Ferrostatin-1 Inhibits Toll-Like Receptor 4/NF-κB Signaling to Alleviate Intervertebral Disc Degeneration in Rats.

Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, is implicated in intervertebral disc degeneration (IDD). The current study explored the role of Fer-1 in IDD via the toll-like receptor 4 (TLR4)/NF-κB signaling pathway. IDD-related gene expression microarray GSE124272 and high-throughput sequencing data set GSE175710 were obtained through the Gene Expression Omnibus database. Differentially expressed genes in IDD were identified, followed by implementation of protein-protein interaction network analysis and receiver operating characteristic curve analysis. The main pathways in IDD were obtained through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses, and target genes of Fer-1 were obtained through PubChem and PharmMapper websites. Finally, GPX4, FTH, and TLR4 expression was determined in a IDD rat model. Three key co-expression modules involved in IDD were obtained through Weighted Gene Co-Expression Network Analysis. Thirteen differentially expressed genes were found to be associated with IDD, and eight key genes (TLR4, BCL2A1, CXCL1, IL1R1, NAMPT, SOCS3, XCL1, and IRAK3) were found to affect IDD. These eight key genes had the diagnostic potential for IDD. The NF-κB signaling pathway was shown to play a predominant role in IDD development. Network pharmacologic analysis indicated a role of Fer-1 in suppressing ferroptosis and ameliorating IDD via the TLR4/NF-κB signaling pathway, which was verified by an in vivo animal experiment. The study showed that Fer-1 down-regulates TLR4 to inactivate NF-κB signaling pathway, suppressing ferroptosis and ultimately alleviating IDD in rats.

BMP7 ameliorates intervertebral disc degeneration in type 1 diabetic rats by inhibiting pyroptosis of nucleus pulposus cells and NLRP3 inflammasome activity.

BACKGROUND: Accumulating evidence indicates that intervertebral disc degeneration (IDD) is associated with diabetes mellitus (DM), while the underlying mechanisms still remain elusive. Herein, the current study sought to explore the potential molecular mechanism of IDD in diabetic rats based on transcriptome sequencing data. METHODS: Streptozotocin (STZ)-induced diabetes mellitus type 1 (T1DM) rats were used to obtain the nucleus pulposus tissues for transcriptome sequencing. Next, differentially expressed genes (DEGs) in transcriptome sequencing data and GSE34000 microarray dataset were obtained and intersected to acquire the candidate genes. Moreover, GO and KEGG enrichment analyses were performed to analyze the cellular functions and molecular signaling pathways primarily regulated by candidate DEGs. RESULTS: A total of 35 key genes involved in IDD of T1DM rats were mainly enriched in the extracellular matrix (ECM) and cytokine adhesion binding-related pathways. NLRP3 inflammasome activation promoted the pyroptosis of nucleus pulposus cells (NPCs). Besides, BMP7 could affect the IDD of T1DM rats by regulating the inflammatory responses. Additionally, NPCs were isolated from STZ-induced T1DM rats to illustrate the effects of BMP7 on IDD of T1DM rats using the ectopic expression method. Both in vitro and in vivo experiments validated that BMP7 alleviated IDD of T1DM rats by inhibiting NLRP3 inflammasome activation and pyroptosis of NPCs. CONCLUSION: Collectively, our findings provided novel mechanistic insights for understanding of the role of BMP7 in IDD of T1DM, and further highlighted BMP7 as a potential therapeutic target for preventing IDD in T1DM.

Long-term results of trans-scaphoid perilunate fracture dislocations treated by open reduction and internal fixation.

