Fan-Rong Wu

Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes.

Acid-sensing ion channels (ASICs) are cationic channels that are activated by extracellular acidification and implicated in pain perception, ischemic stroke, mechanosensation, learning, and memory. It has been shown that ASIC1a is an extracellular pH sensor in the central and peripheral nervous systems, but its physiological and pathological roles in non-neural cells are poorly understood. We demonstrated a novel physiological function of ASIC1a in rat articular chondrocytes. The expression of ASIC1a mRNA and protein in rat articular chondrocytes was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The distribution of ASIC1a protein located in articular chondrocytes was determined by using immunofluorescence cell staining. The possible molecular mechanisms of articular chondrocytes pH sensing, as assessed by recording intracellular calcium ([Ca2+]i) in chondrocytes, were analyzed by using the laser scanning confocal microscopy technique. The cell injure following acid exposure was analyzed with lactate dehydrogenase release assay and electron microscopy. mRNA and protein expression showed that ASIC1a was expressed abundantly in these cells. In cultured chondrocytes, extracellular pH 6.0 increased intracellular calcium in the presence of extracellular Ca2+. The ASIC1a-specific blocker PcTX venom significantly reduced this increase in [Ca2+]i, and inhibited acid-induced articular chondrocyte injury. However, the increase in [Ca2+]i and articular chondrocyte injury were not observed in the absence of extracellular Ca2+. These findings show that increased [Ca2+]i, mediated via ASIC1a, might contribute to acidosis-induced articular chondrocyte injury.

Blockade of acid-sensing ion channels protects articular chondrocytes from acid-induced apoptotic injury

ObjectiveAcid-sensing ion channels (ASICs) are members of the degenerin/epithelial sodium channel (DEG/ENaC) protein superfamily and play a critical role in acid-induced cell injury. In this study, we examined whether drugs such as amiloride that block ASICs could attenuate acid-induced apoptotic injury to articular chondrocytes.MethodsArticular chondrocytes were isolated from Sprague–Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. Articular chondrocyte viability assay was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis of chondrocytes was observed by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method as well as propidium iodide labeling methods. Intracellular calcium ([Ca2+]i) was analyzed by a Ca2+-imaging method. In addition, the expression levels of calpain and calcineurin in articular chondrocytes were examined by real-time PCR and immunocytochemical staining. The activity of caspase-3 was evaluated by spectrophotometric assays.ResultsPositive staining for glycosaminoglycan and collagen II was seen in articular chondrocytes. Blocking acid-sensing ion channels significantly decreased the cell death percentage and increased cell viability following acid exposure. After pretreated with amiloride, acid-induced [Ca2+]i rises were reduced. Amiloride also inhibited calpain and calcineurin expression levels in acid-induced chondrocytes, and inhibited caspase-3 activity.ConclusionThe data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.

Interleukin-23 as a potential therapeutic target for rheumatoid arthritis

Cytokine-mediated immunity plays a crucial role in the pathogenesis of various autoimmune diseases, including rheumatoid arthritis (RA). Increasing evidence has revealed the importance of IL-23, which closely resembles IL-12 structurally and immunologically, in linking innate and adaptive immunity. IL-23, a newly identified heterodimeric pro-inflammatory cytokine, is composed of a p40 subunit in common with IL-12 and a unique p19 subunit. Recent evidence suggests that IL-23, rather than IL-12, is the crucial factor in the pathogenesis of various immune-mediated disorders. In addition, recent studies have explored the role of IL-23 in patients with RA. An elevated expression of IL-23 has been demonstrated in the synovial fibroblasts and plasma of patients with RA. Moreover, an association between IL-23 and IL-23R polymorphisms with susceptibility to RA has been reported. Therefore, the targeting of IL-23 or the IL-23 receptor has been proposed as a potential therapeutic approach for RA. In this review we will discuss the biological features of IL-23, and summarize recent advances in our understanding of the role of IL-23 in the pathogenesis and treatment of RA.

Role of interleukin-22 in liver diseases.

IntroductionInterleukin (IL)-22, originally referred to as IL-TIF for IL-10-related T cell-derived inducible factor, is a member of the IL-10-like cytokine family. IL-22 is highly expressed by Th17 cells and is tightly linked to chronic inflammation, including inflammatory bowel disease and local intestinal inflammation among others.Materials and methodsA PubMed and Web of Science databases search was performed for studies providing evidences on the role of IL-22 in liver diseases.ConclusionIL-22 plays an important role in ameliorating liver injury in many rodent models by targeting hepatocytes that express high levels of IL-22 receptor 1 and IL-10 receptor 2. This review concisely summarizes the role of IL-22 in the development progression of liver disease of different etiologies. It is focused mainly on the IL-22 intracellular signaling and its influence on liver diseases.

