Erianin: A Direct NLRP3 Inhibitor With Remarkable Anti-Inflammatory Activity.

Erianin (Eri) is the extract of Dendrobium chrysotoxum Lindl. The NLRP3 inflammasome is a multiprotein complex that plays key roles in a wide variety of chronic inflammation-driven human diseases. Nevertheless, little is known about the protection of Eri against NLRP3 inflammasome-related diseases. In this study, we demonstrated that Eri inhibited NLRP3 inflammasome activation in vitro and in vivo. Mechanistically, Eri directly interacted with NLRP3, leading to inhibition of NLRP3 inflammasome assembly. Eri associated with the Walker A motif in the NACHT domain and suppressed NLRP3 ATPase activity. In mouse models, Eri had therapeutic effects on peritonitis, gouty arthritis and type 2 diabetes, via NLRP3. More importantly, Eri was active ex vivo for synovial fluid cells and monocytes from patients with IAV infection and gout. Eri may serve as a potential novel therapeutic compound against NLRP3-driven diseases.

Efficacy of fire needle on acute gouty arthritis induced by monosodium urate in rat.

OBJECTIVE: To explore the therapeutic effect and inflammatory mechanism of fire needles on gouty arthritis. METHODS: Sixty male Sprague-Dawley rats were divided into four groups, that is, control group, the model group, the colchicine group, and the fire needle treatment group. Acute gouty arthritis was prepared by injection of monosodium urate in the ankle joint. The inflammation-related protein [toll-like receptor 4 (TLR4), pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-1ß, myeloid differentiation factor 88 (MyD88), nuclear factorkappa B (NF-κB), phosphorylated NF-κB (p-NF-κB), caspase-1, and p-caspase-1] in swollen tissues, the inflammation-related mRNAs indicators (TLR4, NLRP3, NF-κB, and caspase-1) and the inflammatory factors [IL-1ß, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP)] in the serum were detected by Western blot, real-time quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. RESULTS: The fire needle treatment could reduce joint swelling, increase mechanical pain threshold and decrease the inflammation index score in the fire needle treatment group. It could also significantly decreased the protein expression of IL-1ß, TLR4, MyD88, p-NF-κB and NLRP3 in joint tissue, markedly downregulated the mRNA levels of TLR4 and NLRP3 in joint tissue, and significantly reduce serum levels of IL-1ß, IL-6, IL-8, TNF-α, and CRP. CONCLUSION: The fire needle treatment had positive effect of treating gouty arthritis, and its under- lying mechanism might be associated with NF-κB activation and related inflammatory response.

[Cathepsin-B involved in effect of electroacupuncture by inhibiting the activation of NLRP3 inflammasome in rats with acute gouty arthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on Cathepsin-B in the synovium of the knee joint of acute gouty arthritis(AGA) rats, so as to explore the mechanism of EA in the treatment of AGA. METHODS: A total of 60 male Wistar rats were randomly divided into normal control,model, medication and EA groups, with 15 rats in each group. Rat model of AGA was established by injection of 0.2 mL sodium urate crystal suspension into the left knee joint cavity. The rats in the medication group were treated with colchicine by gavage(0.3 mg·kg-1·d-1), and the rats in the EA group were treated with EA at the left "Sanyinjiao" (SP6) and "Zusanli"(ST36) for 10 min each time, once a day for a week. The Coderre gait grading standard was used to score the gait of rats. The pathological morphology of synovial tissue of the left knee joint was observed by H.E. staining. The expression levels of Cathepsin-B protein and Nod-like receptor pyrin domain 3(NLRP3), apoptosis-associated speck-like protein containing CARD (ASC),Caspase-1, interleukin-1ß(IL-1ß) and IL-18 mRNAs were detected by Western blot and real-time fluorescence quantitative PCR, respectively. RESULTS: Compared with the normal control group, the degree of synovitis infiltration in the model group was more serious. And the gait score，the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3,ASC,Caspase-1, IL-1ß,IL-18 were significantly increased (P<0.01).After the interventions, the degree of inflammatory infiltration was mild, The gait score, the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3 and ASC,Caspase-1，IL-1ß,IL-18 were significantly decreased in both medication and EA groups in contrast to the model group (P<0.01, P<0.05). Compared with medication group, the mRNA expression levels of Caspase-1 and IL-18 in the EA group were increased (P<0.05). CONCLUSION: EA may inhibit the activation of NLRP3 inflammasome by reducing the activity of Cathepsin-B in the synovium of the knee joint, so as to treat AGA.

Simiaosan alleviates the symptoms of gouty arthritis via the NALP3/IL­1ß pathway.