PURPOSE: The paper holds the research purpose of confirming the long-term results of trans-scaphoid perilunate fracture dislocations (TSPFD) under the treatment of open reduction and internal fixation. METHODS: Anteroposterial-lateral radiographs of the patient's wrist were taken before and after surgery. We use a dorsal approach for all cases. Postoperative clinical and radiographic assessments were performed routinely. The scapholunate angle (SLA), estradiol angle (RLA), as well as lunotriquetral distance (LTD) assisted in the radiographic assessment. Clinical assessment was performed using the Krimmer score, modified Mayo wrist score (MWS), active flexion extension arc (FEA), radial deviation and ulnar deviation arc (RUDA) and grip strength. A visual analog scale (VAS) assisted in the pain evaluation, the VAS score ranges from 0 to 10. RESULTS: Twenty-two TSPFD patients due to the wrist trauma received operative treatment and we retrospectively analyzed the surgical results, together with evaluating their clinical and radiological follow-up. These patients held a mean age of 30 years old. Herzberg's perilunate fracture-dislocation classification was taken into account to find that 19 males and 3 females suffered dorsal dislocation. The fellow-up time lasted 98.3 months on average. All cases obtained sufficient union after open reduction and internal fixation. The last follow-up found the median of grip strength was 20.00 (interquartile range, 20.00-21.25), which was 84.5% of the normal side. The modified Mayo wrist score evaluation scale considered 12 cases as excellent, and 10 good. The median of VAS and Krimmer scores at the final follow-up were 1.50 (interquartile range, 0.75-2.00) and 100.00 (interquartile range, 100.00-100.00), respectively, higher relative to the pre-operation (P < 0.001). No patients showed nerve damage preoperatively or postoperatively, or pin tract infection in any of the patient. CONCLUSIONS: It is necessary to diagnose such complicated biomechanical damage in early stage and adopt the open reduction and stable fixation for treatment; appropriate treatment can contribute to a functionally adequate and anatomically integrated wrist.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

Scutellarein Ameliorated Chondrocyte Inflammation and Osteoarthritis in Rats.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disorder characterized by the gradual degradation of joint cartilage and local inflammation. This study aimed to investigate the anti-OA effect of scutellarein (SCU), a single-unit flavonoid compound obtained from Scutellaria barbata D. Don, in rats. METHODS: The extracted rat chondrocytes were treated with SCU and IL-1ß. The chondrocytes were divided into control group, IL-1ß group, IL-1ß+SCU 50 µmol/L group, and IL-1ß+SCU 100 µmol/L group. Morphology of rat chondrocytes was observed by toluidine blue and safranin O staining. CCK-8 method was used to detect the cytotoxicity of SCU. ELISA, qRT-PCR, Western blotting, immunofluorescence, SAß-gal staining, flow cytometry, and bioinformatics analysis were applied to evaluate the effect of SCU on rat chondrocytes under IL-1ß intervention. Additionally, anterior cruciate ligament transection (ACL-T) was used to establish a rat OA model. Histological changes were detected by safranin O/fast green, hematoxylin-eosin (HE) staining, and immunohistochemistry. RESULTS: SCU protected cartilage and exhibited anti-inflammatory effects via multiple mechanisms. Specifically, it could enhance the synthesis of extracellular matrix in cartilage cells and inhibit its degradation. In addition, SCU partially inhibited the nuclear factor kappa-B/mitogen-activated protein kinase (NF-κB/MAPK) pathway, thereby reducing inflammatory cytokine production in the joint cartilage. Furthermore, SCU significantly reduced IL-1ß-induced apoptosis and senescence in rat chondrocytes, further highlighting its potential role in OA treatment. In vivo experiments revealed that SCU (at a dose of 50 mg/kg) administered for 2 months could significantly delay the progression of cartilage damage, which was reflected in a lower Osteoarthritis Research Society International (OARSI) score, and reduced expression of matrix metalloproteinase 13 (MMP13) in cartilage. CONCLUSION: SCU is effective in the therapeutic management of OA and could serve as a potential candidate for future clinical drug therapy for OA.

Sakuranetin reduces inflammation and chondrocyte dysfunction in osteoarthritis by inhibiting the PI3K/AKT/NF-κB pathway.

