Acid-sensing ion channel 1a mediates acid-induced pyroptosis through calpain-2/calcineurin pathway in rat articular chondrocytes.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

Hashimoto's thyroiditis induces neuroinflammation and emotional alterations in euthyroid mice.

BACKGROUND: Although studies have reported an increased risk for mood disorders in Hashimoto's thyroiditis (HT) patients even in the euthyroid state, the mechanisms involved remain unclear. Neuroinflammation may play a key role in the etiology of mood disorders in humans and behavioral disturbances in rodents. Therefore, this study established a euthyroid HT model in mice and investigated whether HT itself was capable of triggering neuroinflammation accompanied by emotional alterations. METHODS: Experimental HT was induced by immunizing NOD mice with thyroglobulin and adjuvant twice. Four weeks after the last challenge, mice were tested for anxiety-like behavior in the open field and elevated plus maze tests and depression-like behavior in the forced swimming and tail suspension tests. Then, animals were sacrificed for thyroid-related parameter measure as well as detection of cellular and molecular events associated with neuroinflammation. The changes in components of central serotonin signaling were also investigated. RESULTS: HT mice showed intrathyroidal monocyte infiltration and rising serum thyroid autoantibody levels accompanied by normal thyroid function, which defines euthyroid HT in humans. These mice displayed more anxiety- and depressive-like behaviors than controls. HT mice further showed microglia and astrocyte activation in the frontal cortex detected by immunohistochemistry, real-time RT-PCR, and transmission electron microscopy (TEM). These observations were also accompanied by enhanced gene expression of proinflammatory cytokines IL-1ß and TNF-α in the frontal cortex. Despite this inflammatory response, no signs of neuronal apoptosis were visible by the TUNEL staining and TEM in the frontal cortex of HT mice. Additionally, IDO1 and SERT, key serotonin-system-related genes activated by proinflammatory cytokines, were upregulated in HT mice, accompanied by reduced frontal cortex serotonin levels. CONCLUSIONS: Our results are the first to suggest that HT induces neuroinflammation and alters related serotonin signaling in the euthyroid state, which may underlie the deleterious effects of HT itself on emotional function.

Interleukin-1ß and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel.

The acute-phase proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1ß and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1ß and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1ß and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1ß and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1ß and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca2+ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1ß and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.

Crassifoside H improve the depressive-like behavior of rats under chronic unpredictable mild stress: Possible involved mechanisms.

Crassifoside (CH) is a novel chlorine-containing compound isolated from rhizomes of Curculigo glabrescens. This study aimed to explore the antidepressant-like effect of CH and involved mechanisms. A rat depression model was established using chronic unpredictable mild stress (CUMS) paradigm. Behavioral tests including sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST) were used to evaluate the antidepressant-like effect of CH. The levels of plasma corticosterone (CORT) and corticotrophin-releasing factor (CRF) in hypothalamus were measured to determine the activity of hypothalamic pituitary-adrenal (HPA) axis. Protein expression of 5-hydroxytryptamine 1A (5-HT1A) receptor, brain-derived neurotrophic factor (BDNF), as well as the total and phosphorylated extracellular signal-regulated kinase (ERK)1/2 in hippocampus were also analyzed by Western blotting. The CH administration effectively ameliorated the depressive-like behaviors of CUMS rats, as indicated by the increased sucrose intake in SPT, reduced immobility time in FST, and the increased rearing and grooming numbers, spent more time in inner zone and less time in outer zone in OFT. CH improved CUMS-induced HPA axis hyperactivity by reduced plasma CORT and CRH expression in hypothalamus. Moreover, CH reversed CUMS-induced decrease of 5-HT1A receptor expression, and up-regulated BDNF and phosphorylated-ERK1/2 levels in hippocampus. These findings suggest that CH improved depressive behaviors of CUMS rats by modulating of HPA axis dysfunction, increasing 5-HT1A receptor expression, and activating BDNF-ERK signaling pathway.

