Tatarinan T, an α-asarone-derived lignin, attenuates osteoclastogenesis induced by RANKL via the inhibition of NFATc1/c-Fos expression.

We have previously reported that the lignin-like compounds, Tatarinan O (TO) and Tatarinan N (TN), extracted from the roots of Acorus tatarinowii Schott, inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. In the present study, the potential function of the α-asarone-derived lignins, Tatarinan T (TT) and Tatarinan A (TA), to regulate RANKL-induced osteoclastogenesis was investigated, and it was found that only early treatment with TT may inhibit RANKL-triggered formation of osteoclasts and resorption. The results revealed repressed expression levels of several osteoclast marker genes, including ATPase H+ -transporting V0 subunit d2 (Atp6v0d2), αvß3 integrin, and osteoclast-associated receptor (OSCAR), following TT treatment during osteoclastogenesis. Moreover, TT reduced the expression levels of the core transcription elements, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and c-Fos. However, western blotting analysis showed that TT treatment did not alter nuclear factor-κΒ (NF-κB) activation or mitogen-activated protein kinase (MAPK) or Syk/Btk/phospholipase Cγ2 (PLCγ2) phosphorylation. Taken together, these results suggest the potential of TT in the treatment of diseases of increased bone resorption.

The Protective Effects of HJB-1, a Derivative of 17-Hydroxy-Jolkinolide B, on LPS-Induced Acute Distress Respiratory Syndrome Mice.

Acute respiratory distress syndrome (ARDS),which is inflammatory disorder of the lung, which is caused by pneumonia, aspiration of gastric contents, trauma and sepsis, results in widespread lung inflammation and increased pulmonary vascular permeability. Its pathogenesis is complicated and the mortality is high. Thus, there is a tremendous need for new therapies. We have reported that HJB-1, a 17-hydroxy-jolkinolide B derivative, exhibited strong anti-inflammatory effects in vitro. In this study, we investigated its impacts on LPS-induced ARDS mice. We found that HJB-1 significantly alleviated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNF-α, IL-1ß and IL-6 in BALF. In addition, HJB-1 markedly suppressed LPS-induced IκB-α degradation, nuclear accumulation of NF-κB p65 subunit and MAPK phosphorylation. These results suggested that HJB-1 improved LPS-induced ARDS by suppressing LPS-induced NF-κB and MAPK activation.

Calycosin Suppresses RANKL-Mediated Osteoclastogenesis through Inhibition of MAPKs and NF-κB.

Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-κB and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis.

AIM: To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na(+)/H(+) exchanger 1 (NHE1) and calpain, intracellular free Ca(2+)level ([Ca(2+)](i)), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE(-/-)) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 µg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated. RESULTS: LPS treatment increased NHE1 activity and [Ca(2+)](i) in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca(2+)-dependent protease calpain. Amiloride (1-10 µmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca(2+)](i). and calpain activity. In the presence of the Ca(2+) chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 µmol/L) or the calpain inhibitor ZLLal (50 µmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE(-/-) mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression. CONCLUSION: LPS stimulates NHE1 activity, increases [Ca(2+)](i), and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.

[Effect of Antibiotic-Degrading Bacteria on Maturity and Bacterial Community Succession During Pig Manure Composting].

The inoculation of antibiotic-degrading bacteria into manure could promote the removal of antibiotics during composting. However, knowledge on the impact of inoculating these antibiotic-degrading bacteria on the composting process and indigenous microbial community succession is still limited. This study assessed the antibiotic removal efficiency in pig manure after inoculating a microbial inoculum with antibiotic-degrading bacteria as the key component. The effect of inoculating this microbial inoculum on the physicochemical dynamics and the succession of the manure bacterial community during composting was also analyzed. The results showed that the antibiotic degradation in pig manure reached 81.95% after inoculating the microbial inoculum. When compared with that in the control, the total concentration of antibiotic residues in manure with the microbial agent inoculated was decreased by 42.18%. During composting, inoculating the microbial inoculum accelerated the temperature rise of compost, favored water loss, and alleviated the release of NH3 and H2S. Moreover, the total nutrient content (nitrogen, phosphorus, and potassium) in the final compost and the germination index of radish seeds increased by 6.80% and 68.33%, respectively, after inoculating this microbial inoculum. Furthermore, inoculating the microbial inoculum increased the content of stable organic carbon in the final compost and decreased the content of recalcitrant substances such as cellulose and hemicellulose. The analysis of the manure bacterial community showed that inoculating the microbial inoculum increased the relative abundances of Actinomycetes and Firmicutes in the compost. In particular, the thermophilic bacteria that was positively related to the compost temperature was increased significantly (P<0.01) after inoculating the microbial inoculum, whereas the relative abundance of pathogenic bacteria was correspondingly decreased. Network analysis of the bacterial coexistence pattern showed that inoculating this microbial inoculum also changed the interaction pattern of indigenous manure bacterial communities, which greatly reduced the complexity and connectivity of the bacterial interaction and improved the ecological relationship between beneficial bacteria and other bacterial communities. The effect of this microbial inoculum on the interaction with manure bacterial community laid a foundation for the establishment of a new and healthier composting bacterial community. This study provides a scientific basis for the application and development of multifunctional antibiotic-degrading microbial agents in manure treatments.

