Relationship between the pyroptosis of fibroblast­like synoviocytes and HMGB1 secretion in knee osteoarthritis.

High mobility group box 1 (HMGB1) is an important downstream product of pyroptosis in macrophages, and it serves a vital role in numerous inflammatory diseases. Previous studies have reported that HMGB1 is released by fibroblast­like synoviocytes (FLSs) that are activated by inflammatory cytokines in knee osteoarthritis (KOA); however, the mechanism via which FLS promotes HMGB1 secretion in KOA remains unknown. According to our previous study, pyroptosis occurs in FLSs of patients with KOA and is mediated by Nod­like receptor protein (NLRP)1 or NLRP3 inflammasomes. However, the specific relationship between HMGB1 secretion and FLS pyroptosis requires further investigation. In the present study, the association between HMGB1 secretion and FLS pyroptosis was investigated in vitro and in vivo. In this study, western blotting, ELISA and reverse transcription­quantitative PCR were used to measure expression levels of proteins and mRNA. Caspase­1 activity assay and Hoechst 33342/PI double staining were used to observe the pyroptosis of FLSs. Hematoxylin and eosin staining was used to observe the destruction of cartilage in KOA. Increased expression levels of pyroptosis­related proteins and HMGB1 in the synovium of rat anterior cruciate ligament transection­induced KOA models were identified, and these changes were significantly mitigated via the intra­articular injection of a caspase­1 inhibitor. In vitro, FLSs were treated with lipopolysaccharide (LPS) + ATP to induce pyroptosis, and HMGB1 secretion was subsequently measured. LPS + ATP significantly increased the expression levels of pyroptosis­related proteins and HMGB1 in FLSs, and these effects were significantly mitigated by small interfering RNAs targeting NLRP1, NLRP3, apoptosis­associated speck­like protein with a caspase­recruitment domain or caspase­1. Therefore, the present results indicated that NLRP1/NLRP3 inflammasome­mediated and caspase­1­dependent FLS pyroptosis increased HMGB1 secretion in KOA. These findings may provide a therapeutic strategy to decrease synovial inflammatory responses during KOA progression.

Chrysin Attenuates the NLRP3 Inflammasome Cascade to Reduce Synovitis and Pain in KOA Rats.

PURPOSE: Our recent reports have revealed that inhibiting NLRP3 activation reduces synovial inflammation and fibrosis in knee osteoarthritis (KOA). Synovial inflammation is involved the entire process of KOA and promotes the progression of KOA. Natural flavonoid Chrysin from Scutellariae Radix, a traditional Chinese medicine, exhibits multifarious biological activities and potentially has protective activity against osteoarthritis. However, the mechanism of Chrysin in the treatment of synovial inflammation remains elusive. The purpose of our research was to explore the anti-inflammatory effects of Chrysin on KOA, which was induced by monoiodoacetic acid (MIA) in rats by targeting the NLRP3 inflammasome in the hopes of identifying an effective drug to treat KOA. METHODS: The MIA-induced KOA model was used to evaluate the cold pain threshold and paw withdrawal threshold (PWT) of joints after MIA (40 mg/mL) injection into the knee joints. Microscopically, we used LPS (5 ug/mL) and ATP (4 mmol/L) to stimulate fibroblast-like synovial cells (FLSs) to explore the underlying mechanisms and effects of Chrysin. Two staining methods, H&E and Sirius Red, were applied to assess histopathological changes in synovial membranes. Cellular signal transduction was determined by qRT-PCR and WB. Cytokine expression (inflammatory cytokines and pain-related cytokines) was detected by ELISA. The degree of chronic inflammatory pain was evaluated by c-Fos immunofluorescence. RESULTS: The results showed that Chrysin not only attenuated synovial inflammation but also reduced the secretion of pain-related factors and increased the PWT and cold pain threshold in rats. Chrysin also inhibited NLRP3 inflammasome activation and increased IL-1ß levels to alleviate the synovitis. CONCLUSION: Chrysin can relieve knee synovial inflammation and improve pain behavior in KOA rats, which may be related to the ability of Chrysin to inhibit NLRP3 inflammasome activation. Therefore, Chrysin may be developed as a new drug for the treatment of KOA.

Casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting HIF-1α/NLRP3 inflammasome signaling.