Acute, but not chronic, stress increased the plasma concentration and hypothalamic mRNA expression of NUCB2/nesfatin-1 in rats.

Nesfatin-1, a newly discovered satiety peptide, has recently been reported to be involved in the stress response. Stress-induced expression of nesfatin-1 has been reported and few studies focus on its expression in the hypothalamus, which is the center of the stress response. To test our hypothesis that peripheral and hypothalamic nesfatin-1 overexpression should play an important role in the stress response and the associated hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis, acute stress (AS) was induced using water avoidance stress (WAS), and chronic unpredictable mild stress (CUMS) was also induced using 3 consecutive weeks of 7 different stressors. The behavior of CUMS rats was evaluated by an open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST). The activity of the HPA axis was detected by measurement of the plasma corticosterone concentration and hypothalamic mRNA expression of corticotropin-releasing-hormone (CRH). The plasma concentration and hypothalamic mRNA expression of nesfatin-1 were measured with an enzyme-linked immunosorbent assay (ELISA) and real-time fluorescent quantitative PCR, respectively. The results showed that both AS and CUMS increased the plasma corticosterone concentration and hypothalamic CRH mRNA expression. Depression-like behavior was induced in CUMS rats, as indicated by a decreased movement distance, frequency of rearing and grooming in the OFT, and sucrose preference index and increased immobility in the FST. Moreover, the AS rats showed increased plasma concentration and hypothalamic mRNA expression of nesfatin-1, which were positively correlated with the plasma corticosterone concentration and hypothalamic CRH expression, respectively. These results indicated that acute stress, but not chronic stress, increased the plasma concentration and hypothalamic mRNA expression of NUCB2/nesfatin-1 in rats.

[Chemical constituents from Bidens bipinnata].

To investigate the chemical constituents of the whole plants of Bidens bipinnata, the separation and purification of constituents were performed by chromatography on macroporous resin, silica gel, MCI and Sephadex LH-20. Their structures were elucidated by spectroscopic data as quercetin (1), quercetin-3-0-alpha-L-rhamnoside (2), keampferol-3-O-beta-D-glucopyranoside (3), keampferol-3-O-alpha-L-rhamnoside (4), 3', 5-dyhydroxy-3, 6, 4'-trimethoxyl -7-O-beta-D-glucopyranoside flavonoid (5), 7, 8, 3', 4'-tetraflavanone(6), (2S)- and (2R)-isookanin-7-O-beta-D- glucopyranoside (7a/7b), (2S)- and (2R)-3'-methoxy-isookanin-8-O-beta-D-glucopyranoside (8a/8b), 6, 7, 3', 4'-tetrahydroxyaurone(9), maritimetin (10), esculetin (11), 3-O-caffeoyl-2-methyl-d-erythrono-1, 4-lactone (12), (7S, 8R) balanophonin-4-O-beta-D-glucopyranoside (13), eugenyl-O-beta-apiofuranosyl-( 1"-6') -O-beta-glucopyranoside (14), and (+)-syringaresinol-4'-O-beta-D-glucopyranoside (15). Compounds 8, 13, 14, and 15 were isolated from this genus for the first time. Compounds 1 and 6 were potent inhibitors against HSC-T6 cells in vitro and compounds 1, 2, 6, and 7 were capable of decreasing the inflammatory cytokine production of macrophage cells in vitro.

Antidepressant effects of abscisic acid mediated by the downregulation of corticotrophin-releasing hormone gene expression in rats.