Osteoarthritis (OA) is a prevalent degenerative disease that impairs limb function, and its pathogenesis is closely related to inflammation. Sakuranetin (SK) is a cherry flavonoid phytoalexin with potent anti-inflammatory, anti-oxidant, and ant-ifungal properties. In recent studies, flavonoid and phytoalexin-related medicines have shown promise in the treatment of OA. However, the effects of SK on chondrocyte inflammation and the chondrogenesis process have remained unexplored, as have its functions in OA treatment. This study sought to confirm the therapeutic effects of SK in the OA rat model and reveal the potential mechanisms for protecting chondrocytes. The relevant mechanisms of SK were analyzed by network pharmacology analysis. Chondrocytes were subjected to IL-1ß intervention to simulate an inflammatory environment and received SK treatment. Then, anabolism, catabolism, and inflammatory markers were detected by western blot, qPCR, elisa, and immunofluorescence. Chondrogenic ability was evaluated by micromass and 3D culture assays. The rats were treated with destabilization of the medial meniscus (DMM) surgery to establish an OA model and SK intra-articular injections subsequently. Histological staining, immunohistochemistry, and micro-CT were performed to analyze the structural and morphological changes of cartilage and subchondral bone. In chondrocytes, IL-1ß treatment reduced chondrogenic ability, promoted catabolism, and exacerbated inflammation by triggering the PI3K/AKT/NF-κB pathway, whereas SK treatment partially rescued these negative effects. In vivo, SK treatment effectively alleviated the degeneration of cartilage and subchondral bone, thereby delaying the progression of OA. In summary, SK alleviates chondrocyte inflammation and promotes chondrogenesis by inhibiting the PI3K/AKT/NF-κB pathway, thereby improving OA progression.

Carveol alleviates osteoarthritis progression by acting on synovial macrophage polarization transformation: An in vitro and in vivo study.

Osteoarthritis (OA) is a heterogeneous disease that affects the entire joint. Its pathogenesis involves hypertrophy and hyperplasia of synovial cells and polarization infiltration of macrophages, in which macrophages, as a potential target, can delay the progression of the disease by improving the immune microenvironment in OA. To investigate the role and regulatory mechanism of Carveol in cartilage and synovial macrophage reprogramming and crosstalk during the development of OA. RAW264.7 mouse macrophage cell line was mainly used to stimulate macrophages to polarization towards M1 and M2 by LPS, IL4+IL13, respectively. Different concentrations of Carveol were given to intervene, and macrophage culture medium was collected to intervene mouse C57BL6J chondrocytes. ROS assay kit, western blotting, cellular immunofluorescence, scanning microscope and section histology were used to evaluate the effect of Carveol on anti-M1-polarization, M2-polarization promotion and cartilage protection. The mouse destabilization of medial meniscus (DMM) model was observed by micro-CT scan and histology. We found that CA could inhibit the increase of macrophage inflammation level under the intervention of LPS and promote the production of M2 anti-inflammatory substances under the intervention of IL-4+IL13. In addition, Carveol activated NRF2/HO-1/NQO1 pathway and enhanced ROS clearance in chondrocytes under the intervention of macrophage culture medium. The phosphorylation of I-κBα is inhibited, which further reduces the phosphorylation of P65 downstream of nuclear factor-κB (NF-κB) signaling pathway. In addition, Carveol inhibits mitogen activated protein kinase (MAPK) signaling molecules P-JNK, P-ERK and P-P38, and inhibits the production of inflammatory mediators. In vivo, Carveol can reduce osteophytes and bone spurs induced by DMM, reduce hypertrophy of synovial cells, reduce infiltration of macrophages, inhibit subchondral bone destruction, and reduce articular cartilage erosion. Our study suggests that synovial macrophages are potential targets for OA treatment, and Carveol is an effective candidate for OA treatment.

