Suppression of NUPR1 in fibroblast-like synoviocytes reduces synovial fibrosis via the Smad3 pathway.

BACKGROUND: Synovial fibrosis is a common complication of knee osteoarthritis (KOA), a pathological process characterized by myofibroblast activation and excessive extracellular matrix (ECM) deposition. Fibroblast-like synoviocytes (FLSs) are implicated in KOA pathogenesis, contributing to synovial fibrosis through diverse mechanisms. Nuclear protein 1 (NUPR1) is a recently identified transcription factor with crucial roles in various fibrotic diseases. However, its molecular determinants in KOA synovial fibrosis remain unknown. This study aims to investigate the role of NUPR1 in KOA synovial fibrosis through in vivo and in vitro experiments. METHODS: We examined NUPR1 expression in the murine synovium and determined the impact of NUPR1 on synovial fibrosis by knockdown models in the destabilization of the medial meniscus (DMM)-induced KOA mouse model. TGF-ß was employed to induce fibrotic response and myofibroblast activation in mouse FLSs, and the role and molecular mechanisms in synovial fibrosis were evaluated under conditions of NUPR1 downexpression. Additionally, the pharmacological effect of NUPR1 inhibitor in synovial fibrosis was assessed using a surgically induced mouse KOA model. RESULTS: We found that NUPR1 expression increased in the murine synovium after DMM surgical operation. The adeno-associated virus (AAV)-NUPR1 shRNA promoted NUPR1 deficiency, attenuating synovial fibrosis, inhibiting synovial hyperplasia, and significantly reducing the expression of pro-fibrotic molecules. Moreover, the lentivirus-mediated NUPR1 deficiency alleviated synoviocyte proliferation and inhibited fibroblast to myofibroblast transition. It also decreased the expression of fibrosis markers α-SMA, COL1A1, CTGF, Vimentin and promoted the activation of the SMAD family member 3 (SMAD3) pathway. Importantly, trifluoperazine (TFP), a NUPR1 inhibitor, attenuated synovial fibrosis in DMM mice. CONCLUSIONS: These findings indicate that NUPR1 is an antifibrotic modulator in KOA, and its effect on anti-synovial fibrosis is partially mediated by SMAD3 signaling. This study reveals a promising target for developing novel antifibrotic treatment.

Gallic acid ameliorates synovial inflammation and fibrosis by regulating the intestinal flora and its metabolites.

Gallic acid (GA) has been found by a large number of studies to have pharmacological effects such as antioxidant and anti-inflammatory properties. However, the underlying therapeutic mechanisms are not fully understood.. Studies have shown that altering the intestinal flora affects host metabolism and effectively mediates the development of synovitis. The aim of this study was to explore the pharmacological effects of GA in the treatment of synovial inflammation and anti-synovial fibrosis in knee osteoarthritis (KOA) and the underlying mechanisms by macrogenomics combined with off-target metabolomics. We established a synovitis model via in vivo and in vitro experiments to observe the effect of GA intervention on synovitis. Moreover, we collected serum and feces from rats and analyzed the changes in intestinal flora by macro-genome sequencing and the changes in metabolites in the serum by untargeted metabolomics. We found that GA reduced the levels of IL-1ß, IL-6, and TNF-α, and decreased the protein expression levels of α-SMA, TGF-ß, and Collagen I in synovial tissues and cells, and the composition and function of the intestinal flora were similarly altered. Combined with macrogenomic pathway enrichment analysis and metabolic pathway enrichment analysis, these findings revealed that GA impacts Bacteroidia and Muribaculaceae abundance, and via the following metabolic pathways: sphingolipid metabolism, glycerophospholipid metabolism, and arginine biology.to ameliorate synovial inflammation and fibrosis in KOA. The therapeutic effect of GA on KOA synovitis and fibrosis is partly attributed to the alleviation of metabolic disorder and the rebalancing of the intestinal flora. These results provides a rationale for the therapeutic application of GA in the treatment of synovitis.

Role of microRNAs in pituitary gonadotrope cells.