BACKGROUND: Corticotrophin-releasing hormone (CRH) is considered to be the central driving force of the hypothalamic-pituitary-adrenal axis, which plays a key role in the stress response and depression. Clinical reports have suggested that excess retinoic acid (RA) is associated with depression. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share a similar molecular structure. Here, we proposed that ABA also plays a role in the regulation of CRH activity sharing with the RA signaling pathway. METHODS: [3H]-ABA radioimmunoassay demonstrated that the hypothalamus of rats shows the highest concentration of ABA compared with the cortex and the hippocampus under basal conditions. RESULTS: Under acute stress, ABA concentrations increased in the serum, but decreased in the hypothalamus and were accompanied by increased corticosterone in the serum and c-fos expression in the hypothalamus. Moreover, chronic ABA administration increased sucrose intake and decreased the mRNA expression of CRH and retinoic acid receptor alpha (RARα) in the hypothalamus of rats. Furthermore, ABA improved the symptom of chronic unpredictable mild stress in model rats, as indicated by increased sucrose intake, increased swimming in the forced swim test, and reduced mRNA expression of CRH and RARα in the rat hypothalamus. In vitro, CRH expression decreased after ABA treatment across different neural cells. In BE(2)-C cells, ABA inhibited a series of retinoid receptor expression, including RARα, a receptor that could facilitate CRH expression directly. CONCLUSIONS: These results suggest that ABA may play a role in the pathogenesis of depression by downregulating CRH mRNA expression shared with the RA signaling pathway.

Imbalance of leptin pathway and hypothalamus synaptic plasticity markers are associated with stress-induced depression in rats.

Increasing evidences have indicated that chronic stress is a contributing risk factor in the development of psychiatric illnesses including depression. The mechanisms of their psychopathology are multifaceted and include, besides others, alterations in neuroendocrine function and brain plasticity. In the present study, we investigated the behavior of stressed animals by the sucrose preference test, open field test (OFT), forced swimming test (FST), and tail-suspension test (TST). The response of hypothalamic-pituitary-adrenal (HPA) axis, leptin pathway, and synaptic plasticity markers in the hypothalamus were also detected. Our data demonstrated that chronic unpredictable mild stress (CUMS) could induce depression-like behavior in rat model, accompanied with the hyperactivity of HPA axis. The serum leptin level and hypothalamic mRNA expression of leptin receptor (LEPR) were both decreased. Results of Pearson test showed that the decreased serum leptin level was negatively related with the locomotion and rearing frequency in the open-field test, and the hypothalamic mRNA expression of LEPR was inversely related to serum CORT concentration. Moreover, our results showed that the mRNA expression of synaptotagmin I and synapsin I was both increased in the hypothalamus of CUMS rats, providing new evidence for the synaptic plasticity change in the hypothalamus of depressive rats. Furthermore, our results demonstrated that the mRNA expression of synaptotagmin I, but not synapsin I, was correlated with the depression-like behaviors and HPA axis hyperactivity in CUMS rats. Together with our previous results, the current findings suggested that a CUMS rat model could be effectively used to study molecular mechanisms underling the depressive symptomatology.

Antiinflammatory and immunoregulatory effects of total glucosides of Yupingfeng powder.

BACKGROUND: Yupingfeng, a traditional Chinese complex prescription, has been used efficaciously in China for the cure and prevention of inflammatory diseases related to immunodeficiency such as allergic rhinitis and chronic bronchitis. However, the active components of this prescription remain unclear. The present study focused on investigating the antiinflammatory and immunoregulatory effects of the glucosidic extract from Yupingfeng. METHODS: We tested animal models for ear swelling induced by dimethylbenzene in mice; palm swelling induced by carregeenin and granuloma induced by cotton pellet in rats; level of haemolysin, antibody generation by the splenic cells, delayed hypersensitivity and T cell subsets in spleen of immunosuppressed mice. RESULTS: Glucosidic extract of 24 mg/kg, 48 mg/kg and 96 mg/kg significantly inhibited mice's ear swelling induced by dimethylbenzene. Similarly glucosidic extract of 16 mg/kg, 32 mg/kg and 64 mg/kg inhibited rats' palm swelling induced by carregeenin and granuloma induced by cotton pellet. Glucosidic extract of 24 mg/kg, 48 mg/kg and 96 mg/kg improved the IgM level in serum and level of haemolysin in splenocytes in mice immunosuppressed by cyclophosphamide. Delayed hypersensitivity in mice suppressed by cyclophosphamide was enhanced by glucosidic extract of 24 mg/kg, 48 mg/kg and 96 mg/kg. These results suggested that Yupingfeng could recover humoral and cellular immune function in mice with immunosuppression. Glucosidic extract of 48 mg/kg and 96 mg/kg significantly resisted the immunosuppressive mice ear swelling and maintained it at nearly normal level. The enhanced, delayed hypersensitivity actions of glucosidic extract, suppressed by cyclophosphamide, might be brought about by inducing TH cell and regulating T lymphocytes subset. CONCLUSIONS: The glucosidic extract from Yupingfeng has antiinflammatory and immunoregulation action, suggesting that these glucosides are the principal active components of the traditional Chinese prescription Yupingfeng.