Pharmacological modulation of gp130 signalling enhances Achilles tendon repair by regulating tenocyte migration and collagen synthesis via SHP2-mediated crosstalk of the ERK/AKT pathway.

Tendon injuries typically display limited reparative capacity, often resulting in suboptimal outcomes and an elevated risk of recurrence or rupture. While cytokines of the IL-6 family are primarily recognised for their inflammatory properties, they also have multifaceted roles in tissue regeneration and repair. Despite this, studies examining the association between IL-6 family cytokines and tendon repair remained scarce. gp130, a type of glycoprotein, functions as a co-receptor for all cytokines in the IL-6 family. Its role is to assist in the transmission of signals following the binding of ligands to receptors. RCGD423 is a gp130 modulator. Phosphorylation of residue Y759 of gp130 recruits SHP2 and SOCS3 and inhibits activation of the STAT3 pathway. In our study, RCGD423 stimulated the formation of homologous dimers of gp130 and the phosphorylation of Y759 residues without the involvement of IL-6 and IL-6R. Subsequently, the phosphorylated residues recruited SHP2 kinase, activating the downstream ERK and AKT pathways. These mechanisms ultimately promoted the migration ability of tenocytes and matrix synthesis, especially collagen I. Moreover, RCGD423 also demonstrated significant improvements in collagen content, alignment of collagen fibres, and biological and biomechanical function in a rat Achilles tendon injury model. In summary, we demonstrated a promising gp130 modulator (RCGD423) that could potentially enhance tendon injury repair by redirecting downstream signalling of IL-6, suggesting its potential therapeutic application for tendon injuries.

sGC agonist BAY1021189 promotes thoracic aortic dissection formation by accelerating vascular smooth muscle cell phenotype switch.

Thoracic aortic dissection (TAD) is common but lethal cardiovascular disease with high mortality. This study aimed to expound whether and how sGC-PRKG1 signaling pathway might promote the formation of TAD. Our work identified two modules with high relevance to TAD using WGCNA method. Combined with previous studies, we focused on the participation of endothelial NOS (eNOS) in the progression of TAD. Through immunohistochemistry, immunofluorescence and western blot we verified that eNOS expression was elevated in the tissues of patients and mice with aortic dissection, and the phosphorylation Ser1177 of eNOS was activated. In a BAPN-induced TAD mouse model, sGC-PRKG1 signaling pathway promotes TAD formation by inducing vascular smooth muscle cells (VSMCs) phenotype transition, which was demonstrated as a decrease in markers of the contractile phenotype of VSMCs such as αSMA, SM22α, and Calponin. These results were also verified by experiments in vitro. To explore the further mechanism, we conducted immunohistochemistry, western blot and quantitative RT-PCR (qPCR), the results of which indicated that sGC-PRKG1 signaling pathway was activated when TAD occurred. In conclusion, our current study revealed that sGC-PRKG1 signaling pathway could promote TAD formation by accelerating VSMCs phenotype switch.

Imaging analysis and predictive nomogram construction for degenerative lumbar spondylolisthesis with severe clinical symptom based on propensity score matching.

Intervertebral disc degeneration, local lumbar segmental morphology changes, and atrophy of multifidus muscle have been considered to be associated with degenerative lumbar spondylolisthesis. However, there remains a great deal of controversy. To further investigate their relationship with degenerative lumbar spondylolisthesis, we conducted a retrospective study that included 67 patients with degenerative spondylolisthesis and 182 control subjects. Propensity score matching was employed to match the case group and the control group. Disc height was evaluated by the anterior disc height index (DHIA) and posterior disc height index (DHIP). Local lumbar segmental morphology was assessed by segmental lordosis (SL). The fatty infiltration and atrophy of multifidus muscle was evaluated by multifidus muscle net content (MFNC). Our results indicate that DHIA, DHIP, SL, and MFNC in the case group were significantly lower than in the control group. Furthermore, the DHIA, DHIP, and MFNC of the slipped segment (L4/5) were lower than those of the non-slipped segment (L3/4). Correlation analysis showed a high relationship between DHIA and MFNC and the degree of degenerative lumbar spondylolisthesis. Logistic regression analysis revealed that DHIA and MFNC might act as protective factors against the development of degenerative lumbar spondylolisthesis. Additionally, a prognostic nomogram was developed and validated to assess the likelihood of patients with severe symptoms requiring surgical intervention.