Previous studies have suggested that the herbal medicine simiaosan has beneficial effects on gouty arthritis (GA), for which conventional Western medicines are insufficient (particularly in cases of multiple episodes). The objective of the present study was to investigate the mechanism by which simiaosan alleviated the symptoms of GA. Sprague­Dawley rat models of acute GA were successfully established, as verified by pathological analyses. Additionally, an NLR family pyrin domain containing 3 (NLRP3) overexpression vector was constructed and a high transfection efficiency was confirmed by reverse transcription PCR. The following five treatment groups were established: i) Normal control; ii) model + saline; iii) model + simiaosan; iv) model + NALP3­overexpressing adenovirus + simiaosan; and v) model + empty vector adenovirus + simiaosan. The samples from mice in each group were subjected to hematoxylin and eosin (H&E) staining for assessing the histopathological changes, enzyme­linked immunosorbent assays for determining IL­1ß and TGF­ß1 levels and western blotting for evaluating NALP3 expression. H&E staining indicated that simiaosan could reduce the infiltration of inflammatory cells, while NALP3 overexpression aggravated the inflammatory response in tissues. Expression levels of IL­1ß, TGF­ß1 and NALP3 were significantly higher in the model and the model + NALP3­overexpressing adenovirus + simiaosan groups compared with the normal control group. Levels of IL­1ß, TGF­ß1 and NALP3 were significantly lower in the model + simiaosan and model + empty vector adenovirus + simiaosan groups compared with the model group. These results indicated that the effects of simiaosan were mediated through NALP3 inhibition. Therefore, the herbal medicine simiaosan was revealed to possess an ability to alleviate the symptoms of GA by regulating the NALP3/IL­1ß signaling pathway.

Efficacy of Xixiancao (Herba Siegesbeckiae Orientalis) on interactions between nuclear factor kappa-B and inflammatory cytokines in inflammatory reactions of rat synovial cells induced by sodium urate.

OBJECTIVE: To investigate the interaction between nuclear factor kappa-B (NF-κB) and inflammatory cytokines in synovial cell inflammatory responses induced by sodium urate, and to evaluate the efficacy of Xixiancao (Herba Siegesbeckiae Orientalis) on these interactions. METHODS: The interactions between NF-κB and inflammatory cytokines/mediators in synovial cells in acute gouty arthritis were investigated. We observed the expressions of NF-κB, interleukin (IL)-1ß, IL-8, and tumor necrosis factor alpha (TNF-α) in synovial cells at different timepoints in an in vitro model of synovial cell inflammatory responses induced by sodium urate and in an in vivo model of gouty arthritis. Changes in the expressions of NF-κB, IL-1ß, IL-8, and TNF- in synovial cells of all experimental groups were compared and observed after treatment with different doses of Xixiancao (Herba Siegesbeckiae Orientalis) and colchicine. The interactions between NF-κB and IL-1ß, IL-8, and TNF-α were analyzed. Pathological changes in synovial tissues were observed in rats with acute gouty arthritis. RESULTS: Compared with the blank group, the expression levels of NF-κB, IL-1ß, IL-8, and TNF-α were increased significantly at different timepoints in the in vitro model of synovial cell inflammatory responses induced by sodium urate, and in the in vivo model of gouty arthritis. Compared with the model group, the expressions of NF-κB, IL-1ß, IL-8, and TNF-α in synovial cells induced by sodium urate were decreased in the different Xixiancao (Herba Siegesbeckiae Orientalis) dose groups and the colchicine group. The effect was more obvious in the high dose Xixiancao (Herba Siegesbeckiae Orientalis) group. The expression of NF-κB in synovial cells was positively correlated with the expressions of IL-1ß, IL-8, and TNF-. Histopathological examination of synovial tissues in the high dose Xixiancao (Herba Siegesbeckiae Orientalis) group and Colchicine group showed that the characteristics of acute gouty arthritis were reduced, and there was a trend towards a positive correlation between NF-κB and inflammatory cytokine expressions. CONCLUSION: The activation of NF-κB is associated with the activation of IL-1ß, IL-8, and TNF-α during the pathogenesis of acute gouty arthritis, leading to the continuation and enhancement of the inflammatory response. Expressions of IL-1ß, IL-8, and TNF-α in synoviocytes during acute gouty arthritis effectively inhibit local inflammation.