Purple tea water extract blocks RANKL-induced osteoclastogenesis through modulation of Akt/GSK3ß and Blimp1-Irf8 pathways.

A number of studies demonstrated that some tea extracts exert inhibitory effects on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). However, the effect of purple tea, a famous tea in China, on osteoclastogenesis remains unclear. In this study, a water-based purple tea extract (PTE) was found to suppress osteoclast formation, osteoclastic resorption pit area formation, and F-actin ring formation within RANKL-stimulated bone marrow macrophages (BMMs). Furthermore, our results demonstrated that PTE could inhibit expression of master transcription factors NFATc1 and c-Fos and their target genes DC-STAMP, Ctsk, and Atp6v0d2. Western blot analysis revealed that PTE treatment led to reduced RANKL-induced phosphorylation of Akt and GSK3ß without altering transient activation of NF-κB and MAPKs (p38, JNK, ERK1/2) signaling. In addition, the results demonstrated that PTE treatment of RANKL-stimulated BMMs could down-regulate Blimp1 expression and up-regulate Irf8 expression. In summary, these results suggest that PTE treatment of RANKL-stimulated BMMs inhibited osteoclast differentiation via modulation of Blimp1-Irf8 and Akt/GSK3ß signaling pathways. Aligning with our in vitro results, in vivo PTE administration ameliorated bone loss in LPS-treated mice. Taken together, the results presented in this work suggest that PTE treatment possesses anti-osteolytic activity.

Agrimophol suppresses RANKL-mediated osteoclastogenesis through Blimp1-Bcl6 axis and prevents inflammatory bone loss in mice.

Excessive activity of osteoclasts causes many bone-related diseases, such as rheumatoid arthritis and osteoporosis. Agrimophol (AGR), a phenolic compound, originated from Agrimonia pilosa Ledeb. In prior studies, AGR is reported to possess schistosomicidal and mycobactericidal activities. However, no reports covered its anti-osteoclastogenesis characteristic. In this study, we found that AGR inhibited RANKL-induced osteoclastogenesis, bone-resorption, F-actin ring formation, and the mRNA expression of osteoclast-associated genes such as CTSK, TRAP, MMP-9, and ATP6v0d2 in vitro. In addition, AGR suppressed RANKL-induced expression of c-Fos and NFATc1. However, AGR treatment did not affect NF-κB activation and MAPKs phosphorylation in RANKL-stimulated BMMs, which implicated that AGR might not influence the initial expression of NFATc1 mediated by NF-κB and MAPKs signaling. Our results further indicated that AGR did not alter phosphorylation levels of GSK3ß and the expression of calcineurin, which implicated that AGR treatment might not interfere with phosphorylation and de-phosphorylation of NFATc1 mediated by GSK3ß and calcineurin, respectively. B-lymphocyte-induced maturation protein-1 (Blimp1), which was regarded as a transcriptional repressor of negative regulators of osteoclastogenesis, was markedly attenuated in the presence of AGR, leading to the enhanced expression of B-cell lymphoma 6 (Bcl-6). Meanwhile, Blimp1 knockdown in BMMs by siRNA strongly enhanced the expression of Bcl6 and reduced NFATc1 induction by RANKL. These findings suggested that AGR inhibited RANKL-induced osteoclast differentiation through Blimp1-Bcl-6 signaling mediated modulation of NFATc1 and its target genes. Consistent with these in vitro results, AGR exhibited a protective influence in an in vivo mouse model of LPS-induced bone loss by suppressing excessive osteoclast activity and attenuating LPS-induced bone destruction. Hence, these results identified that AGR could be considered as a potential therapeutic agent against bone lysis disease.

Maqui berry exhibited therapeutic effects against DSS-induced ulcerative colitis in C57BL/6 mice.