The gonadotrope cells within the pituitary control vital processes of reproduction by producing follicle stimulating hormone (FSH) and luteinizing hormone (LH). Both external stimuli and internal regulatory factors contribute to the regulation of gonadotrope development and function. In recent years, growing evidences indicate that microRNAs (miRNAs), which regulate gene expression post-transcriptionally, play critical roles in multiple processes of gonadotrope development and function, including the syntheses of α or ß subunits of FSH and LH, the secretion of LH, the regulation of GnRH signaling, and the maintenance of gonadotrope cell kinetics. Here, we review recent advances of miRNAs' expression, functions and mechanisms approached by using miRNA knockout mouse models, in silico analysis and the in vitro cultures of primary pituitary cells and gonadotrope-derived cell lines. By summarizing and discussing different roles of miRNAs in gonadotropes, this minireview helps to gain insights into the complex molecular network in gonadotropes and reproduction.

Total glucosides of white paeony capsule alleviate articular cartilage degeneration and aberrant subchondral bone remodeling in knee osteoarthritis.

Knee osteoarthritis (KOA) is a prevalent degenerative joint disease that is primarily managed by improving the destroyed cartilage and reversing subchondral bone remodeling. Total glucosides of white paeony (TGP) capsule primarily contains extracts from the white peony root and has been shown to have various pharmacological effects, but its role in KOA still requires comprehensive evaluation. In this study, we aimed to investigate the protective effect of TGP on knee cartilage and subchondral bone, as well as elucidate the underlying molecular mechanisms. The effect of TGP on KOA progression was evaluated in the destabilization of the medial meniscus (DMM)-induced KOA model of mouse and interleukin (IL)-1ß-induced KOA model of primary mouse chondrocytes. In vivo and in vitro experiments demonstrated that TGP had a protective effect on the cartilage. Treatment with TGP could induce the synthesis of critical elements in the cartilage extracellular matrix and downregulate the synthesis of degrading enzymes in the extracellular matrix. Regarding the underlying mechanisms, TGP inhibited the phosphorylation and nuclear translocation of p65 by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. In addition, TGP could reduce the secretion of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α). Moreover, it has a sustained effect on coupled subchondral bone remodeling through regulation of the OPG/RANKL/RANK pathway. In conclusion, TGP may protect articular cartilage by downregulating the NF-κB signaling pathway and may support coupled subchondral bone remodeling by regulating OPG/RANKL/RANK signaling pathway in the DMM-induced KOA model of mouse, suggesting a new therapeutic potential for KOA treatment.

Activation of vascular endothelial cells by synovial fibrosis promotes Netrin-1-induced sensory nerve sprouting and exacerbates pain sensitivity.

Synovial fibrosis is one of the most dominant histopathological changes in osteoarthritis of the knee (KOA), and activation of vascular endothelial cells in synovial fibrosis is both an important factor in mediating pain in KOA and a major contributor to the generation of pain signals. At the same time, angiogenesis and nerve fibres are more likely to underlie the pathology of pain induced by synovial fibrosis. In the present study, we established a co-culture model of human umbilical vein endothelial cells (HUVECs) with dorsal root ganglion (DRG) and detected tissue and cellular Netrin-1, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), growth-associated protein-43 (GAP43), colorectal cancer deleted (DCC), uncoordinated 5 (UNC5), and the related expression of calcitonin gene-related peptide (CGRP), substance P (SP) and nerve growth factor (NGF) in supernatant by ELISA to investigate the intervention of vascular endothelial cell activation on sensory nerve sprouting exacerbating peripheral pain sensitivity and to investigate the effect of Netrin-1 from the perspective of Netrin-1 secretion to illustrate its effector mechanism.

Agnuside Alleviates Synovitis and Fibrosis in Knee Osteoarthritis through the Inhibition of HIF-1α and NLRP3 Inflammasome.