Anti-inflammatory effect of triterpenoic Acids of Eriobotrya japonica (Thunb.) Lindl. Leaf on rat model of chronic bronchitis.

This study was designed to investigate the anti-inflammatory effect of Triterpenoic Acids from Eriobotrya japonica (Thunb.) Lindl. (TAL) on chronic bronchitis (CB) in rats. CB model was established by combination of Bacillus Calmette-Guerin (BCG, 5 mg/kg, injected through the caudal vein) and lipopolysaccharide (LPS, 1 g/L, injected through endotracheal intubation). Rats with CB model were treated with TAL (50, 150 and 450 mg/kg) for 3 weeks. The leukocytes in bronchoalveolar lavage fluid (BALF) were counted after Wright staining, the levels of cytokine tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and IL-10 in the supernatants of lung homogenate were assessed by enzyme-linked immunosorbent assay (ELISA), and the protein expression of nuclear factor kappaB (NF-kappaB) and intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelium were tested by immunohistochemical staining. As compared to the normal and sham groups, the total number of leukocyte, the differential counts of neutrophils and alveolar macrophage (AM) in BALF, the levels of TNF-alpha and IL-8 in the supernatants of lung homogenate, and the expression of NF-kappaB and ICAM-1 on bronchial epithelium in CB rats were significantly increased, while the level of IL-10 was decreased. TAL (50, 150 and 450 mg/kg) attenuated these alterations in model CB rats, which indicates that TAL has anti-inflammatory effect in the rats with CB.

Preventive effects of total flavonoids of Litsea coreana leve on hepatic steatosis in rats fed with high fat diet.

AIM OF THE STUDY: To evaluate the protective effects of total flavonoids of Litsea Coreana leve (TFLC) on rat high fat diet-induced hepatic steatosis model. MATERIALS AND METHODS: Rats were given either a high fat diet alone or the same diet plus TFLC for 4 weeks. RESULTS: TFLC improved liver histology with reduced serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as decreased the over accumulation lipids in serum and liver. TFLC increased serum levels of leptin and insulin, while decreased serum TNFalpha level in high fat diet fed rat. Furthermore, TFLC was found increased the expression of peroxisome proliferator-activated receptor alpha (PPARalpha) in high fat diet fed rat liver. These benefits were associated with increased superoxide dismutase (SOD) and decreased malondialdehyde (MDA) in high fat diet fed rat liver. CONCLUSIONS: TFLC exerts protective effects against hepatic steatosis in rats fed with high fat diet possibly through its antioxidant actions, improving the adipocytokines release and increasing the expression of PPARalpha.

Preliminary study of total flavonoids from Litsea coreana Levl. on experimental adjuvant-induced arthritis in rats.

