect on skeletal muscle. However, there are no studies related to whether CUR improves KOA-induced quadriceps femoris muscle atrophy. We established a model of KOA in rats. Rats in the experimental group were fed CUR for 5 weeks. Changes in autophagy levels, reactive oxygen species (ROS) levels, and changes in the expression of the Sirutin3 (SIRT3)-superoxide dismutase 2 (SOD2) pathway were detected in the quadriceps femoris muscle of rats. KOA led to quadriceps femoris muscle atrophy, in which autophagy was induced and ROS levels were increased. CUR increased SIRT3 expression, decreased SOD2 acetylation and ROS levels, inhibited the over-activation of autophagy, thereby alleviating quadriceps femoris muscle atrophy and improving KOA. CUR has a protective effect against quadriceps femoris muscle atrophy, and KOA is alleviated after improvement of quadriceps femoris muscle atrophy, with the possible mechanism being the reduction of ROS-induced autophagy via the SIRT3-SOD2 pathway.

Vagotomy accelerates the onset of symptoms during early disease progression and worsens joint-level pathogenesis in a male rat model of chronic knee osteoarthritis

ObjectiveLow vagal tone is common in osteoarthritis (OA) comorbidities and results in greater peripheral inflammation. Characterizing vagal tone's role in OA pathogenesis may offer insights into OA's influences beyond the articular joint. We hypothesized that low vagal tone would accelerate onset of OA-related gait changes and worsen joint damage in a rat knee OA model. MethodsKnee OA was induced in male Sprague Dawley rats by transecting the medial collateral ligament and medial meniscus. Then, left cervical vagus nerve transection (VGX, n ​= ​9) or sham VGX (non-VGX, n ​= ​6) was performed. Gait and tactile sensitivity were assessed at baseline and across 12 weeks, with histology and systemic inflammation evaluated at endpoint. ResultsAt week 4, VGX animals showed limping gait characteristics through shifted stance times from their OA to non-OA limb (p ​= ​0.055; stance time imbalance ​= ​1.6 ​± ​1.6%) and shifted foot strike locations (p ​< ​0.001; spatial symmetry ​= ​48.4 ​± ​0.835%), while non-VGX animals walked with a balanced and symmetric gait. Also at week 4, while VGX animals had a mechanical sensitivity (50% withdrawal threshold) of 13.97 ​± ​7.70 compared to the non-VGX animal sensitivity of 29.74 ​± ​9.43, this difference was not statistically significant. Histologically, VGX animals showed thinner tibial cartilage and greater subchondral bone area than non-VGX animals (p ​= ​0.076; VGX: 0.80 ​± ​0.036 ​mm2; non-VGX: 0.736 ​± ​0.066 ​mm2). No group differences in systemic inflammation were observed at endpoint. ConclusionsVGX resulted in quicker onset of OA-related symptoms but remained unchanged at later timepoints. VGX also had thinner cartilage and abnormal bone remodeling than non-VGX. Overall, low vagal tone had mild effects on OA symptoms and joint remodeling, and not at the level seen in common OA comorbidities.

Hypobaric hypoxia aggravates osteoarthritis via the alteration of the oxygen environment and bone remodeling in the subchondral zone.

A high prevalence of osteoarthritis (OA) has been observed among individuals living at high altitudes, and hypobaric hypoxia (HH) can cause bone mass and strength deterioration. However, the effect of HH on OA remains unclear. In this study, we aimed to explore the impact of HH on OA and its potential mechanisms. A rat knee OA model was established by surgery, and the rats were bred in an HH chamber simulating a high-altitude environment. Micro-computed tomography (Micro-CT), histological analysis, and RNA sequencing were performed to evaluate the effects of HH on OA invivo. A hypoxic co-culture model of osteoclasts and osteoblasts was also established to determine their effects on chondrogenesis invitro. Cartilage degeneration significantly worsened in the HH-OA group compared to that in the normoxia-OA (N-OA) group, 4 weeks after surgery. Micro-CT analysis revealed more deteriorated bone mass in the HH-OA group than in the N-OA group. Decreased hypoxia levels in the cartilage and enhanced hypoxia levels in the subchondral bone were observed in the HH-OA group. Furthermore, chondrocytes cultured in a conditioned medium from the hypoxic co-culture model showed decreased anabolism and extracellular matrix compared to those in the normoxic model. RNA sequencing analysis of the subchondral bone indicated that the glycolytic signaling pathway was highly activated in the HH-OA group. HH-related OA progression was associated with alterations in the oxygen environment and bone remodeling in the subchondral zone, which provided new insights into the pathogenesis of OA.