Knee osteoarthritis (KOA) is a disabling chronic inflammatory disease that is closely associated with synovium tissue hypoxia and synovial fibrosis. Casticin, a compound purified from the Chinese herb Viticis Fructus, has been proved effective in preventing inflammation and fibrosis in previous studies. However, the effect of casticin on synovial fibrosis in KOA is not clear. In present study, we aimed to investigate how did casticin affect synovial fibrosis on monoiodoacetic acid (MIA)-induced KOA in rats. The MIA-induced knee osteoarthritis model and lipopolysaccharide (LPS) stimulated primary synovial fibroblasts inflammation model were established. Pathological and morphological changes in synovial tissue were observed by H&E and sirius red staining. The hypoxia of synovium was detected by pimonidazole staining and immunohistochemistry of hypoxia-inducible factors 1α (HIF-1α). The levels of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome components, fibrogenic markers (TGF-ß, COL1A1 and TIMP1) and inflammatory cytokines were examined by western blotting, qRT-PCR or ELISA in both KOA rat models and primary synovial fibroblasts. Our data suggested that casticin improved hypoxia and inflammation in synovium tissue, as well the synovial fibrosis in rats. Besides, casticin inhibited the activation of NLRP3 inflammasome in MIA-induced KOA rats and synovial fibroblasts. In conclusion, our findings demonstrated that casticin alleviated MIA-induced KOA by inhibiting of HIF-1α/NLRP3 inflammasome activation. Therefore, casticin could be a potential treatment strategy for KOA.

Interventional effects of the direct application of "Sanse powder" on knee osteoarthritis in rats as determined from lipidomics via UPLC-Q-Exactive Orbitrap MS.

BACKGROUND: Our previous clinical evidence suggested that the direct application of "Sanse powder" the main ingredient of "Yiceng" might represent an alternative treatment for knee osteoarthritis. However, the mechanism underlying its effect is poorly understood. In this study, we investigated the mechanism of the effect of direct "Sanse powder" application for the treatment of knee osteoarthritis (KOA) in rats by using lipidomics. METHODS: KOA rats were established by cutting the anterior cruciate ligament, and the cold pain threshold and mechanical withdrawal threshold (MWT) of seven rats from each group were measured before modelling (0 days) and at 7, 14, 21 and 28 days after modelling. Histopathological evaluation of the synovial tissue was performed by haematoxylin and eosin (H&E) staining after modelling for 28 days. Interleukin-1ß (IL-1ß), pro-interleukin-1ß (pro-IL-1ß) and tumor necrosis factor-α (TNF-α) proteins in synovial tissue were measured by western blot, and the mRNA expression levels of IL-1ß and TNF-α in synovial tissue were measured using Real-time reverse transcription polymerase chain reaction (qRT-PCR), the levels of IL-1ß and TNF-α in rat serum were measured by enzyme-linked immunosorbent assay (ELISA), Serum lipid profiles were obtained by using ultra-performance liquid chromatography combined with quadrupole-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS). RESULTS: The results confirmed that the direct application of "Sanse powder" had a significant protective effect against KOA in rats. Treatment with "Sanse powder" not only attenuated synovial tissue inflammation but also increased the levels of the cold pain threshold and MWT. In addition, the lipidomics results showed that the levels of diacylglycerol (DAG), triacylglycerols (TAGs), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), fatty acid esters of hydroxy fatty acids (FAHFAs), and phosphatidylethanolamine (PE) were restored almost to control levels following treatment. CONCLUSIONS: Lipidomics provides a better understanding of the actions of direct application "Sanse powder" therapy for KOA.

Vanillic Acid Reduces Pain-Related Behavior in Knee Osteoarthritis Rats Through the Inhibition of NLRP3 Inflammasome-Related Synovitis.