Litsea coreana Levl., a traditional Chinese medicine, has long been used for its diverse benefits such as detoxification and detumescence. Total flavonoids from Litsea coreana Levl. (TFLC) are the effective fraction of L. coreana. This study was designed to investigate the anti-inflammatory effects and mechanisms of TFLC against Feund's complete adjuvant (FCA)-induced arthritis in rats. Arthritis was evaluated by secondary paw swelling, polyarthritis index, body weight and histopathologic analysis. Con A- or LPS-stimulated splenocyte proliferation and cytokine (IL-1 and IL-2) production were assessed by MTT assay and activated mouse cell proliferation assay, respectively. The results indicate that therapeutic administration of TFLC (50, 100, 200 mg/kg, ig x 12 days) could significantly suppress secondary arthritis in rats with adjuvant-induced arthritis (AA). In vivo, TFLC (50, 100, 200 mg/kg, ig x 12 days) augmented splenocyte proliferation and increased IL-2 production in splenocytes, while reduced IL-1 activity in peritoneal macrophages (PM(Phi)) of AA rats. In vitro, TFLC at concentrations from 0.005 to 50 microg/ml exerted the same immunoregulatory effects on AA rats as those in vivo. In addition, an attractive feature of TFLC lies in its apparent lack of toxicity. These results suggest that TFLC without toxicity has a significant anti-arthritic effect on AA rats which could be associated with its anti-inflammatory and immunomodulatory properties.

Immunomodulating effects of fractioned polysaccharides isolated from Yu-Ping-Feng-Powder in cyclophosphamide-treated mice.

Yu-Ping-Feng-Powder (YP), a traditional Chinese medicine prescription, is widely applied in China for the cure and prevention of diseases related to immunodeficiency. To test whether the fractioned polysaccharides (YPF-P) isolated from YP have immunomodulating activities, the effects of YPF-P on cyclophosphamide (Cy)-treated mice were studied in relation to phagocytosis of macrophage, splenocyte proliferation, and humoral, and cellular immunity parameters. It was found that YPF-P enhances phagocytic activity, augments ConA- or LPS-stimulated T cell proliferation, increases the quantitative haemolysis of SRBC (QHS) and delayed-type hypersensitivity reaction (DTH) to dinitrofluorobenzene. Hence, YPF-P restored the immuno-competence suppressed by Cy. YPF-P also augmented IL-2 and IFN-gamma production, but failed to increase IL-4 production, which indicates that there is high probability that it enhance Th1 function. These results suggested that YPF-P has immunomodulating effects and that the polysaccharides constitute one of the active components of YP.

Anti-oxidative effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf in chronic bronchitis rats.

The study was to evaluate the effect of triterpene acids of Eriobotrya japonica (Thunb.) Lindl. leaf (TAL) on expression of antioxidative mediators by alveolar macrophages (AM) in rats with chronic bronchitis (CB), CB was induced by endotracheal instillation of lipopolysaccharedes (LPS) followed by Bacillus Calmette-Guérin (BCG) injection through caudal vein 1 week later. Treatment groups received TAL at there different doses (50, 150, or 450 mg/kg daily, intragastrically (i.g.)) or dexamethasone (1.2 mg/kg daily i.g.) for 2 weeks, 7 days after LPS injection. AM were then isolated and incubated. Superoxide dismutase (SOD) and methylene dianiline (MDA) levels in AM were measured by commercial kits; meanwhile, heme oxygenase-1 (HO-1) expression and its mRNA expression in AM were detected by immunocytochemistry and RT-PCR, respectively. HO-1 activity of the lung was also detected by a specific biochemistry reaction. The levels of MDA and HO-1 expressed by cultured AM and the HO-1 activity in the lung of the TAL groups were significantly lower than those from the CB group without treatment (p < 0.01 and p < 0.05, respectively), while the SOD levels were increased in a dose-dependent manner by TAL treatment. These results suggest that TAL inhibits HO-1 expression and MDA production and up-regulates SOD expression in AM from CB rats, which might be one of molecular mechanisms of its anti-inflammatory effects in CB rats.