Dihydromyricetin Inhibits Inflammation of Fibroblast-Like Synoviocytes through Regulation of Nuclear Factor-κB Signaling in Rats with Collagen-Induced Arthritis.

Dihydromyricetin (DMY), the main flavonoid of Ampelopsis grossedentata, has potent anti-inflammatory activity. However, the effect of DMY on chronic autoimmune arthritis remains undefined. In this study, we investigated the therapeutic effects of DMY on collagen-induced arthritis (CIA). Wistar rats were immunized with bovine type II collagen to establish CIA and were then administered DMY intraperitoneally (5, 25, and 50 mg/kg) every other day for 5 weeks. Paw swelling, clinical scoring, and histologic analysis were assessed to determine the therapeutic effects of DMY on the development of arthritis in CIA rats. The results showed that treatment with DMY significantly reduced erythema and swelling in the paws of CIA rats. Pathologic analysis of the knee joints and peripheral blood cytokine assay results confirmed the antiarthritic effects of DMY on synovitis and inflammation. Fibroblast-like synoviocytes (FLSs) were isolated from the synovium of CIA rats and treated with 10 ng/ml interleukin (IL)-1ß DMY significantly inhibited the proliferation, migration, and inflammation of IL-1ß-induced FLSs, whereas it significantly increased IL-1ß-induced FLS apoptosis in a dose-dependent manner (6.25-25 µM). Moreover, DMY suppressed phosphorylation of IκB kinase (IKK) and inhibitor of NF-κB α and subsequently reduced the IL-1ß-induced nucleus translocation of NF-κB in FLSs. Through a molecular docking assay, we demonstrated that DMY could directly bind to the Thr9 and Asp88 residues in IKKα and the Asp95, Asn142, and Gln167 residues in IKKß These findings demonstrate that DMY could alleviate inflammation in CIA rats and attenuate IL-1ß-induced activities in FLSs through suppression of NF-κB signaling.

Mir204 and Mir211 suppress synovial inflammation and proliferation in rheumatoid arthritis by targeting Ssrp1.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial hyperplasia. Mir204 and Mir211 are homologous miRNAs with the same gene targeting spectrum. It is known that Mir204/211 play an important role in protecting osteoarthritis development; however, the roles of Mir204/211 in RA disease have not been determined. In the present study, we investigated the effects and molecular mechanisms of Mir204/211 on synovial inflammation and hyperproliferation in RA. The effects of Mir204/211 on the inflammation and abnormal proliferation in primary fibroblast-like synoviocytes (FLSs) were examined by Mir204/211 gain-of-function and loss-of-function approaches in vitro and in vivo. We identified the structure-specific recognition protein 1 (Ssrp1) as a downstream target gene of Mir204/211 based on the bioinformatics analysis. We overexpressed Ssrp1and Mir204/211 in FLS to determine the relationship between Ssrp1 and Mir204/211 and their effects on synovial hyperplasia. We created a collagen-induced arthritis (CIA) model in wild-type as well as Mir204/211 double knockout (dKO) mice to induce RA phenotype and administered adeno-associated virus (AAV)-mediated Ssrp1-shRNA (AAV-shSsrp1) by intra-articular injection into Mir204/211 dKO mice. We found that Mir204/211 attenuated excessive cell proliferation and synovial inflammation in RA. Ssrp1 was the downstream target gene of Mir204/211. Mir204/211 affected synovial proliferation and decelerated RA progression by targeting Ssrp1. CIA mice with Mir204/211 deficiency displayed enhanced synovial hyperplasia and inflammation. RA phenotypes observed in Mir204/211 deficient mice were significantly ameliorated by intra-articular delivery of AAV-shSsrp1, confirming the involvement of Mir204/211-Ssrp1signaling during RA development. In this study, we demonstrated that Mir204/211 antagonize synovial hyperplasia and inflammation in RA by regulation of Ssrp1. Mir204/211 may serve as novel agents to treat RA disease.

Pharmacology and molecular docking study of cartilage protection of Chinese herbal medicine Fufang Shatai Heji (STHJ) by inhibiting the expression of MMPs in collagen-induced arthritis mice.

BACKGROUND: This study aims to explore whether Fufang Shatai Heji (STHJ), as a mixture collected by a decoction of a variety of Chinese herbal medicines for immune system diseases, can improve the cartilage destruction of rheumatoid arthritis (RA). METHODS: The therapeutic effects of STHJ were studied using collagen induced arthritis (CIA) mice. The improvement effect of STHJ on synovitis and cartilage damage caused by arthritis was studied by joint pathological analysis. The inhibitory effect of STHJ on related degradation enzymes in cartilage was studied by immunohistochemistry and real-time polymerase chain reaction (PCR). The specific targets of STHJ were predicted by molecular docking. RESULTS: After successfully inducing CIA, the paws of the mice showed significant swelling, and athological analysis of the ankle and knee joints also showed significant cartilage destruction and synovial hyperplasia. However, synovial hyperplasia and cartilage destruction were markedly alleviated after administration of STHJ. And after STHJ treatment, the expression of ADAMTS-4, ADAMTS-5, MMP-9 and MMP-13, in the cartilage layer of CIA mice was significantly inhibited. Through molecular docking assays, we proved that acteoside in STHJ could directly bind to the Glu111, Phe110 residues in MMP-9 and glycyrrhizic acid in STHJ bind to the Glu382, Asn433 residues in MMP-13. CONCLUSIONS: Our results suggested that STHJ may alleviate synovial hyperplasia and cartilage destruction in CIA mice and protect cartilage by inhibiting the expression of MMP-9 and other enzymes.