Is 3D-printed Titanium cage a reliable option for 3-level anterior cervical discectomy and fusion in treating degenerative cervical spondylosis?

Background: To assess the clinical and radiographical outcomes of 3-level anterior cervical discectomy and fusion (ACDF) with a 3D-printed titanium cage in treating degenerative cervical spondylosis. Methods: In this study, 25 patients with degenerative cervical spondylosis who underwent 3-level ACDF using a 3D-printed titanium cage from March 2019 to June 2021 were retrospectively enrolled. The patient-reported outcome measures (PROMs) were evaluated by visual analog scale (VAS) for the neck (VAS-neck) and arm pain (VAS-arm), Neck Disability Index (NDI) score, Japanese Orthopedic Association (JOA) score, SF-12 concise health survey, and the Odom criteria. The radiographical parameters, including C2-C7 lordosis, segmental angle, segmental height, and subsidence, were assessed. The mean duration of follow-up was 25.6 months. Results: Bony fusion was achieved in all patients (100%). In three patients (12%) mild dysphagia was observed during the follow-up. The VAS-neck, VAS-arm, NDI score, JOA score, SF-12 score, C2-C7 lordosis, and segmental angle improved noticeably at the latest follow-up. Based on the Odom criteria, 22 patients (88%) reported satisfactory (excellent or good). The mean loss of C2-C7 lordosis and segmental angle between the immediate postoperative and the latest follow-up values were 1.6° ± 0.5° and 1.1° ± 0.5°, respectively. The mean subsidence was 0.9 ± 0.6 mm. Conclusion: In patients with multi-level degenerative cervical spondylosis, 3-level ACDF using the 3D-printed titanium cage can effectively relieve the symptoms, stabilize the spine, and restore segmental height and cervical curvature. It is proven to be a reliable option for patients with 3-level degenerative cervical spondylosis. However, a future comparative study involving a larger population and longer follow-up time may be required to further evaluate the safety, efficacy and outcomes of our preliminary results.

Dihydrocaffeic acid improves IL-1ß-induced inflammation and cartilage degradation via inhibiting NF-κB and MAPK signalling pathways.

Aims: Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods: In vitro, interleukin-1 beta (IL-1ß) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results: DHCA prevented iNOS and IL-6 from being upregulated by IL-1ß. Moreover, the IL-1ß-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1ß-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and MAPK pathways. Furthermore, DHCA mitigated the destruction of articular cartilage in vivo. Conclusion: We present evidence that DHCA alleviates inflammation and cartilage degradation in OA chondrocytes via suppressing the NF-κB and MAPK pathways, indicating that DHCA may be a potential agent for OA treatment.

Decorin: a potential therapeutic candidate for ligamentum flavum hypertrophy by antagonizing TGF-ß1.

Ligamentum flavum hypertrophy (LFH) is the main physiological and pathological mechanism of lumbar spinal canal stenosis (LSCS). The specific mechanism for LFH has not been completely clarified. In this study, bioinformatic analysis, human ligamentum flavum (LF) tissues collection and analysis, and in vitro and in vivo experiments were conducted to explore the effect of decorin (DCN) on LFH pathogenesis. Here, we found that TGF-ß1, collagen I, collagen III, α-SMA and fibronectin were significantly upregulated in hypertrophic LF samples. The DCN protein expression in hypertrophic LF samples was higher than that in non-LFH samples, but the difference was not significant. DCN inhibited the expression of TGF-ß1-induced fibrosis-associated proteins in human LF cells, including collagen I, collagen III, α-SMA, and fibronectin. ELISAs showed that TGF-ß1 can upregulate PINP and PIIINP in the cell supernatant, and this effect was inhibited after DCN administration. Mechanistic studies revealed that DCN suppressed TGF-ß1-induced fibrosis by blocking the TGF-ß1/SMAD3 signaling pathway. In addition, DCN ameliorated mechanical stress-induced LFH in vivo. In summary, our findings indicated that DCN ameliorated mechanical stress-induced LFH by antagonizing the TGF-ß1/SMAD3 signaling pathway in vitro and in vivo. These findings imply that DCN is a potential therapeutic candidate for ligamentum flavum hypertrophy.