Investigation of the mechanism of action of Porana sinensis Hemsl. against gout arthritis using network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Porana sinensis Hemsl. has been widely used to treat joint pain and rheumatoid arthritis in traditional Chinese medicine (TCM). Although evidence exists to support a pharmacological action of P. sinensis for the treatment of gout arthritis (GA), the underlying mechanism of action remains unknown due to it being a multi-component and multi-target agent. AIM OF THE STUDY: To clarify the active compounds and mechanism of P. sinensis against GA. MATERIALS AND METHODS: The present study combined network pharmacology with experiments to clarify the mechanism of P. sinensis against GA. A protein-protein interaction network for gout was constructed to identify the potential drug targets, and molecular docking was subsequently performed to determine whether the protein was a target for the compounds of P. sinensis. KEGG pathway analysis was then conducted to elucidate the pathway involved in the P. sinensis-mediated treatment of gout. A rat model of GA was used to further investigate the mechanism of P. sinensis against GA. RESULTS: The network pharmacology study indicates that coumarins and chlorogenic acids of P. sinensis may serve as additives to GA treatment. P. sinensis played a role in the treatment of GA by regulating the PI3K-Akt, MAPK, NF-kappa B and toll-like receptor pathways and so on. Moreover, experimental validation suggests that P. sinensis extract significantly suppressed the expression of TLR2 and MyD88 mRNA, regulating the release of cytokines (IL-1ß, IL-4 and TGF-ß), lowering lipid peroxidation (MDA) and increasing antioxidant status (SOD). CONCLUSION: The present study clarifies the mechanism of P. sinensis against GA, and provides evidence to support its clinical use.

Anti-inflammatory Effect of Total Saponin Fraction from Dioscorea nipponica Makino on Gouty Arthritis and Its Influence on NALP3 Inflammasome.

OBJECTIVE: To investigate the mechanism of Chinese herbal medicine Dioscorea nipponica for the treatment of monosodium urate crystals-induced gouty arthritis (GA) in rats. METHODS: Sixty male Wistar rats were divided into 6 groups: normal, model, indomethacin and three total saponin (900, 300 and 100 mg/kg) groups. The liver, kidney and serum levels of lysosomal enzymes, antioxidant capacities, and inflammatory factors were measured. In addition, the mRNA and protein levels of the NALP3 inflammasome components in the mononuclear cells of rats' peripheral blood were analyzed using real-time polymerase chain reaction and Western blotting methods, respectively. RESULTS: Total saponins groups could reduce the activities of ß-galactosidase, ß-N acetyl glucosamine enzyme, ß-glucuronidase, acid phosphatase, and malonaldehyde as well as the contents of TNF-α, IL-1ß and IL-8 (all P<0.05). They could also increase the activities of glutathione peroxidase and total superoxide dismutase (both P<0.05). Further studies showed that total saponins groups of high, middle and low doses could all increase the mRNA and protein levels of caspase-1, adapter apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and NALP3 in the mononuclear cells of peripheral blood (all P<0.05). CONCLUSION: Dioscorea nipponica may treat GA by regulating lysosomal enzymes, antioxidant capacities and the NALP3 inflammasome.

miR-155 is dispensable in monosodium urate-induced gouty inflammation in mice.

BACKGROUND: The findings of a previous study by Jin et al. have shown that microRNA (miR)-155 was upregulated in patients with acute gouty arthritis and enhanced the proinflammatory cytokines. There is no direct evidence to support that miR-155 is indeed involved in monosodium urate (MSU)-induced inflammatory responses in vivo. The aim of this study was to investigate the role of miR-155 knock-out (KO) or knock-in (KI) mice in MSU-induced animal models to mimic acute gout. METHODS: MiR-155 expression in cultured bone marrow-derived macrophages (BMDMs) from miR-155 KO, miR-155 KI, and wild-type (WT) mice treated with MSU crystals in vitro was detected by real-time quantitative polymerase chain reaction (qPCR). MiR-155 KO and WT mice were used to induce an acute gouty inflammatory response with MSU crystals including models of foot pad inflammation, ankle arthritis, air pouch inflammation, and peritonitis. Furthermore, the proinflammatory interleukin (IL)-1ß levels in lavage fluids from air pouch and peritoneal cavity models were measured by enzyme-linked immunosorbent assay (ELISA), and tumor necrosis factor (TNF)-α production from BMDMs of miR-155 KI mice treated with MSU were measured by flow cytometry. RESULTS: MiR-155 expression was quickly upregulated in BMDMs from WT mice following MSU treatment in vitro. In comparison with WT mice in vivo, the swelling index of miR-155 KO mice showed no significant difference in the murine foot pad and ankle arthritis models for the indicated different time points. There were similar changes in total cell numbers of lavage fluids in the air pouch and peritoneal cavity models between miR-155 KO and WT mice following MSU crystal injection. Moreover, the IL-1ß levels of lavage fluids in the air pouch and peritonitis models from miR-155 KO mice were almost the same as those from WT mice. TNF-α levels were comparable from BMDMs treated with MSU crystals in vitro between miR-155 KI mice and WT mice. CONCLUSIONS: MiR-155 is dispensable in MSU-induced gouty inflammation in mice. Deletion of miR-155 might not be an effective therapeutic approach to relieve the inflammation in acute gout.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori's anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

Anti-inflammatory effects of traditional mixed extract of medicinal herbs (MEMH) on monosodium urate crystal-induced gouty arthritis.