Mechanism of acupoint penetration acupuncture therapy regulating chondrocyte autophagy via the PI3K/Akt-mTOR pathway in KOA rats

ObjectiveTo investigate whether acupoint penetration acupuncture (APA) could regulate chondrocyte autophagy and apoptosis via the PI3K/Akt-mTOR signaling pathway to reduce cartilage degeneration in knee osteoarthritis (KOA) rats. MethodsKOA was induced in rats via intra-articular injection of sodium iodoacetate resolution. Forty male Sprague-Dawley rats were randomly assigned to blank control, model, APA, electro-acupuncture (EA), and sham model groups (n = 8) and those in the APA and EA groups received their respective therapies. Following completion of the treatment course, histological examinations of cartilage and muscle were conducted. Levels of apoptosis- and autophagy-related factors, including Bax, Bcl-2, mTOR, ULK-1, and Beclin-1 protein, and mRNAs were assessed. Additionally, β-endorphin (β-EP) concentrations in the brain and serum were measured. ResultsHistological analysis revealed that APA alleviated cartilage and muscle damage compared with the model group. APA inhibited cartilage degeneration by modulating the expression of apoptosis- and autophagy-related proteins and mRNA, thus preventing chondrocyte apoptosis. In the APA group, Bax and mTOR protein levels were significantly lower than those in the model group (both P = .024). Conversely, the Bcl-2 expression level was significantly higher than that in the EA group (P = .035). Additionally, ULK-1 expression was significantly lower than that in the EA group (P = .045). The mRNA level of Bax was significantly higher than that in the blank control group (P < .001). However, Beclin-1 levels were significantly higher than those in both the model and EA groups (both P < .001). ELISA results showed a significant decrease in the concentration of β-EP in the brains of the rats in the APA group compared with those in the model group (P = .032). ConclusionsAPA reduced osteoarthritis-related pain and alleviated cartilage damage by upregulating chondrocyte autophagy and down-regulating apoptosis via signaling pathways involving PI3K/Akt-mTOR in KOA rats.

Lipoxin A4 ameliorates knee osteoarthritis progression in rats by antagonizing ferroptosis through activation of the ESR2/LPAR3/Nrf2 axis in synovial fibroblast-like synoviocytes

BackgroundOur previous studies have shown that lipoxin A4 (LXA4) can serve as a potential biomarker for assessing the efficacy of exercise therapy in knee osteoarthritis (KOA), and fibroblast-like synoviocytes (FLSs) may play a crucial role in KOA pain as well as in the progression of the pathology. ObjectiveBy analyzing the GSE29746 dataset and collecting synovial samples from patients with different Kellgren–Lawrence (KL) grades for validation, we focused on exploring the potential effect of LXA4 on ferroptosis in FLSs through the ESR2/LPAR3/Nrf2 axis to alleviate pain and pathological advancement in KOA. MethodsThe association between FLSs ferroptosis and chondrocyte matrix degradation was explored by cell co-culture. We overexpressed and knocked down LPAR3 in vitro to explore its potential mechanism in FLSs. A rat model of monosodium iodoacetate (MIA)-induced KOA was constructed and intervened with moderate-intensity treadmill exercise and intraperitoneal injection of PHTPP to investigate the effects of the LXA4 intracellular receptor ESR2 on exercise therapy. ResultsESR2, LPAR3, and GPX4 levels in the synovium decreased with increasing KL grade. After LXA4 intervention in the co-culture system, GPX4, LPAR3, and ESR2 were upregulated in FLSs, collagen II was upregulated in chondrocytes, and MMP3 and ADAM9 were downregulated. LPAR3 overexpression upregulated the expression of GPX4, Nrf2, and SOD1 in FLSs, while downregulating the expression of MMP13 and MMP3; LPAR3 knockdown reversed these changes. Moderate-intensity platform training improved the behavioral manifestations of pain in KOA rats, whereas PHTPP treatment partially reversed the improvement in synovial and cartilage pathologies induced by platform training. ConclusionLXA4 inhibited FLSs ferroptosis by activating the ESR2/LPAR3/Nrf2 axis, thereby alleviating the pain and pathological progression of KOA. This study brings a new target for the treatment of KOA and also leads to a deeper understanding of the potential mechanisms of exercise therapy for KOA.