Increasing evidence has shown that NLRP3 inflammasome activation participates in chronic aseptic inflammation and is related to tissue fibrosis. Our last study also revealed the vital role of NLRP3 inflammasome, highly associated with tissue hypoxia, in the onset and development of knee osteoarthritis (KOA). In this study, we tried to find a possible benign intervention for that pathological process. Agnuside (AGN), a nontoxic, natural small molecule isolated from the extract of Vitex negundo L. (Verbenaceae), has been demonstrated to have antioxidation, anti-inflammatory, analgesia, and many other properties as an iridoid glycoside, although its specific target is still unclear. Therefore, we established MIA-induced KOA model rats and investigated the effects of AGN oral gavage on oxygen-containing state, NLRP3 inflammasome, synovitis, and fibrosis in KOA. Pimonidazole staining and HIF-1α immunohistochemical assay both showed that AGN at the oral dose of 6.25 mg/kg can effectively relieve local hypoxia in synovial tissue. Besides, we observed a decrease of HIF-1α, caspase-1, ASC, and NLRP3 after AGN intervention, both in the mRNA and protein levels. In addition, rats treated with the AGN showed less inflammatory reaction and fibrosis, not only in the expression of NLRP3, inflammasome downstream factors IL-1ß and IL-18, and fibrosis markers TGF-ß, TIMP1, and VEGF but also in the observation of HE staining, anatomical characteristics, Sirius Red staining, and type I collagen immunohistochemistry. Subsequently, we established LPS-induced models of fibroblast-like synoviocytes (FLSs) mimicking the inflammatory environment of KOA and activating NLRP3 inflammasome. FLSs treated with AGN (3 µM) resulted in a downregulation of HIF-1α and the components required for NLRP3 inflammasome activation. Meanwhile, the content of proinflammatory factors IL-1ß and IL-18 in FLS supernatant was also reduced by AGN. In addition, both mRNA and protein levels of the fibrotic markers were significantly decreased after AGN management. To conclude, this study demonstrates that AGN alleviates synovitis and fibrosis in experimental KOA through the inhibition of HIF-1α accumulation and NLRP3 inflammasome activation. Additionally, not only does it reveal some novel targets for anti-inflammatory and antioxidant effects of AGN but also announces its potential value in treating KOA in humans.

Sanse Powder Essential Oil Nanoemulsion Negatively Regulates TRPA1 by AMPK/mTOR Signaling in Synovitis: Knee Osteoarthritis Rat Model and Fibroblast-Like Synoviocyte Isolates.

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1ß, IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPARγ) and PPARγ coactivator-1α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling.

Inhibition of Synovial Macrophage Pyroptosis Alleviates Synovitis and Fibrosis in Knee Osteoarthritis.

Increasing evidence has shown that macrophage pyroptosis in different tissues participates in chronic aseptic inflammation and is related to tissue fibrosis. Our last studies also revealed the vital role of synovial fibroblast pyroptosis in the onset and development of knee osteoarthritis (KOA). In this study, we aimed to investigate whether synovial macrophage pyroptosis did occur and whether this form of cell death should be related to synovitis and fibrosis of KOA. In the synovial tissue of KOA model rats, we observed a decrease of caspase1, NLRP3, ASC, and GSDMD caused by macrophage depletion in both the mRNA and protein expressions. Besides, rats treated with the specific caspase1 inhibitor Ac-YVAD-CMK showed less inflammatory reaction and fibrosis, not only in the expression of proinflammatory factors IL-1ß, IL-18, and HMGB1 and fibrosis markers TGF-ß, PLOD2, COL1A1, and TIMP1 but also in the observation of HE staining, Sirius Red staining, and the transverse diameters of the right knees. Subsequently, we established an LPS+ATP-induced model in macrophages mimicking the inflammatory environment of KOA and inducing macrophage pyroptosis. Macrophages transfected with caspase1 siRNA showed reduced cell death; meanwhile, the relative expression of pyroptosis-related proteins were also downregulated. In addition, the level of fibrotic markers in synovial fibroblasts were significantly decreased after coculture with siRNA GSDMD-transfected macrophages. To conclude, synovial macrophage pyroptosis may occur in the pathological processes of KOA and inhibition of synovial macrophage pyroptosis alleviates synovitis and fibrosis in KOA model rats.

Protective effects of emodin on subchondral bone and articular cartilage in osteoporotic osteoarthritis rats: A preclinical study.