Maqui berry (Aristotelia chilensis) is an edible berry. The study aimed to explore the therapeutic effect of maqui berry on inflammatory bowel disease. Maqui berry water extract was separated by multiple solvents extraction. The chemical bases, antioxidant and anti-inflammatory properties of different extract fractions were then compared. Dextran sodium sulfate (DSS)-induced ulcerative colitis mice were used for the pharmacological activity test in vivo. Experimental results showed that the ethyl acetate fraction of maqui berry water extract (MWE) was rich in phenols and exhibited good antioxidant and anti-inflammatory activities. MWE considerably reduced the expression of COX2 and IL-6 in LPS-stimulated RAW 264.7 cells. Inflammatory bowel disease index, MDA, NO, i-NOS, and COX2 in colon tissues and MPO, TNF-α, and IL-1ß in blood serums were remarkably decreased in the treatment group compared to in the model group (p < 0.05). Intestinal histopathological damage was significantly alleviated in the treatment group, and the expression of occludin was increased (p < 0.05). MWE treatment alleviated the imbalance of gut microbiota caused by DSS injury. Overall, MWE plays a therapeutic role in ulcerative colitis through its anti-inflammatory effect, reduces immune stress, and regulates gut microbiota.

Anemonin Attenuates RANKL-Induced Osteoclastogenesis and Ameliorates LPS-Induced Inflammatory Bone Loss in Mice via Modulation of NFATc1.

Osteoporosis is a metabolic bone disease characterized by insufficient osteoblastic function and/or excessive osteoclastic activity. One promising strategy for treating osteoporosis is inhibiting excessive osteoclast resorbing activity. Previous studies have revealed that anemonin (ANE), isolated from various types of Chinese natural herbs, has anti-inflammatory and anti-oxidative properties. However, whether ANE regulates osteoclastogenesis is unknown. This study aimed to investigate the potential effect of ANE on osteoclastogenesis and inflammatory bone loss in mice. In in vitro studies, ANE suppressed RANKL-stimulated osteoclast differentiation and function by downregulating the expression of osteoclast master transcriptor NFATc1, as well as its upstream transcriptor c-Fos, by decreasing NF-κB and ERK1/2 signaling. Interestingly, ANE did not change the phosphorylation and degradation of IκB-α and activation of JNK and p38 MAPKs. However, ANE repressed the phosphorylation of MSK-1 which is the downstream target of ERK1/2 and p38 MAPK and can phosphorylate NF-κB p65 subunit. These results implicated that ANE might suppress NF-κB activity via modulation of ERK1/2 mediated NF-κB phosphorylation. In addition, ANE directly suppressed NFATc1 transcription by inhibiting Blimp-1 expression, and the subsequent enhancement of the expression of NFATc1 negative regulators, Bcl-6 and IRF-8. Moreover, in vivo studies were conducted using an LPS-induced inflammatory bone loss mice model. Micro-CT and histology analysis showed that ANE treatment significantly improved trabecular bone parameters and bone destruction. These data indicate that ANE can attenuate RANKL-induced osteoclastogenesis and ameliorate LPS-induced inflammatory bone loss in mice through modulation of NFATc1 via ERK1/2-mediated NF-κB phosphorylation and Blimp1 signal pathways. ANE may provide new treatment options for osteoclast-related diseases.

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes.

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes.

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.

Tatarinan N inhibits osteoclast differentiation through attenuating NF-κB, MAPKs and Ca2+-dependent signaling.

Osteoclasts are multinucleated cells that originate from hemopoietic stem cells. Targeting over activated osteoclasts is thought to be an effective therapeutic approach to osteoporosis. In a previous study, we reported that Tatarinan O, a lignin-like compound, suppressed RANKL-induced osteoclastogenesis. In this study, we further examined the effects on osteoclast formation of three lignin-like compounds including Tatarinan N (TN), Tatarinan U (TU) and Tatarinan V (TV), all containing a common structure of asarone. We found that only TN suppressed RANKL-induced osteoclast differentiation, bone resorption pit formation and F-acting ring formation. TU and TV did not influence RANKL-induced osteoclastogenesis. We also found that TN dose-dependently inhibited the expression of osteoclastogenesis-associated genes, including TRAP, cathepsin K and MMP-9. Furthermore, we found that TN down-regulated the key transcription factor NFATc1 and c-Fos by preventing the activation of NF-κB and phosphorylation of MAPKs including ERK1/2 and p38 but not JNK. TN attenuated calcineurin expression via suppression of the Btk-PLCγ2 cascade and reduction of intracellular Ca2+, modulating NFATc1 activation. Taking together, our results indicated that TN might have therapeutic potential for osteoporosis.

Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils.

OBJECTIVE: To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. SAMPLE POPULATION: Neutrophils isolated from 8 healthy horses. PROCEDURES: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined. RESULTS: Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.

Effects of stimulation of adenosine A2A receptors on lipopolysaccharide-induced production of reactive oxygen species by equine neutrophils.

OBJECTIVE: To assess the anti-inflammatory effects of an adenosine analogue on lipopolysaccharide (LPS)-stimulated equine neutrophils. SAMPLE POPULATION: Neutrophils obtained from 10 healthy horses. PROCEDURES: An adenosine analogue (5'-N-ethylcarboxamidoadenosine [NECA]) was tested for its ability to inhibit production of reactive oxygen species (ROS) in LPS-stimulated equine neutrophils. Selective adenosine receptor antagonists were used to identify the receptor subtype responsible for effects. To assess the mechanism of action of NECA, cAMP concentrations were measured, and effects of dibutyryl cAMP (a stable analogue of cAMP) and rolipram (a type 4 phosphodiesterase inhibitor) were investigated. RESULTS: NECA elicited concentration-dependent inhibition of ROS production that was inhibited by ZM241385, a selective adenosine A(2A) receptor antagonist; this effect of NECA was not affected by the adenosine A(2B) receptor antagonist MRS1706. Also, ZM241385 blocked NECA-induced increases in cAMP concentrations, whereas MRS1706 did not alter this effect of NECA. Rolipram potentiated NECA-induced inhibition of ROS production, and dibutyryl cAMP also inhibited ROS production. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of adenosine A(2A) receptors inhibited ROS production by LPS-stimulated equine neutrophils in a cAMP-dependent manner. These results suggest that stable adenosine A(2A) receptor agonists may be developed as suitable anti-inflammatory drugs in horses.

Sciadopitysin suppresses RANKL-mediated osteoclastogenesis and prevents bone loss in LPS-treated mice.

Previous studies reported that sciadopitysin (Sc), a type of biflavonoids, protects reactive oxygen species (ROS)-mediated osteoblast dysfunction, but its role in osteoclastogenesis remains unclear. In this study, we observed that Sc dose-dependently suppressed RANKL-induced osteoclastogenesis and bone resorption. Our results indicated that Sc treatment strongly reduced RANKL-induced osteoclast-specific genes expression, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP) and MMP-9. Furthermore, Sc apparently attenuated RANKL-increased expressions of c-Fos and NFATc1. Meanwhile, Sc also strikingly inhibited the activation of NF-κB without altering the phosphorylation of MAPKs (p38, JNK and ERK1/2). Finally, our study demonstrated that Sc administration could reverse the bone loss in LPS-induced mice model. This study suggests that Sc inhibits RANKL-induced osteoclastogenesis and bone loss by inhibiting NF-κB activation and reducing the expression of c-Fos and NFATc1. Therefore, Sc might be benefit for RANKL-mediated osteolytic bone diseases.

Activation of adenosine A1 receptor modulates dopamine D1 receptor activity in stably cotransfected human embryonic kidney 293 cells.

The antagonistic interactions between adenosine A1 receptors and dopamine D1 receptors were studied in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In the cotransfected cells, but not in control cells only transfected with dopamine D1 receptors, adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 10 microM) increased the Kd of dopamine D1 receptor antagonist [N-methyl-3H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H]SCH23390) without affecting the Bmax. Moreover, CPA induced a concentration-dependent decrease in the affinity of dopamine D1 receptors for the agonist (+/-)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) and inhibited dopamine D1 receptor-mediated cyclic AMP response element recruitment. Furthermore, pertussis toxin treatment completely counteracted the effects of low concentrations of CPA but only partially counteracted the effects of high concentrations of CPA. These results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the second messenger generation. Furthermore, the antagonistic interactions between these two receptors induced by low concentrations of CPA might have a different manner with those induced by high concentrations of CPA.

[Interaction Between Sulfonamide Antibiotics Fates and Chicken Manure Composting].