Guasha improves knee osteoarthritis by inhibiting chondrocyte apoptosis and regulating expression of autophagy-related genes and proteins in rats.

To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA. A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining. After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01). Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.

Targeting STAT6-mediated synovial macrophage activation improves pain in experimental knee osteoarthritis

BackgroundPain from osteoarthritis (OA) is one of the top causes of disability worldwide, but effective treatment is lacking. Nociceptive factors are released by activated synovial macrophages in OA, but depletion of synovial macrophages paradoxically worsens inflammation and tissue damage in previous studies. Rather than depleting macrophages, we hypothesized that inhibiting macrophage activation may improve pain without increasing tissue damage. We aimed to identify key mechanisms mediating synovial macrophage activation and test the role of STAT signaling in macrophages on pain outcomes in experimental knee OA.MethodsWe induced experimental knee OA in rats via knee destabilization surgery, and performed RNA sequencing analysis on sorted synovial tissue macrophages to identify macrophage activation mechanisms. Liposomes laden with STAT1 or STAT6 inhibitors, vehicle (control), or clodronate (depletion control) were delivered selectively to synovial macrophages via serial intra-articular injections up to 12 weeks after OA induction. Treatment effects on knee and hindpaw mechanical pain sensitivity were measured during OA development, along with synovitis, cartilage damage, and synovial macrophage infiltration using histopathology and immunofluorescence. Lastly, crosstalk between drug-treated synovial tissue and articular chondrocytes was assessed in co-culture.ResultsThe majority of pathways identified by transcriptomic analyses in OA synovial macrophages involve STAT signaling. As expected, macrophage depletion reduced pain, but increased synovial tissue fibrosis and vascularization. In contrast, STAT6 inhibition in macrophages led to marked, sustained improvements in mechanical pain sensitivity and synovial inflammation without worsening synovial or cartilage pathology. During co-culture, STAT6 inhibitor-treated synovial tissue had minimal effects on healthy chondrocyte gene expression, whereas STAT1 inhibitor-treated synovium induced changes in numerous cartilage turnover-related genes.ConclusionThese results suggest that STAT signaling is a major mediator of synovial macrophage activation in experimental knee OA. STAT6 may be a key mechanism mediating the release of nociceptive factors from macrophages and the development of mechanical pain sensitivity. Whereas therapeutic depletion of macrophages paradoxically increases inflammation and fibrosis, blocking STAT6-mediated synovial macrophage activation may be a novel strategy for OA-pain management without accelerating tissue damage.

NGF Signaling Exacerbates KOA Peripheral Hyperalgesia via the Increased TRPV1-Labeled Synovial Sensory Innervation in KOA Rats.

Knee osteoarthritis (KOA) pain is caused by nociceptors, which are actually sensory nerve fiber endings that can detect stimuli to produce and transmit pain signals, and high levels of NGF in synovial tissue led to peripheral hyperalgesia in KOA. The purpose of this study is to investigate how sensory nerve fibers respond to the NGF/TrKA signal pathway and mediate the peripheral hyperalgesia in KOA rats. Forty SD male rats were randomly divided into 4 groups: normal, KOA, KOA + NGF, and KOA + siRNA TrKA. KOA model rats were induced by anterior cruciate ligament transection (ACLT). Mechanical and cold withdrawal thresholds (MWT and CWT) were measured 4 times in each group. The synovial tissues were harvested on day 28, and the expressions of NGF, TrKA, TRPV1, IL-1β, and PGP9.5 were determined using western blot, qPCR, and immunofluorescence staining. The primary rat fibroblast-like synoviocytes (FLSs) and DRG cells were divided into 4 groups as in vivo. The expressions of NGF, TrKA, TRPV1, and CGRP in vitro were determined using western blot and qPCR. KOA and intra-articular injection with NGF protein increased both mRNA and protein levels, not only TRPV1, PGP 9.5, and IL-1β in the synovial tissue, but also TRPV1, PGP 9.5, and S100 in the DRG tissue, while above changes were partly reversed after siRNA TrKA intervention. Besides, siRNA TrKA could improve peripheral hyperalgesia and decreased the TRPV1 positive nerve fiber innervation in synovial tissue. The results in vitro were consistent with those in vivo. This study showed the activation of the NGF/TrKA signaling pathway in KOA promoted the release of pain mediators, increased the innervation of sensory nerve fibers in the synovium, and worsened peripheral hyperalgesia. It also showed increased TRPV1 positive sensory innervation in KOA was mediated by NGF/TrKA signaling and exacerbated peripheral hyperalgesia.