BACKGROUND: Osteoporotic osteoarthritis (OP-OA) is a severe pathological form of OA, urgently requiring precise management strategies and more efficient interventions. Emodin (Emo), an effective ingredient found in the traditional Chinese medicine rhubarb, has been dEmonstrated to promote osteogenesis and inhibit extracellular matrix degradation. In this study, we aimed to investigate the interventional effects of Emo on the subchondral bone and cartilage of the knee joints in OP-OA model rats. METHODS: Thirty-two SD rats were randomly and equally divided into sham, OP-OA, Emo low-dose, and Emo high-dose groups. Micro-CT scanning was conducted to examine the bone microstructure of the rat knee joints. H&E and Safranin O and Fast Green staining (SO&FG) were performed for the pathomorphological evaluation of the rat cartilage tissues. ELISA was used to estimate the rat serum expression levels of inflammatory factors, including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Additionally, the CCK-8 assay was utilized for determining the viability of Emo-treated BMSCs. Western blot and real-time PCR analyses were also employed to measure the bone formation indexes and cartilage synthesis and decomposition indexes. Lastly, the osteogenic and chondrogenic differentiation efficiency of the BMSCs was investigated via Alizarin Red and Alcian Blue staining. RESULTS: Emo intervention alleviated the bone microstructural disruption of the subchondral bone and articular cartilage in the OP-OA rats and up-regulated the expression of bone and cartilage anabolic metabolism indicators, decreased the expression of cartilage catabolism indicators, and diminished the expression of inflammatory factors in the rat serum (P<0.05). Furthermore, Emo reversed the decline in the osteogenic and chondrogenic differentiation ability of the BMSCs (P<0.05). CONCLUSION: Emo intervention mitigates bone loss and cartilage damage in OP-OA rats and promotes the osteogenic and chondrogenic differentiation of BMSCs.

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

Objectives: 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. Methods: 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-1ß, 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. Results: KOA and intra-articular injection with NGF protein increased both mRNA and protein levels, not only TRPV1, PGP 9.5, and IL-1ß 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. Conclusion: 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.

Casticin ameliorates osteoarthritic cartilage damage in rats through PI3K/AKT/HIF-1α signaling.

Knee osteoarthritis (KOA) is a chronic, disabling knee joint lesion in which degeneration and defects in articular cartilage are the most important features. Casticin (CAS) is a flavonoid extracted from the Chinese herb Vitex species that has anti-inflammatory and antitumor effects. The aim of this study was to investigate the therapeutic and mechanistic effects of CAS on cartilage damage in KOA. A KOA rat model was established by anterior cruciate ligament transection (ACLT), and cartilage morphological changes were assessed by histological analysis and micro-CT scans. Subsequently, chondrocytes were treated with 10 ng/mL IL-1ß to establish an OA model. CCK-8 assays and EdU assays were performed to assess the viability of CAS-treated chondrocytes. Western blotting, flow cytometry and Hoechst 33342/PI Double Stain were used to detect chondrocyte apoptosis. Western blotting, qRT‒PCR and ELISA were used to detect changes in inflammatory mediators. In addition, cartilage matrix-related indices were detected by Western blotting, qRT‒PCR and immunofluorescence (IF) analysis. Immunohistochemistry (IHC) and Western blotting were performed to detect the expression of p-PI3K, p-AKT and HIF-1α in vivo and in vitro. Micro-CT, pathological sections and related scores showed that CAS improved the alterations in bony structures and reduced cartilage damage and osteophyte formation in the ACLT model. In vivo, CAS attenuated IL-1ß-induced cartilage matrix degradation, apoptosis and the inflammatory response. In addition, CAS inhibited the expression of the PI3K/AKT/HIF-1α signaling pathway in the ACLT animal model and IL-1ß cell model. CAS may ameliorate cartilage damage in OA by inhibiting the PI3K/AKT/HIF-1α signaling pathway, suggesting that CAS is a potential strategy for the treatment of OA.

Berbamine ameliorates DSS-induced colitis by inhibiting peptidyl-arginine deiminase 4-dependent neutrophil extracellular traps formation.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with immune dysregulation affecting colon inflammatory response. Recent studies have highlighted that neutrophil extracellular traps (NETs) play an important role in the pathogenesis of UC. Berbamine (BBM), one of the bioactive ingredients extracted from Chinese herbal medicine Berberis vulgaris L, has attracted intensive attentions due to its significant anti-inflammatory activity and a marketing drug for treating leukemia in China. However, the exact role and potential molecular mechanism of BBM against UC remains elusive. In the present study, our results showed that BBM could markedly improve the pathological phenotype and the colon inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. Then, comprehensive approaches combining network pharmacology and molecular docking analyses were employed to predict the therapeutic potential of BBM in treating UC by peptidyl-arginine deiminase 4 (PAD4), a crucial molecule involved in NETs formation. The molecular docking results showed BBM had a high affinity for PAD4 with a binding energy of -9.3 kcal/mol Moreover, PAD4 expression and NETs productions, including citrullination of histone H3 (Cit-H3), neutrophil elastase (NE), myeloperoxidase (MPO) in both neutrophils and colonic tissue were reduced after BBM administration. However, in the mice with DSS-induced colitis pretreated with GSK484, a PAD4-specific inhibitor, BBM could not further reduce disease related indexes, expression of PAD4 and NETs productions. Above all, the identification of PAD4 as a potential target for BBM to inhibit NETs formation in colitis provides novel insights into the development of BBM-derived drugs for the clinical management of UC.