An in vitro and in vivo study: Valencene protects cartilage and alleviates the progression of osteoarthritis by anti-oxidative stress and anti-inflammatory effects.

BACKGROUND: Osteoarthritis (OA) is a heterogeneous disease involving the whole joint. The pathogenesis involves oxidative stress levels and chronic inflammation, and Valencene (VA) has excellent anti-inflammatory and antioxidant stress abilities. PURPOSE: The objective was to study the effects of VA therapy on combating oxidative stress and to evaluate the protective effect of chondrocytes to alleviate the progression of OA. METHODS: C57BL6J mouse chondrocytes were used as the primary cells in this study. Mouse chondrocytes were stimulated with IL-1ß, and VA was administered in different concentrations. Reactive oxygen species (ROS) assay kits, western blotting, cellular immunofluorescence, and scanning microscopy were used to evaluate VA's antioxidant stress mechanism, anti-inflammatory effect, and cartilage protective ability. The mouse arthritis model constructed by destabilization of medial meniscus (DMM) was observed by micro-CT scan and histology after different treatments. RESULTS: We found that VA can reverse the rise of ROS under IL-1ß, the degeneration of the cartilage extracellular matrix, and the production of inflammatory mediators. In terms of mechanism, VA activated NRF2/HO-1/NQO1 pathway, thus enhancing ROS clearance. The phosphorylation of IκBα is inhibited, which further reduces the downstream phosphorylation of P65 in nuclear factor-κB (NF-κB) signaling. In addition, VA inhibited mitogen-activated protein kinase (MAPK) signaling molecules P-JNK, P-ERK, and P-P38, inhibiting the production of inflammatory mediators and thus inhibiting Aggrecan and Collagen Type II （COL2）degeneration. In vivo, VA reduced DMM-induced osteophytes and spurs, suppressed subchondral bone destruction, and reduced articular cartilage erosion. CONCLUSION: Our study demonstrated that VA is an effective candidate for OA treatment.

A comprehensive analysis of single-cell RNA transcriptome reveals unique SPP1+ chondrocytes in human osteoarthritis.

Osteoarthritis (OA) has become the most common degenerative disease in the world, which brings a serious economic burden to society and the country. Although epidemiological studies have shown that the occurrence of osteoarthritis is associated with obesity, sex, and trauma, the biomolecular mechanisms for the development and progression of osteoarthritis remain ambiguous. Several studies have drawn a connection between SPP1 and osteoarthritis. SPP1 was first found to be highly expressed in osteoarthritic cartilage, and later more studies have shown that SPP1 is also highly expressed in subchondral bone and synovial in OA patients. However, the biological function of SPP1 remains unclear. Single-cell RNA sequencing (scRNA-seq) is a novel technique that reflects gene expression at the cellular level, making it better depict the state of different cells than ordinary transcriptome data. However, most of the existing chondrocyte scRNA-seq studies focus on the occurrence and development of OA chondrocytes and lack analysis of normal chondrocyte development. Therefore, to better understand the mechanism of OA, scRNA-seq analysis of a larger cell volume containing normal and osteoarthritic cartilage is of great importance. Our study identifies a unique cluster of chondrocytes characterized by high SPP1 expression. The metabolic and biological characteristics of these clusters were further investigated. Besides, in animal models, we found that the expression of SPP1 is spatially heterogeneous in cartilage. Overall, our work provides novel insight into the potential role of SPP1 in OA, which sheds light on understanding the role of SPP1 in OA, promoting the progress of the treatment and prevention in the field of OA.