Modulation of gut microbiota by crude mulberry polysaccharide attenuates knee osteoarthritis progression in rats

Mulberry (Morus alba L.), a kind of common fruits widely cultivated worldwide, has been proven various biological activities. However, its potential role in the progression of knee osteoarthritis (KOA) remains unclear. This study aims to investigate the potential protective effects of crude polysaccharide extracted from mulberry fruit, referred to as a complex blend of polysaccharides and other unidentified extracted impurities, on KOA progression. The KOA rats were established by injection of 1 mg sodium monoiodoacetate into knee, and administrated with crude mulberry polysaccharide (Mup) by gastric gavage for 4 weeks. Furthermore, intestinal bacteria clearance assay (IBCA) and fecal microbiota transplantation were conducted for the evaluation of the effect of gut microbiota (GM) on KOA. Our findings demonstrated that Mup, particularly at a dosage of 200 mg/kg, effectively improved abnormal gait patterns, reduced the level of inflammation, mitigated subchondral bone loss, restored compromised joint surfaces, alleviated cartilage destruction, and positively modulated the dysregulated profile of GM in KOA rats. Moreover, IBCA compromised the protective effects of Mup, while transplantation of fecal bacteria from Mup-treated rats facilitated KOA recovery. Collectively, our study suggested that Mup had the potential to ameliorate the progression of KOA, potentially through its modulation of GM profile.

Confirmation of pain-related neuromodulation mechanism of Bushen Zhuangjin Decoction on knee osteoarthritis

Ethnopharmacological relevanceBushen Zhuangjin Decoction (BZD) are an herbal compound commonly used to treat osteoarthritis (OA) in China. Aim of the studyThis study aimed to verify the mechanism of Bushen Zhuangjin Decoction in relieving the pain of knee osteoarthritis. Materials and methodsNetwork pharmacology evaluation was used to discover the potential targets of BZD to relieve pain in KOA. The therapeutic effects of BZD treatment on KOA pain using histomorphology, behavioral assessments, suspension chip analysis, and ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) assays. The functional magnetic resonance imaging was used to explore the effects of BZD treatment on brain function associated to KOA. ResultsNetwork pharmacological analysis revealed the association between the analgesic effect of BZD on KOA and the pain signaling neurotransmitter 5-HT. Subsequently, we conducted experiments to verify the therapeutic effect of BZD on pain in KOA animal models. Behavioral tests demonstrated that the pain threshold of knee osteoarthritis rats decreased in PWT and PWL, but BZD was able to increase the pain threshold. Histopathological staining indicated thinning of the cartilage layer and sparse trabeculae in the subchondral bone. Suspension chip analysis revealed a significant increase in pro-inflammatory factors of IL-1α, IL-5, IL-12, IL-17A, RANTES, TNF-α and M-CSF in KOA, along with a significant decrease in anti-inflammatory factor of IL-13. However, BZD treatment decreased the expression of pro-inflammatory factors and increased the content of anti-inflammatory factor. UHPLC-MS/MS analysis showed a significant decrease in the serum levels of GABA, E, GSH, Kyn, Met, and VMA in KOA, which were significantly increased by BZD. Conversely, the serum levels of TrpA, TyrA, Spd, and BALa were significantly increased in KOA and significantly decreased by BZD. ELISA and Western blot analysis showed increased expression of subchondral bone pain-related neuropeptides SP, CGRP, TH, NPY, VEGFA, 5-HT3 in KOA, which were decreased in BZD. Functional magnetic resonance imaging demonstrated that BZD exerts its therapeutic effect on KOA by modulating the activity and functional connections of the cortex, hypothalamus, and hippocampus. ConclusionsThis study confirmed the significant role of pain-related neuromodulation mechanisms in the analgesic therapy of BZD and provides a theoretical foundation for using BZD as a traditional Chinese medical treatment for KOA pain.