Based on aerobic manure composting with or without the addition of a mixture of sulfadimethoxine SM2 and sulfamonomethoxine SMM (1:1, m/m), changes in the physic-chemical properties of manure compost, the microbial community physiological profiles, the antibiotics concentration and the abundances of five antibiotic resistance genes (ARGs) during the composting were tracked. The results indicated that the introduction of sulfonamide antibiotics led to inhibition on the basal respiration of manure compost during the early composting period, delayed the formation of thermophilic temperature and reduced the conversion of nutrients such as organic matter, ammonia nitrogen and nitrate nitrogen. Meanwhile, the introduction of sulfonamide antibiotics dramatically affected the physiological profile of microbial community in manure in the middle stage of composting. HPLC-MS/MS results showed that both SMM and SM2 in manure were completely degraded within 14 days, while the degradation rate of SMM was faster than that of SM2. For both composting treatments with or without addition of exogenous antibiotics, the relative abundance of sull and sul2 showed an initial decline in the first 14 or 21 days and a slight increase thereafter. The addition of exogenous antibiotics showed insignificant enhancement on increasing the relative abundance of sul1 and IntI1 in manure, but resulted in an apparent increase in sul2 relative abundance. Although the fates of tetQ and tetW during composting were different from that of sulfonamide ARGs, the introduction of sulfonamide antibiotics into manure increased the relative abundance of tetracycline ARGs. Redundancy analysis indicated that composting temperature correlated negatively with sul1, sul2 and IntI1 relative abundance in manure but had no obvious relationship with tetQ and tetW relative abundance. All the ARGs detected in this work correlated negatively with C/N ratio and the nitrate nitrogen concentration of manure compost but positively correlated with pH, moisture and ammonia nitrogen concentration of manure compost.

Tatarinan O, a lignin-like compound from the roots of Acorus tatarinowii Schott inhibits osteoclast differentiation through suppressing the expression of c-Fos and NFATc1.

Osteoclasts (OC) are large multinucleated cells derived from monocyte/macrophage precursors. Suppressing osteoclastogenesis is considered as an effective therapeutic approach to erosive bone disease. The root of Acorus tatarinowii Schott, a well-known traditional Chinese medicine was used to treat rheumatosis and other inflammatory disease. However, the effects of tatarinan O (TO), one of the lignin-like compounds isolated from the roots of Acorus tatarinowii Schott during bone development are still unclear. In the present study, we explored the effect of TO on RANKL-induced osteoclastogenesis in vitro. TO was found to suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages (BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genes, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. Hence, for the first time we revealed that TO dose-dependently inhibited osteoclastogenesis from RANKL-stimulated mouse BMMs via decreasing the expression of NFATc1 and c-Fos.

Ethyl pyruvate protects PC12 cells from dopamine-induced apoptosis.

Pyruvate acid can protect cells against oxidative damage. However, its instability limits its usefulness as a therapeutic agent. In this study, we examined the effect of ethyl pyruvate, an aliphatic ester derived from pyruvate acid, on dopamine-induced cytotoxicity in rat pheochromocytoma PC12 cells. The results demonstrated that dopamine induced apoptosis in PC12 cells accompanied with increases of intercellular reactive oxygen species, nuclear translocation of nuclear transcription factor kappa B (NF-kappaB) and expression of p53 and decrease of mitochondrial transmembrane potential. Ethyl pyruvate markedly reduced the dopamine-induced production of reactive oxygen species, nuclear translocation of NF-kappaB, upregulation of p53, loss of mitochondrial transmembrane potential and apoptosis in PC12 cells. The results suggested that ethyl pyruvate might protect PC12 cells against dopamine by suppressing intercellular oxidative stress and modulating key signal pathways of apoptosis, and that ethyl pyruvate might be used as a potential therapeutic agent for Parkinson's disease.

17-Hydroxy-jolkinolide A inhibits osteoclast differentiation through suppressing the activation of NF-κB and MAPKs.

Osteoclasts (OC) are bone-specific multinucleated giant cells (MNCs) derived from the monocyte/macrophage hematopoietic lineage cells. Inhibiting osteoclast formation is considered as an effective therapeutic approach for the treatment of the pathological bone loss. In this study, we investigated effects of 17-hydroxy-jolkinolide A (HJA), an ent-abietane diterpenoid isolated from the dried root of Euphorbia fischeriana, on osteoclastogenesis induced by RANKL. The results showed that HJA significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). HJA also prevented bone resorption by mature osteoclasts in a dose-dependent manner. In addition, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cts K) and MMP-9, was significantly inhibited by HJA. Furthermore, HJA also significantly inhibited RANKL-induced activation of NF-κB and phosphorylation of MAPK. Our results indicate that HJA has an inhibitory role in the bone loss by preventing osteoclast formation as well as its bone resorptive activity. Therefore, HJA may be useful as a therapeutic reagent for bone loss-associated diseases.