Dexamethasone-loaded thermo-sensitive hydrogel attenuates osteoarthritis by protecting cartilage and providing effective pain relief.

BACKGROUND: We utilized the destabilization of medial meniscus (DMM)-induced mice to illustrate the osteoarthritis (OA) suppressing and pain-relieving effects of a novel prolonged-release intra-articular (IA)-dexamethasone-loaded thermo-sensitive hydrogel (DLTH). METHODS: The effects of temperature and pH on DLTH formation and in vitro DLTH release profile were assessed. C57BL/6J mice were randomly divided into three groups: Ctrl group, Model group and DLTH group. The DLTH group received joint injections of 10 µL DLTH (1 mg/kg) into the right knee once a week from week 2 to week 11. We performed micro-computed tomography (Micro-CT) and histological analyses of safranin O-fast green, hematoxylin and eosin, and tartrate-resistant acid phosphatase in knee joints. We also carried out immunohistochemical (IHC) staining for matrix metalloproteinase-9 (MMP-9), MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) in cartilage and Ki-67 in synovia. Pain behavioral testing was carried out in all mice. The serum content of prostaglandin E2 (PGE2) and real-time polymerase chain reaction (PCR) of inflammatory cytokines and pain-related factors in dorsal root ganglia (DRGs) were evaluated. RESULTS: It took 20 minutes to form DLTH at pH 7.0 and 37 °C. The cumulative release profiles of dexamethasone (Dex) from DLTH at 37 °C revealed a rapid release in the first 24 h and a sustained slow release for 7 days. In vivo study illustrated that DLTH attenuated OA bone destruction and ameliorated synovitis and progression of OA in DMM-induced mice. The chondroprotective effects of DLTH were mediated by decreased expressions of MMP-9, MMP-13, and ADAMTS-5. The results showed that IA-DLTH exerted pain-relieving effects in OA mice. Upregulation of nociceptive response time (NRT) and downregulations of serum PGE2, inflammatory factors, and pain-related mediators in DRGs of mice in the DLTH group were recorded. CONCLUSIONS: Data presented in this study elucidated that DLTH exhibited a long and lasting Dex release and it is a potential sustainable drug delivery system (DDS) to treat OA locally. IA-DLTH injection exerted chondroprotective and pain-relieving effects in DMM-induced arthritis. The involvement of MMP-9, MMP-13, ADAMTS-5, and inflammatory and pain-related factors, may account for the suppression of OA progression and pain.

Metformin inhibits inflammation and bone destruction in collagen-induced arthritis in rats.

BACKGROUND: Metformin (MF) is a widely used biguanide oral hypoglycemic agent, which has obvious anti-inflammatory and immunomodulatory effects. However, the mechanism of MF on rheumatoid arthritis (RA) remains uncertain. In this study, we investigated the therapeutic effects of MF on collagen-induced arthritis (CIA). METHODS: CIA was induced in rats by intradermal injection of a mixture of bovine type II collagen and incomplete Freund's adjuvant (IFA) on day 0 and day 7 through the base of the tail. Intraperitoneal injection of MF (100 mg/kg) was given every 3 days, from day 14 for 3 weeks. The effects of MF on arthritis-induced systemic inflammation and synovitis were studied by pathological analysis of the knee joint and serological examination of peripheral blood in CIA rats. The bone protection effect of MF was studied by microscopic computed tomography (micro-CT) and histological analysis of the knee joint. The effects of MF on chondrocytes in CIA rats were studied by detecting the relevant pro-apoptotic mediators in the chondrocytes. RESULTS: After administration of MF in CIA rats, systemic inflammation and synovitis caused by arthritis were significantly suppressed. Histomorphometry and micro-CT analysis of the knee joint revealed that MF can protect bone by inhibiting the changes of trabecular bone in CIA rats. Histological analysis of the knee joint found that MF can inhibit osteoclast formation and degradation of the cartilage layer matrix. Detection of the relevant pro-apoptotic mediators in chondrocytes revealed that MF can significantly inhibit the apoptosis of chondrocytes in CIA rats. CONCLUSIONS: Our study showed that MF can inhibit systemic inflammation and synovitis and plays a role in bone protection by inhibiting cartilage layer matrix degradation, osteoclast formation, and chondrocyte apoptosis.

Dexamethasone-Loaded Thermosensitive Hydrogel Suppresses Inflammation and Pain in Collagen-Induced Arthritis Rats.