Infiltration of LPAR5+ macrophages in osteosarcoma tumor microenvironment predicts better outcomes.

Introduction: Tumor microenvironment (TME) has been shown to be extensively involved in tumor development. However, the dynamic change of TME components and their effects are still unclear. Here, we attempted to identify TME-related genes that could help predict survival and may be potential therapeutic targets. Methods: Data was collected from UCSC Xena and GEO database. ESTIMATE and CIBERSORT algorithms were applied to estimate the components and the proportions of TIICs in TME. We analyzed the gene expression differences of immune components and stromal components, respectively, and finally got the overlapped DEGs. Through protein-protein interaction (PPI) network and univariate Cox regression analysis based on shared DEGs, we screened out and validated the TME-related genes. Focusing on this gene, we analyzed the expression and prognostic value of this gene, and investigated its relationship with immune cells by correlation analysis, single cell analysis, immunohistochemistry and immunofluorescence analysis. Results: Through a series analysis, we found that the proportion of immune and stromal components was an important prognostic factor, and screened out a key gene, LPAR5, which was highly correlated with prognosis and metastasis. And the expression of LPAR5 was positively correlated with immune cells, especially macrophages, indicating LPAR5+ macrophages played an important role in tumor microenvironment of osteosarcoma. Meanwhile, the genes in LPAR5 high expression group were enriched in immune-related activities and pathways, and differentially expressed genes between LPAR5+ macrophages and LPAR5- macrophages were enriched in the biological processes associated with phagocytosis and antigen presentation. What' more, we found that LPAR5 was mainly expressed in TME, and high LPAR5 expression predicting a better prognosis. Conclusion: We identified a TME-related gene, LPAR5, which is a promising indicator for TME remodeling in osteosarcoma. Particularly, LPAR5+ macrophages might have great potential to be a prognostic factor and therapeutic target for osteosarcoma.

MCC950, the NLRP3 Inhibitor, Protects against Cartilage Degradation in a Mouse Model of Osteoarthritis.

Osteoarthritis (OA), characterized by chronic systemic low-level inflammation and cartilage degeneration, is a type of arthritis closely associated with aging. Inflammation and aging play a pivotal role in the occurrence and progression of OA. NLRP3 inflammasome is involved in many inflammatory and aging diseases, and NLRP3 inhibitor MCC950 has anti-inflammatory and antisenescence effects on some diseases such as Alzheimer's disease. In the present study, we found that NLRP3 protein was upregulated in human and mouse OA cartilage. Moreover, NLRP3 and Caspase1 expression induced by IL-1ß in chondrocytes was blocked by MCC950. In addition, MCC950 inhibited the expression of inflammatory mediators, matrix-degrading enzymes, senescence marker protein P16 (INK4A), and ß-galactosidase, as well as excessive production of ROS. Meanwhile, MCC950 promoted autophagy-related protein expression and autophagy flux under the inflammatory condition. However, autophagy inhibitor 3-MA reversed anti-inflammatory and anticatabolic effects of MCC950. In in vivo experiments, intra-articular administration of MCC950 further showed its protective effect on cartilage degeneration. Bioinformatic analysis and in vitro experimental results revealed that MCC950 might play a protective role in cartilage by regulating Nrf2/HO-1/NQO1, PI3k/Akt/mTOR, P38/MAPK, and JNK/MAPK pathways. In conclusion, our work demonstrated that NLRP3 inhibitor MCC950 might serve as a promising strategy for OA treatment.

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BACKGROUND: Procambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation. RESULTS: In this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant. CONCLUSIONS: The high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.