Alleviation of peripheral sensitization by quadriceps insertion of cog polydioxanone filaments in knee osteoarthritis rats

A recently established therapeutic strategy, involving the insertion of biodegradable cog polydioxanone filaments into the quadriceps muscles using the Muscle Enhancement and Support Therapy (MEST) device, has demonstrated significant efficacy in alleviating knee osteoarthritis (OA) pain. This study investigated changes in peripheral sensitization as the potential mechanism underlying MEST-induced pain relief in monoiodoacetate (MIA) induced OA rats. The results revealed that MEST treatment potently reduces MIA-induced sensitization of L3/L4 dorsal root ganglion (DRG) neurons, the primary nociceptor pathway for the knee joint. This reduction in DRG sensitization, as elucidated by voltage-sensitive dye imaging, is accompanied by a diminished overexpression of TRPA1 and NaV1.7, key nociceptor receptors involved in mechanical pain perception. Importantly, these observed alterations strongly correlate with a decrease in mechanically-evoked pain behaviors, providing compelling neurophysiological evidence that MEST treatment alleviates OA pain by suppressing peripheral sensitization.

Evaluation of the therapeutic potential of infrapatellar fat pad adipose-derived stem cells and their secretome for regenerating knee articular cartilage in a rat model of osteoarthritis.

Mesenchymal stem cell (MSC) therapy has ameliorative effects for treating knee osteoarthritis (KOA) disease. Moreover, there is a growing interest in using MSCs-derived secretome (Sec) containing trophic factors secreted by MSCs for KOA treatment. Recently, some studies have suggested that the combination of MSCs and Sec has the potential to treat the diseases. This study aimed to evaluate the ameliorative effects of combined administration of infrapatellar fat pad (IPFP)-derived MSCs, a type of adipose-derived stem cells (ASCs), for treating degenerated cartilage in a rat model of KOA. IPFP-ASCs were isolated from the IPFP of male rats. Sec was obtained from IPFP-ASCs in the fourth passage. Eight weeks after the induction of KOA by collagenase II, the rats were divided into 5 groups (n=5), including a control group with no treatment, and four experimental groups that received sodium hyaluronate (Hyalgan®, Hya), ASCs, Sec, and IPFP-ASCs+Sec, respectively by an infrapatellar injection. To perform the pathological and radiological evaluations, the animals were sacrificed 8 weeks later. Our findings indicated that combined administration of the IPFP-ASCs and Sec statistically (P<0.05) improved scores of medial tibial and femoral condyles and medial fabella osteophytes. Also, it statistically (P<0.05) enhances the cartilage surface, matrix, cell distribution and population viability, and subchondral bone indices. No statistical difference was observed between IPFP-ASCs+Sec and IPFP-ASCs. Administration of IPFP-ASCs+Sec has a therapeutic potential to treat KOA in rats. However, there is no difference in the combined administration of IPFP-ASCs and Sec with IPFP-ASCs alone.

Ski ameliorates synovial cell inflammation in monosodium iodoacetate-induced knee osteoarthritis

Knee osteoarthritis (KOA) is one of the most common degenerative diseases and is characterized by cartilage degeneration, synovial inflammation, joint stiffness and even loss of motor function. In the clinical treatment of arthritis, conventional analgesic and anti-inflammatory drugs have great side effects. We have evaluated the possibility of the endogenous transcription regulator Ski as an anti-inflammatory alternative in OA through experimental studies in animal models and in vivo and in vitro. Male Sprague‒Dawley rats were injected with monosodium iodoacetate (MIA) into the knee joints to induce symptoms identical to those of human OA. We isolated knee synovial tissue under sterile conditions and cultured primary synovial cells. In vitro, Ski inhibits the proinflammatory factors IL-1β, IL-6 and TNF-α mRNA and protein expression in lipopolysaccharide (LPS)-stimulated fibroblast-like synoviocytes (FLSs) and U-937 cells. In addition, Ski attenuates or inhibits OA-induced synovial inflammation by upregulating the protein expression of the anti-inflammatory factor IL-4 and downregulating the protein expression of downstream molecules related to the NF-κB inflammatory signaling pathway. In vivo, Ski downregulated proinflammatory factors and p–NF–κB p65 in KOA synovial tissue and alleviated pain-related behaviors in KOA rats. These experimental data show that Ski has strong anti-inflammatory activity. Ski is an endogenous factor, and if used in the clinical treatment of OA, the side effects are small. However, the anti-inflammatory mechanism of Ski must be further studied.