Protective effect of total flavonoids from Bidens bipinnata L. against carbon tetrachloride-induced liver injury in mice

Bidens bipinnata L. is well known in China as a traditional Chinese medicine. This study was designed to evaluate the hepatoprotective activity of the total flavonoids of B. bipinnata L. (TFB) against carbon tetrachloride (CCI4)‐induced acute liver injury in mice and to determine its mechanism of action. Oral administration of TFB at doses of 50, 100 and 200 mg kg−1 for 7 days significantly reduced the elevated relative values of liver weight, serum transaminases (alanine aminotransferase and aspartate aminotransferase) and the hepatic morphologic changes induced by CCl4 in mice. In addition, TFB markedly inhibited CCl4‐induced lipid peroxidation and enhanced the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase. Moreover, pretreatment with TFB suppressed nitric oxide production and nuclear factor‐kB activation in CCl4‐treated mice. The results suggest that TFB has significant hepatoprotective activity and its mechanism is related, at least in part, to its antioxidant properties. Further research is required to investigate the detailed mechanism of the protective effect of TFB on acute liver injury.

Inhibition of acid-sensing ion channel 1a in hepatic stellate cells attenuates PDGF-induced activation of HSCs through MAPK pathway

Acid-sensing ion channels (ASICs), a group of Na+-selective and Ca2+-permeant ligand-gated cation channels, can be transiently activated by extracellular acid. Among seven subunits of ASICs, acid-sensing ion channel 1a (ASIC1a), which is responsible for Ca2+ transportation, is elevated in response to inflammation, tumor, and ischemic injury in central nervous system and non-neuronal tissues. In this study, we demonstrated for the first time the presence of ASIC1a in rat liver and hepatic stellate cells (HSCs). Furthermore, the expression of ASIC1a was increased in primary HSCs and liver tissues of CCl4-treated rats, suggesting that ASIC1a may play certain role in liver fibrosis. Interestingly, we identified that the level of ASIC1a was significantly elevated in response to platelet-derived growth factor (PDGF) induction in a time- and dose-dependent manner. It was also established that Ca2+-transporting ASIC1a was involved in acid-induced injury of different cell types. Moreover, inhibition or silencing of ASIC1a was able to inhibit PDGF-induced pro-fibrogenic effects of activated rat HSCs, including cell activation, de novo synthesis of extracellular matrix components through mitogen-activated protein kinase signaling pathway. Collectively, our studies identified that ASIC1a was expressed in rat liver and HSCs and provided a strong evidence for the involvement of the ASIC1a in the progression of hepatic fibrosis.

Recombinant human endostatin inhibits adjuvant arthritis by down-regulating VEGF expression and suppression of TNF-α, IL-1β production

ObjectiveThe aim of this study was to examine the effect of recombinant human endostatin (rhEndostatin) on adjuvant arthritis (AA) in rats and its possible mechanisms.MethodsRhEndostatin was subcutaneously administrated to AA rats after immunization. The progression of AA was assessed by the macroscopic arthritis scoring system of paws. Histological examination of the synovial tissues was examined by hematoxylin and eosin staining. The expression level of vascular endothelial growth factor (VEGF) mRNA and proteins in the synovial tissues was evaluated by realtime PCR and immunohistochemistry, respectively. Fibroblast-like synoviocytes (FLS) were isolated from synovial tissues. Cell proliferation assay was evaluateded with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The levels of tumour necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) in culture medium was examined by radioimmno assay.ResultsRhEndostatin attenuated the severity of arthritis on both second hind paw volume and polyarthritis score, as well as improved the arthritic status histologically in AA rats. Simultaneously, rhEndostatin can inhibit the expression of VEGF in synovial tissues. The proliferation of FLS and TNF-α, IL-1β production from culture medium was significantly inhibited by rhEndostatin.ConclusionOur data suggest that rhEndostatin inhibits adjuvant arthritis by down-regulating VEGF expression and suppression of TNF-α, IL-1β production.

A novel retinoic acid analog, 4-amino-2-trifluoromethyl-phenyl retinate, inhibits gastric cancer cell growth