Objectives: Synovitis plays an important role in knee osteoarthritis (KOA) pain. The activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in fibroblast-like synoviocytes (FLSs) promotes KOA development. In this study, we aimed to investigate whether vanillic acid (VA), a monomer derived from Chinese herbal medicines, could target NLRP3 inflammasome-related synovitis to reduce pain. Methods: Rats in the KOA and KOA + VA groups were injected with monosodium iodoacetate (MIA) in the knee to induce KOA. From day 14, the KOA + VA group was given VA at 30 mg/kg every day via gastric intubation. FLSs were collected from the synovial tissues. We examined both the protein and gene expression of caspase-1, apoptosis-associated speck-like protein with a caspase recruitment domain (ASC), NLRP3, components of the NLRP3 inflammasome, and interleukin-1ß (IL-1ß) and IL-18 in vivo and in vitro. Results: The upregulation of caspase-1, ASC, and NLRP3 in the KOA model were reduced by VA. VA also lowered the level of IL-1ß and IL-18 in the KOA model. In addition, VA relieved pain-related behavior of KOA model rats and downregulated the pain mediators CGRP, NGF, and TrkA in FLSs. Interestingly, we also observed reduced synovial fibrosis in the animal experiments. Conclusion: Our research showed that VA reduces synovitis and pain-related behaviors in a rat model of KOA, which provides the basis for further investigations into the potential therapeutic impact of VA in KOA.

Meta-analysis of the association of IL1-RN variable number of tandem repeats polymorphism with osteoarthritis risk.

OBJECTIVE: The aim of this meta-analysis was to clarify the role of Interleukin-1 receptor antagonist gene (IL1-RN) Variable Number of Tandem Repeats (VNTR) polymorphism on the risk of OA by means of meta-analysis. METHODS: Eligible articles were retrieved from PubMed, Web of science and Google scholar with a total of 1187 OA cases and 2659 controls. The strength of the association between the IL1-RN VNTR polymorphism and the risk of OA was assessed by odds ratios (ORs) with the corresponding 95% confidence interval (CI) for each study. RESULTS: The meta-analysis of seven published studies retrieved from the literature search showed a significantly increased OA risk in the recessive model analysis (22 vs 2L + LL: Pb = 0.18, I2 = 32.8, OR(95% CI) = 1.50(1.12, 2.02), P = 0.007), the additive model analysis (22 vs LL: Pb = 0.08, I2 = 46.8, OR(95% CI) = 1.56(1.15, 2.12), P = 0.004) and in the allele contrast model (2 vs L: Pb = 0.02, I2 = 58.8, OR(95% CI) = 1.20(1.05, 1.36), P = 0.007). By subgroup analysis, the IL1-RN VNTR polymorphism was found to be significantly associated with OA susceptibility in Caucasian and Hospital based case-control study (HCC) groups. CONCLUSION: This meta-analysis showed that IL1-RN VNTR polymorphism may increase the susceptibility to OA. More studies with detailed information are needed to validate our conclusion. LEVEL OF EVIDENCE: Level III, diagnostic study.

Transient Receptor Potential Ankyrin 1 (TRPA1) Mediates Lipopolysaccharide (LPS)-Induced Inflammatory Responses in Primary Human Osteoarthritic Fibroblast-Like Synoviocytes.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal and non-neuronal cells. Recently, emerging evidences suggested the crucial role of TRPA1 in the disease progression of osteoarthritis (OA). Therefore, we aimed to investigate whether TRPA1 mediate lipopolysaccharide (LPS)-induced inflammatory responses in primary human OA fibroblast-like synoviocytes (OA-FLS). The expression of TRPA1 in LPS-treated OA-FLS was assessed by polymerase chain reaction (PCR) and western blot (WB), and the functionality of TRPA1 channel by Ca2+ influx measurements. Meanwhile, production of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, matrix metalloproteinase (MMP)-1, and MMP-3 in LPS-treated cells was measured by immunoassay. Histological observation after inhibition of TRPA1 was also performed in rats with LPS-induced inflammatory arthritis. After being induced by LPS, the gene and protein expression of TRPA1 was increased in the time-dependent or dose-dependent manner. Meanwhile, Ca2+ influx mediated by TRPA1 in human OA-FLS was also enhanced. In addition, pharmacological inhibition and gene silencing of TRPA1 downregulated the production of IL-1ß, TNF-α, IL-6, MMP-1, and MMP-3 in LPS-treated FLS. Finally, synovial inflammation and cartilage degeneration were also reduced by the TRPA1 antagonist. We found the LPS caused the increased functional expression of TRPA1, the activation of which involved in LPS-reduced inflammatory responses in primary human OA-FLS, and the inhibition of TRPA1 produces protective effect in LPS-induced arthritis.