The mechanism of palmatine-mediated intestinal flora and host metabolism intervention in OA-OP comorbidity rats.

Background: ErXian decoction is a Chinese herbal compound that can prevent and control the course of osteoarthritis (OA) and osteoporosis (OP). OP and OA are two age-related diseases that often coexist in elderly individuals, and both are associated with dysregulation of the gut microbiome. In the initial study, Palmatine (PAL) was obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and network pharmacological screening techniques, followed by 16S rRNA sequencing and serum metabolomics of intestinal contents, to explore the mechanism of PAL in the treatment of OA and OP. Methods: The rats selected for this study were randomly divided into three groups: a sham group, an OA-OP group and a PAL group. The sham group was intragastrically administered normal saline solution, and the PLA group was treated with PAL for 56 days. Through microcomputed tomography (micro-CT), ELISA, 16S rRNA gene sequencing and non-targeted metabonomics research, we explored the potential mechanism of intestinal microbiota and serum metabolites in PAL treatment of OA-OP rats. Results: Palmatine significantly repair bone microarchitecture of rat femur in OA-OP rats and improved cartilage damage. The analysis of intestinal microflora showed that PAL could also improve the intestinal microflora disorder of OA-OP rats. For example, the abundance of Firmicutes, Bacteroidota, Actinobacteria, Lactobacillus, unclassified_f_Lachnospiraceae, norank_f_Muribaculaceae, Lactobacillaceae, Lachnospiraceae and Muribaculaceae increased after PAL intervention. In addition, the results of metabolomics data analysis showed that PAL also change the metabolic status of OA-OP rats. After PAL intervention, metabolites such as 5-methoxytryptophol, 2-methoxy acetaminophen sulfate, beta-tyrosine, indole-3-carboxylic acid-O-sulfate and cyclodopa glucoside increased. Association analysis of metabolomics and gut microbiota (GM) showed that the communication of multiple flora and different metabolites played an important role in OP and OA. Conclusion: Palmatine can improve cartilage degeneration and bone loss in OA-OP rats. The evidence we provided supports the idea that PAL improves OA-OP by altering GM and serum metabolites. In addition, the application of GM and serum metabolomics correlation analysis provides a new strategy for uncovering the mechanism of herbal treatment for bone diseases.

L-carnitine alleviates synovitis in knee osteoarthritis by regulating lipid accumulation and mitochondrial function through the AMPK-ACC-CPT1 signaling pathway.

BACKGROUND: Knee osteoarthritis (KOA) is a disability-associated condition that is rapidly growing with the increase in obesity rates worldwide. There is a pressing need for precise management and timely intervention in the development of KOA. L-carnitine has been frequently recommended as a supplement to increase physical activity in obese individuals due to its role in fatty acid metabolism, immune disorders, and in maintaining the mitochondrial acetyl-CoA/CoA ratio. In this study, we aimed to investigate the anti-inflammatory effects of L-carnitine on KOA and delineate a potential molecular mechanism. METHODS: Lipopolysaccharide-stimulated primary rat fibroblast-like synoviocytes (FLS) were treated with an AMP-activated protein kinase (AMPK) inhibitor or siRNA and carnitine palmitoyltransferase 1 (CPT1) siRNA to examine the synovial protective effects of L-carnitine. An anterior cruciate ligament transection model of rats was treated with an AMPK agonist (metformin) and CPT1 inhibitor (etomoxir) to define the therapeutic effects of L-carnitine. RESULTS: L-carnitine displayed a protective effect against synovitis of KOA in vitro and in vivo experiments. Specifically, L-carnitine treatment can reduce synovitis by inhibiting AMPK-ACC-CPT1 pathway activation and showed an increase in fatty acid ß-oxidation, a lower lipid accumulation, and a noticeable improvement in mitochondrial function. CONCLUSIONS: Our data suggested that L-carnitine can mitigate synovitis in FLS and synovial tissue, and the underlying mechanism may be related to improving mitochondrial function and reducing lipid accumulation via the AMPK-ACC-CPT1 signaling pathway. Therefore, L-carnitine may be a potential treatment strategy for KOA.