PURPOSE: To overcome negative adverse effects and improve therapeutic index of dexamethasone (Dex) in rheumatoid arthritis (RA), we developed a novel sustained release formulation-intra-articular injectable dexamethasone-loaded thermosensitive hydrogel (DLTH) with chitosan-glycerin-borax as carrier for the remission of inflammation and pain. The focus of this article is to explore both anti-inflammatory and pain-relieving effects of DLTH joint injection in bovine type-II collagen-induced arthritis (CIA) rats. METHODS: Wistar rats were randomized into three groups, including the normal group (n=6), the model group (n=6) and the DLTH group (n=10). Joint injection of DLTH (1mg/kg Dex per rat) was injected on day 12 in the DLTH group twice a week for three weeks. Clinical signs of body weight, paw swelling and arthritis scores, histologic analysis, hind paw mechanical withdrawal threshold (MWT), plantar pressure pain threshold (PPT) were taken into consideration. Serum contents of IL-17A, prostaglandin E2 (PGE2), prostacyclin 2 (PGI2) and prostaglandin D2 (PGD2), real-time polymerase chain reaction (PCR) analysis of inflammatory factors and pain-related mediators in synovium and dorsal root ganglia (DRG), Western blotting of NF-κB in synovium were all evaluated. RESULTS: Paw swelling, arthritis scores and joint inflammation destruction were all attenuated in the DLTH-treated group. Results showed that DLTH not only down-regulated serum IL-17A, but also mRNA levels of inflammatory factors and NGF, and key proteins contents of the NF-κB pathway in synovium. Increases of MWT and PPT in DLTH-treated rats elucidated pain-reducing effects of DLTH. Elevated serum PGD2 levels and declines of serum PGE2 and PGI2, and inflammatory and pain-related genes in DRGs in the DLTH group were also recorded. CONCLUSION: These data elucidated that DLTH joint injection impeded synovial inflammation processes through down-regulating transcription activity of NF-κB pathway, and intra-articular DLTH may aid in the regulation of RA pain through regulating inflammation and pain conduction process.

DMY protects the knee joints of rats with collagen-induced arthritis by inhibition of NF-κB signaling and osteoclastic bone resorption.

Collagen-induced arthritis (CIA) is a widely used animal model for studying rheumatoid arthritis (RA), which manifests serious joint dysfunction, progressive bone erosion and articular cartilage destruction. Considering that joint damage in RA is caused by systemic inflammation and dihydromyricetin (DMY), the main flavonoid of Ampelopsis Michx, possesses anti-inflammatory properties, in the present study we have investigated the potential capability of DMY to inhibit inflammation-mediated joint damage and explore the underlying mechanisms. A rat model of RA induced by CIA was administered with DMY for 5 weeks. Prior to histological analysis, the knee joints were scanned by microcomputed tomography (µCT) to detect bone damage. Articular cartilage destruction was assessed by Alcian blue and Toluidine blue staining and the pathological alteration of osteoblasts and osteoclasts in joints was evaluated by hematoxylin-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. The effects of DMY on osteoblast differentiation and osteoclast formation in vitro were investigated. Consistent with the in vivo results, DMY had no significant effect on osteoblast differentiation but an inhibitory effect on osteoclast formation. Furthermore, we determined that the mechanism of the DMY-suppressed osteoclast formation was blocking the phosphorylation of I-κB kinase (IKK) so as to hinder the activation of nuclear factor-κB (NF-κB). Collectively, DMY could ameliorate knee joint damage, especially in articular cartilage, which is the weight-bearing region, by inhibiting osteoclast formation through NF-κB signaling.

The Traditional Chinese Medicine Fufang Shatai Heji (STHJ) Enhances Immune Function in Cyclophosphamide-Treated Mice.

Fufang Shatai Heji (STHJ) is a mixture of traditional Chinese medicines, such as Radix Adenophorae, Radix Pseudostellariae, and Radix Astragali. STHJ is commonly used to treat diseases caused by low immune function, for example, Sjögren's syndrome (SS). The primary objective of this study was to assess the immunopotentiating effect of STHJ using an immunosuppressive mouse model receiving cyclophosphamide (CTX). Following CTX treatment, STHJ was administered by oral gavage for 30 consecutive days. The percentage of specific lymphocyte subpopulations in the spleen was measured by flow cytometry. Levels of inflammatory factors in serum were detected by enzyme-linked immunosorbent assays (ELISAs). The administration of STHJ significantly elevated thymus and spleen indices, increased B cell and natural killer (NK) cell activities, and decreased CD8+ T, CD8+CD122+ T, NKT, and γδT cell activities in the CTX-treated mice. In addition, STHJ upregulated the expression of interleukin- (IL-) 2, IL-6, and tumor necrosis factor-α (TNF-α) and downregulated IL-10 expression in CTX-treated mice. In conclusion, STHJ effectively remitted CTX-induced immunosuppression by modulating the balance of lymphocyte subsets and cytokines. Our results suggest STHJ treatment could be used as an effective therapeutic approach to improve immune function in patients with low immunity.