The Combined Intraosseous Administration of Orthobiologics Outperformed Isolated Intra-articular Injections in Alleviating Pain and Cartilage Degeneration in a Rat Model of MIA-Induced Knee Osteoarthritis

Background: Intra-articular (IA) platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) injections have shown efficacy and safety in treating osteoarthritis (OA). However, the effectiveness and mechanisms of combined intraosseous (IO) administration of these orthobiologics have yet to be explored. Purpose/Hypothesis: The purpose of this study was to evaluate the effect on pain, cartilage, synovium/infrapatellar fat pad (IFP), and subchondral bone in rat knee OA, comparing isolated IA with combined IA and IO (IA+IO) injections of PRP or BMAC. It was hypothesized that combined injections would be superior to sole IA injections. Study Design: Controlled laboratory study. Methods: A total of 48 rats were divided into 6 groups: sham (only joint puncture during OA induction with IA+IO saline injection treatment) and 5 groups with OA induction, control (IA+IO saline injection), PRP (IA PRP+IO saline injection), BMAC IA (IA BMAC+IO saline injection), PRP IA+IO (IA+IO PRP injection), and BMAC IA+IO (IA+IO BMAC injection). OA was induced by IA injection of monosodium iodoacetate (MIA). Rats were administered different orthobiologics according to their grouping 3 weeks after the MIA injection. Pain changes were evaluated using the weightbearing ratio assay at weeks 3, 4, 5, 7, and 9 after OA induction. Rats were euthanized at week 9 for gross, radiological, histological, immunohistochemical, and immunofluorescence assessments of cartilage, synovium, and subchondral bone. Results: Compared with the control group, all orthobiologics injection groups had reduced joint pain. Compared with IA injection, IA+IO injections provided superior pain relief by suppressing calcitonin gene-related peptide and substance P in both the synovium/IFP and subchondral bone. IA+IO injections slowed the progression of subchondral bone lesions by inhibiting CD31hiEmcnhi vessel formation and excessive osteoclast and osteoblast turnover while preserving subchondral bone microarchitecture, slowing cartilage degeneration. However, IA+IO injections did not outperform isolated IA injections in reducing synovitis and synovium/IFP fibrosis. Compared with PRP, BMAC exhibited superior inhibition of pain-related mediators, but no significant differences were observed in synovitis suppression, infrapatellar fat pad fibrosis, and subchondral bone protection. Conclusion: IA+IO injections of orthobiologics were more effective in relieving pain, slowing cartilage degeneration, and inhibiting abnormal vascularization and remodeling compared with isolated IA injections. BMAC showed superior pain relief in the synovium/IFP and subchondral bone compared with PRP. Further research is needed to optimize PRP and BMAC components for enhanced efficacy in OA management. Clinical Relevance: Our findings contribute to advancing the understanding of pain relief mechanisms and support the endorsement of IO injection of orthobiologics for the treatment of OA and joint pain.

Research of the analgesic effects and central nervous system impact of electroacupuncture therapy in rats with knee osteoarthritis

It aimed to observe the effects of TongDu TiaoShen (TDTS) electroacupuncture (EA) on the analgesia and central system of knee osteoarthritis (KOA) rats and explore its mechanism. SD rats were rolled into the blank group, model group (KOA), control group (duloxetine 500 mg/kg/d, Ctrl), conventional EA group, and TDTS-EA group. Radiometric pain measurements and the Lequesne MG scale were used to evaluate the behavioral performance of the rats. Dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), β-endorphin (β-EP), and leucine-enkephalin (L-ENK) were detected in the midbrain and spinal cord of lumbar enlargement. Interleukin (IL)-1β protein expression was detected by Western blot. The incubation period of thermal pain and foot contraction was decreased in the KOA group versus blank group, the Lequesne MG score was increased, DA, NE, 5-HT, β-EP, and L-ENK in the midbrain and spinal cord were increased, and synovial tissue IL-1β protein expression was increased (P < 0.05). EA group and TDTS-EA group had an increased incubation period of thermal pain contraction, decreased Lequesne MG score, decreased DA, NE, etc. In the midbrain, increased 5-HT and NE in the spinal cord, and decreased IL-1β in the synovial tissue versus KOA group (P < 0.05). The Lequesne MG score and midbrain DA, NE, 5-HT, β-EP, and synovial tissue IL-1β expression were decreased in TDTS-EA group versus EA group (P < 0.05). EA can effectively improve the behavioral score of KOA and participate in central analgesia by regulating central DA, NE, 5-HT, β-EP, and L-ENK.