Chrysin ameliorates synovitis and fibrosis of osteoarthritic fibroblast-like synoviocytes in rats through PERK/TXNIP/NLRP3 signaling.

Objective: Synovitis and fibrosis are common pathological features of knee osteoarthritis (KOA). The interaction of synovitis and fibrosis can promote KOA progression. Chrysin (CHR), a natural flavonoid, may treat inflammation and prevent fibrosis. However, the effect and mechanism of CHR in KOA synovitis and fibrosis remains unclear. Methods: The KOA model was established in male SD rats by anterior cruciate ligament transection (ACLT), and histological analysis was used to evaluate synovitis and fibrosis. IL-6, IL-1ß and TNF-α mRNA expression in synovial tissue was measured by qRT‒PCR. Immunohistochemistry (IHC) was performed to detect GRP78, ATF-6 and TXNIP expression in vivo. Synovial fibroblasts (SFs) were treated with TGF-ß1 to stimulate the inflammatory response and fibrosis. CCK-8 assays were used to detect the viability of CHR-treated SFs. The IL-1ß level was detected by immunofluorescence analysis. Coimmunoprecipitation (Co-IP) and double immunofluorescence colocalization were used to detect the physiological interaction between TXNIP and NLRP3. The expression of fibrosis-related mediators and PERK/TXNIP/NLRP3 signaling molecules was detected by western blotting and qRT-PCR. Results: Four weeks after CHR treatment, pathological sections and associated scores showed that CHR improved synovitis and fibrosis in the ACLT model. In vitro, CHR attenuated the TGF-ß1-induced inflammatory response and fibrosis in SFs. Moreover, CHR suppressed the expression of synovial fibrosis markers and PERK/TXNIP/NLRP3 signaling molecules in the synovial tissue of rats with ACLT and cultured SFs. More importantly, we found that CHR inhibited TXNIP-NLRP3 interactions in TGF-ß-induced SFs. Conclusion: Our findings indicate that CHR can ameliorate synovitis and fibrosis in KOA. The underlying mechanism may be related to the PERK/TXNIP/NLRP3 signaling pathway.

Calycosin mitigates chondrocyte inflammation and apoptosis by inhibiting the PI3K/AKT and NF-κB pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao Gancao Decoction (SG-Tang), originated from the Treatise on Febrile Diseases, is often used to treat OA pain symptoms. Whereas its efficacy has been verified by several clinical studies, the underlying mechanism remained unclear. Network pharmacology and UPLC-QTOF-MS analysis found that calycosin could be regarded as the active components of SG-Tang in treating OA. However, the effect of calycosin on cartilage destruction and the pathogenesis of OA are not known. Therefore, we evaluated the benefits of calycosin for OA and revealed the underlying mechanisms. AIM OF STUDY: Using network pharmacology, UPLC-QTOF-MS analysis and experiments, the active components of SG-Tang were analyzed to explore their potential therapeutic mechanism in OA. MATERIALS AND METHODS: The components of SG-Tang were detected by UPLC-QTOF-MS, and the possible active components and mechanism of SG-Tang in the treatment of OA were screened by network pharmacology. The OA mouse model was constructed by DMM. In total, 30 mice were randomly divided into three groups: Sham, DMM, and DMM + Calycosin. H&E, safranin O/fast green staining and the OARSI scores were used to evaluate joint injury in mice. In addition, OA models were established using chondrocytes treated with 10 ng/mL IL-1ß. Treatment groups were treated with 100, 200 or 400 µM calycosin. CCK-8 assay was used for assessing the cytotoxic effects of calycosin. TUNEL staining and Western blotting were used to detect chondrocyte apoptosis. In addition, PI3K/Akt and NF-κB signaling pathway-related markers and cartilage matrix-related indicators were also detected. RESULTS: In vivo studies showed that calycosin inhibited IL-1ß-induced IL-6 and TNF-α production, as well as iNOS and COX-2 expression. Meanwhile, calycosin could inhibit IL-1ß-induced degradation of cartilage matrix, including downregulation of MMP3, MMP-13, collagen II and aggrecan. NF-κB and PI3K/AKT were also inhibited by calycosin in OA chondrocytes. Furthermore, calycosin inhibited IL-1ß-induced apoptosis in mouse chondrocytes. In a mouse model of OA, our results suggest that calycosin has a chondroprotective effect. CONCLUSIONS: According to this study, calycosin may act as a protective agent against OA by inhibiting the PI3K/AKT and NF-κB pathways. Furthermore, this study suggested that calycosin is a potential candidate for the treatment of OA.