Korean oriental medicine prescription is widely used for the treatment of gouty diseases. In the present study, we investigated anti-inflammatory effects of modified Korean herbal formulation, mixed extract of medicinal herbs (MEMH), and its modulatory effects on inflammatory mediators associated with gouty arthritis. Both in vitro and in vivo studies were carried out to assess the anti-inflammatory efficacy of MEMH on monosodium urate (MSU) crystals-induced gouty inflammation. MSU crystals stimulated human chondrosarcoma cell line, SW1353, and human primary chondrocytes were treated with MEMH in vitro. The expression levels of pro-inflammatory mediators and metalloproteases were analyzed. The effect of MEMH on NFκB signaling pathway in SW1353 cells was examined. Effect of MEMH on the mRNA expression level of pro-inflammatory mediators and chemotactic factor from human monocytic cell line, THP-1, was also analyzed. The probable role of MEMH in the differentiation process of osteoblast like cells, SaOS-2, after MSU treatment was also observed. To investigate the effects of MEMH in vivo, MSU crystals-induced ankle arthritic model was established. Histopathological changes in affected joints and plasma levels of pro-inflammatory mediators (IL-1ß and TNFα) were recorded. MEMH inhibited NFκB signaling pathway and COX-2 protein expression in chondrocytes. MSU-induced mRNA expressions of pro-inflammatory mediators and chemotactic cytokines were suppressed by MEMH. In MSU crystals-induced ankle arthritic mouse model, administration of MEMH relieved inflammatory symptoms and decreased the plasma levels of IL-1ß and TNFα. The results indicated that MEMH can effectively inhibit the expression of inflammatory mediators in gouty arthritis, demonstrating its potential for treating gouty arthritis.

SHEsisPlus, a toolset for genetic studies on polyploid species.

Currently, algorithms and softwares for genetic analysis of diploid organisms with bi-allelic markers are well-established, while those for polyploids are limited. Here, we present SHEsisPlus, the online algorithm toolset for both dichotomous and quantitative trait genetic analysis on polyploid species (compatible with haploids and diploids, too). SHEsisPlus is also optimized for handling multiple-allele datasets. It's free, open source and also designed to perform a range of analyses, including haplotype inference, linkage disequilibrium analysis, epistasis detection, Hardy-Weinberg equilibrium and single locus association tests. Meanwhile, we developed an accurate and efficient haplotype inference algorithm for polyploids and proposed an entropy-based algorithm to detect epistasis in the context of quantitative traits. A study of both simulated and real datasets showed that our haplotype inference algorithm was much faster and more accurate than existing ones. Our epistasis detection algorithm was the first try to apply information theory to characterizing the gene interactions in quantitative trait datasets. Results showed that its statistical power was significantly higher than conventional approaches. SHEsisPlus is freely available on the web at http://shesisplus.bio-x.cn/. Source code is freely available for download at https://github.com/celaoforever/SHEsisPlus.

Regulation of the hypoxanthine phosphoribosyltransferase gene: in vitro and in vivo approaches.

The hypoxanthine phosphoribosyltransferase (HPRT) locus is a constitutively expressed housekeeping gene characterized by a notably higher level of expression in the mammalian brain. The enzyme it encodes is key to purine salvage in humans and is the basis for the X-linked recessive disorder, Lesch-Nyhan syndrome (LNS). Methylation in the promoter plays a critical, if not fully understood, role in transcriptional silencing of the locus on the inactive chromosome, possibly by conferring structural stability. In vivo footprinting assays of the promoter region have shown protein interaction with multiple Spl-binding sites, a possible AP2 site, and a potentially novel binding site. In vitro studies of HPRT promoter deletion constructs have identified a minimal promoter element necessary for maximal transcription and a position-dependent, orientation-independent repressor element (HPRT-NE) that functions on heterologous promoters. Regulatory intron elements have also been observed. Studies on transgenic mice bearing HPRT promoter constructs have shown that the minimal promoter element is insufficient for in vivo expression and that HPRT-NE is responsible for conferring neuronal specificity. HPRT-mice possess metabolic defects similar to LNS patients, but fail to develop human behavioral abnormalities, perhaps because of species differences in purine metabolism. A neuronal-specific protein complex appears to be necessary for activator function of HPRT-NE, while a ubiquitously expressed complex may be responsible for repression. Sequence analysis Indicates that the latter complex may depend on the multifunctional transcription factor YY1 for binding. A fuller understanding of HPRT gene regulation will hopefully provide insight into the transcriptional mechanisms controlling the expression of housekeeping and brain-specific genes.