Retinoic acid (RA) analogs have been used in the treatment of a variety of cancers; however, their application is limited due to serious therapy-related sequelae. In the present study, the effects of a novel RA analog, 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), on the growth of gastric cancer cells were evaluated. Three gastric cancer cell lines, AGS, MKN-74 and SC-M1, were treated with either all‑trans retinoic acid (ATRA) or ATPR, and their growth and distribution in different cell cycle phases were assessed using an MTT assay and propidium iodide (PI) staining followed by flow cytometry. The binding affinity of ATPR to the retinoic acid receptors, retinoic acid receptor-α (RAR-α) and retinoid X receptor-α (RXR-α), was determined using ligand-binding assays. Activator protein-1 (AP-1) activity was measured using a luciferase reporter assay. Western blot analysis was used to determine cyclin E, Bcl-2 and Bax protein expression. ATPR preferentially bound RXR-α (0.04 nM) as compared with RAR-α (20.96 nM). Although both ATRA and ATPR inhibited the growth of AGS, MKN-74 and SC-M1 cells in a dose-dependent manner, a significantly greater inhibitory effect was observed with treatment with 5 and 500 µM ATPR for 3 days (P<0.05). In addition, ATPR (50 µM), but not ATRA, significantly increased the population of AGS and MKN-74 cells in the subG1 phase and decreased the Bcl-2/Bax ratio (P<0.05). Furthermore, in MNK-74 and SC-M1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 5 or 10 µM of ATPR significantly suppressed the activity of the AP-1 reporter as compared to treatment with ATRA (P<0.05). Thus, ATPR inhibits cancer cell proliferation to a greater extent compared to ATRA, possibly through the RXR-mediated inhibition of AP-1 activity.

Inhibition of ASICs reduces rat hepatic stellate cells activity and liver fibrosis: An in vitro and in vivo study

Hepatic fibrosis is a chronic inflammation‐associated disease, which is involved in the infiltration of inflammatory cells and releasing of proinflammatory cytokines. In the pathological process, protons are released by damaged cells and acidosis is considered to play a critical role in cell injury. Although the underlying mechanism (s) remain ill‐defined, ASICs (acid‐sensing ion channels) are assumed to be involved in this process. The diuretic, amiloride, is neuroprotective in models of cerebral ischemia, a property attributable to the inhibition of central ASICs by the drug. However, the effect of inhibition of ASICs by amiloride in the liver fibrotic process remains unclear. We found that amiloride (25, 50, or 100 μM) could restrain acid‐induced HSCs at pH6 in vitro. In vivo experiments showed that amiloride could significantly alleviate liver injury, decreasing levels of profibrogenic cytokines, collagen deposition, and reducing pathological tissue damage. In summary, amiloride inhibits hepatic fibrosis in vivo and in vitro, which is probably associated with the downregulation of ASICs.

Targeting interleukin-33 in rheumatoid arthritis

In a recent article in Rheumatol Int, Matsuyama et al. reported the change in serum and synovial fluid levels of interleukin (IL)-33 before and after treatment with TNF inhibitors. They found that serum levels of IL-33 after treatment decreased significantly in responders to TNF inhibitors, while they did not change in non-responders. Moreover, sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors [1]. This study is interesting, which provides valuable data on the role of IL-33 in rheumatoid arthritis (RA). The decrease in IL-33 levels may be a useful marker with which to assess the efficacy of TNF inhibitors in the treatment of RA. Recently, there is increasing evidence in both humans and mouse models that IL-33 plays a role in RA development and progression [2]. Through immunohistochemical analyses, Palmer et al. [3] confirmed that IL-33 protein was highly expressed in the synovium of patients with RA. Low levels of IL-33 mRNA and protein were found in human RA synovial fibroblasts, but levels were strongly induced by IL-1b and/or TNF-a. Using the murine model of collagen-induced arthritis (CIA), IL-33 potently enhanced the development of CIA and attendant articular inflammation. Mast cells played a pivotal role in the proinflammatory effect of IL-33 in CIA. Moreover, data showed that IL-33 can enhance autoantibody-mediated articular inflammation via promoting mast cell degranulation and proinflammatory cytokine production [4]. All these findings suggest that IL-33 has a major role in the development of RA. Encouragingly, several recent discoveries regarding the effects of targeting IL-33 in autoimmune diseases have lately been reported. Mu et al. [5] indicated that the level of IL-33 was abnormally elevated in RA serum and infliximab could reduce the serum IL-33 levels in RA patients. In addition, suppression of IL-33 receptor expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNF-a therapy of inflammation [6]. Interestingly, mice lacking ST2 (ST2), the IL-33 receptor a-chain, not only displayed decreased collageninduced arthritis development but also exhibited significantly attenuated proinflammatory cytokine production (IL-17, TNFa and IFN-c) [4]. Consistent with this, ST2 mice developed attenuated autoantibody-induced arthritis (AIA) and reduced expression of articular proinflammatory cytokines [7]. Taken together, these findings hinted that specific blockade of the IL33/ST2 pathway may be an effective therapy for RA. In summary, although it is well accepted that IL-33 is a significant potential contributor to the pathogenesis of RA, further studies are needed to clarify the role of IL-33 in inflammatory processes in various inflammatory, infectious and autoimmune diseases. The development of therapeutic agents targeting IL-33 could result in significant, innovative therapies for the treatment of RA and other autoimmune diseases. Therefore, much more work is required, especially in human systems, to comprehensively explore the therapeutic potential of IL-33 in RA and other autoimmune diseases.