Characteristics of sensory innervation in synovium of rats within different knee osteoarthritis models and the correlation between synovial fibrosis and hyperalgesia.

Introduction: Knee osteoarthritis (KOA) showed synovial fibrosis and hyperalgesia, although the correlation between the two is unclear. Besides, the specific changes of sensory innervation in animal models are still controversial, which makes it difficult to choose the modeling methods for KOA pain research. Objectives: Study the characteristics of sensory innervation within three commonly used KOA rat models and the correlation between synovial fibrosis and hyperalgesia. Methods: KOA models were induced by destabilization of medial meniscus (DMM), anterior cruciate ligament transection (ACLT), and monoiodoacetate (MIA), respectively. Mechanical, cold and thermal withdrawal threshold (MWT, CWT and TWT) were measured. The harvested tissues were used for pathological sections, immunofluorescence and quantitative analysis. Results: KOA synovium showed more type I collagen deposition, increased expression of CD31, VEGF and TGF-ß. These changes were most pronounced in surgical models, with DMM presenting the most prominent at Day 14 and ACLT at Day 28. Day 14, changes in mechanical hyperalgesia and cold hyperalgesia were most typical in DMM model and statistically different from MIA. There was a negative correlation between the percentage of type I collagen and MWT value (r = -0.88), as well as CWT value (r = -0.95). DMM synovium showed more axonal staining, upregulated CGRP, TRPV1, NGF and Netrin1 compared with MIA. Above changes were also observed at Day 28, but ACLT replaced DMM as the most typical. In DRG, only the levels of CGRP and NGF were different among KOA models at Day 14, and the highest in DMM, which was statistically different compared with MIA. Conclusions: This study described the details of sensory innervation in different KOA model of rats, and the degree of synovial fibrosis was positively correlated with the pain sensitivity of KOA model rats. Additionally, surgical modeling especially ACLT method is more recommended for KOA pain research.

GCTOF-MS Combined LC-QTRAP-MS/MS Reveals Metabolic Difference Between Osteoarthritis and Osteoporotic Osteoarthritis and the Intervention Effect of Erxian Decoction.

Purpose: OP and OA are chronic bone diseases with high incidence in the middle-aged and elderly populations. The latest research shows that the pathological environment of OP may be involved in the aggravation of the pathological process of OA, and the pathological state of OP plays an important role in the aggravation of OA pathology. EXD is a traditional Chinese medicine decoction that has been used to treat osteoporosis. Therefore, we further study whether OA will be aggravated in the OP environment and whether EXD can alleviate OA by intervening in the OP environment. The purpose of this study was to analyze the effect of OP on OA metabolites by using metabolomic methods and to explore the intervention mechanism of EXD on osteoporotic OA. Method: Thirty-two SD rats were randomly divided into normal group, OA group, OP-OA group, and EXD group. EXD was administered by gavage. Histopathological evaluation of cartilage tissue was performed using Saffron fast green and HE staining. Western blot and qRT-PCR were used to detect the expression levels of chondrogenesis genes SOX9, COL2A1, and COMP in cartilage tissue. GC-TOFMS and LC-QTRAP-MS/MS metabolomics methods were used to analyze the changes of metabolites in serum samples of rats in each group. Result: The slice results showed that the cartilage damage in the OP-OA group was more serious than that in the OA group, which was significantly relieved after EXD intervention, indicating that the cartilage damage in the OP-OA group was more severe than that in the OA group and further reduced the protein and gene expressions of cartilage markers SOX9, COL2A1, and COMP. Thirty-seven substances were identified, and gentiopicroside, emodin, quercetin, and diosmetin were analyzed as possible active components of EXD. EXD treatment significantly reduced cartilage damage and reversed the expression of these markers. Metabolomics showed that EXD attenuated cartilage destruction by modulating the expression of cystine, chenodeoxycholate, and D-Turanose, involving glycolysis/gluconeogenesis, pantothenate, and CoA biosynthesis metabolic pathways. Conclusion: The OP environment may promote the progression of OA through metabolic factors. The benign intervention of EXD in osteoporotic OA involves cystine, chenodeoxycholate, and D-Turanose, and their associated glycolysis/gluconeogenesis, pantothenate, and CoA biosynthesis metabolic pathways. Therefore, we have a deep understanding of the metabolic-related intervention of EXD in osteoporotic OA and are eager to better understand the mechanism of multi-targeted intervention of EXD in bone metabolic lesions.

Cedrol alleviates the apoptosis and inflammatory response of IL-1ß-treated chondrocytes by promoting miR-542-5p expression.

Cedrol has been shown to exert anti-tumor, anti-inflammatory, and anti-oxidative effects, but its role in osteoarthritis (OA) is unclear. This study aimed to explore the effect of cedrol in OA. Chondrocytes were isolated from newborn rats and cultured in Dulbecco's modified Eagle's medium (DMEM). Then, Alcian blue staining was used to identify the chondrocytes. IL-1ß and cedrol were used to treat chondrocytes. Cell viability and apoptosis were measured by MTT and flow cytometry assays, respectively. The expressions of miR-542-5p, miR-26b-5p, miR-572, miR-138-5p, miR-328-3p, miR-1254, Bcl-2, Bax, iNOS, COX-2, and MMP-13 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blot. NO and PGE2 levels were detected by ELISA. All the cells extracted from the newborn rats were dyed blue, indicating that the cells were chondrocytes. IL-1ß could reduce the viability and promote apoptosis and inflammatory response of chondrocytes, while cedrol could reverse the effect of IL-1ß. In addition, cedrol could significantly increase the expression of miR-542-5p in IL-1ß-treated chondrocytes. Moreover, miR-542-5p inhibitor could partly reverse the effect of cedrol in the apoptosis and inflammation response of chondrocytes. Cedrol alleviated IL-1ß-induced apoptosis and inflammatory response of chondrocytes by promoting miR-542-5p expression.

Interventional Efects of the Topical of "Sanse Powder" Essential Oils Nanoemulsion on Knee Osteoarthritis in Rats by Targeting the ERS/TXNIP/NLRP3 Signaling Axis.

Purpose: Our recent research is dedicated to finding effective drugs for the treatment of knee osteoarthritis (KOA) from traditional Chinese medicine and trying to make full use of modern science and technology to uncover the mechanisms and targets behind them. Synovial inflammation is one of the key pathological features of KOA, and a growing number of researchers realize that early intervention of synovial inflammation may be able to reverse disease progression. The close association of traditional natural products with modern nanotechnology may be important for improving the anti-synovitis efficacy. The purpose of our research was to explore the anti-synovitis mechanism of NEs-SP-EO that might be associated with the ERS/TXNIP/NLRP3 signalling axis. Methods: Chemical composition of "Sanse Powder" essential oil (SP-EO) and NEs-SP-EO were analyzed by GC-MS. NEs-SP-EO were prepared and characterized by nanoparticle tracking analysis, polydispersity index (PDI), zeta potential (ZP), ultraviolet-visible spectroscopy, and transmission electronic microscopy. The CCK8 assay for cell viability of NEs-SP-EO was performed on fibroblast-like synovial cells (FLSs) and the inflammatory environment was stimulated by LPS to explore the therapeutic mechanisms in vitro. Experiments of NEs-SP-EO in vivo were performed in male SD rats. Results: The GC-MS results showed that 30 compounds were present in SP-EO and 11 components of NEs-SP-EO were identified. The results also showed that the formulation of NEs-SP-EO exhibited suitable particle size, negative charge, and stable system. In vitro and vivo testing, NEs-SP-EO produced anti-synovitis efficacy by reduced the induction of the ERS/TXNIP/NLRP3 signaling axis as well as regulating the overproduction of IL-1ß, IL-18. Conclusion: We have developed a new type of essential oil nanoemulsion from "Sanse Powder" and demonstrated that it can managing synovitis of KOA. Besides, we have initially explored the anti-inflammatory mechanism that may be related to the ERS/TXNIP/NLRP3 signaling axis.