Formononetin improves the inflammatory response and bone destruction in knee joint lesions by regulating the NF-kB and MAPK signaling pathways.

Formononetin (FMN) is a phytoestrogen that belongs to the isoflavone family. It has antioxidant and anti-inflammatory effects, as well as, many other biological activities. Existing evidence has aroused interest in its ability to protect against osteoarthritis (OA) and promote bone remodeling. To date, research on this topic has not been thorough and many issues remain controversial. Therefore, the purpose of our study was to explore the protective effect of FMN against knee injury and clarify the possible molecular mechanisms. We found that FMN inhibited osteoclast formation induced by receptor activator of NF-κB ligand (RANKL). Inhibition of the phosphorylation and nuclear translocation of p65 in the NF-κB signaling pathway plays a role in this effect. Similarly, during the inflammatory response of primary knee cartilage cells activated by IL-1ß, FMN inhibited the NF-κB signaling pathway and the phosphorylation of the ERK and JNK proteins in the MAPK signaling pathway to suppress the inflammatory response. In addition, in vivo experiments showed that both low- and high-dose FMN had a clear protective effect against knee injury in the DMM (destabilization of the medial meniscus) model, and the therapeutic effect of high-dose FMN was stronger. In conclusion, these studies provide evidence of the protective effect of FMN against knee injury.

Overexpression of miR-148a-3p inhibits extracellular matrix degradation and alleviates IL-1ß-induced intervertebral disc degeneration.

Objectives: Recently, studies on microRNAs (miRNAs) and their targets and related genes have provided new therapeutic opportunities for controlling intervertebral disc degeneration (IDD). We aimed to investigate the effects of miR-148a-3p overexpression on IDD progression. Materials and Methods: This study used microRNA microarrays to analyze key regulators of IDD. Q-PCR was used to verify the IL-1ß-induced down-regulation of miR-148a-3p expression both in nucleus pulposus (NP) tissues of IDD patients and in degenerated NP cells (NPCs) of rats. Rat NPC micromass cultures and ex vivo intervertebral disc (IVD) culture models were established, and histological staining was performed to verify the effect of miR-148a-3p on the general morphology and proteoglycan and collagen contents of IVDs. In addition, q-PCR and western blotting analyses were performed to examine the expression of ECM molecules and matrix-degrading enzymes. A luciferase reporter assay was used to confirm the target genes of miR-148a-3p. Results: Our data revealed that miR-148a-3p was down-regulated in IDD. Overexpression of miR-148a-3p had no effect on ACAN or COL2A1 gene expression but decreased MMP3, MMP13, and ADAMTS5 gene expression. The matrix deposited by miR-148a-3p-overexpressing rat NPCs contained high levels of proteoglycans and collagen. The ex vivo experiments verified that agomiR-148a-3p alleviated the NPC matrix degradation induced by IL-1ß. A luciferase reporter assay confirmed that miR-148a-3p directly targeted ADAMTS5 and MMP13. Conclusion: We proved that miR-148a-3p can attenuate ECM loss and protect NP function by inhibiting matrix-degrading enzymes.

SIS3 suppresses osteoclastogenesis and ameliorates bone loss in ovariectomized mice by modulating Nox4-dependent reactive oxygen species.

Osteoporosis is a metabolic disorder of reduced bone mass, accompanied by the deterioration of the bone microstructure, resulting in increased brittleness and easy fracture. Its pathogenesis can be explained by mainly excessive osteoclast formation or bone resorption hyperfunction. Oxidative stress is intricately linked with bone metabolism, and the maturation and bone resorption of osteoclasts respond to intracellular ROS levels. SIS3 is a small-molecule compound that selectively suppresses Smad3 phosphorylation in the TGF-ß/Smad signaling pathway and attenuates the ability to bind to target DNA. Several studies have reported that Smad3 plays a significant role in bone metabolism. However, whether SIS3 can modulate bone metabolism by affecting osteoclastogenesis and the specific molecular mechanisms involved remain unknown. Here, we demonstrated that SIS3 could suppress osteoclastogenesis and ameliorate bone loss in ovariectomized mice. Mechanistically, SIS3 inhibited Smad3 phosphorylation in BMMs, and the deficiency of phosphorylated Smad3 downregulated ROS production and Nox4-dependent expression during osteoclast formation, thereby blocking MAPK phosphorylation and the synthesis of downstream osteoclast marker proteins. Similarly, Nox4 plasmid transfection significantly alleviated osteoclast formation inhibited by SIS3. In addition, we identified the interaction region between Smad3 and Nox4 by ChIP and dual luciferase reporter assays. Collectively, we found that SIS3 could inhibit Smad3 phosphorylation, reduce Nox4-dependent ROS generation induced by RANKL, and prevent osteoclast differentiation and maturation, making it a promising alternative therapy for osteoporosis.

Azilsartan Suppresses Osteoclastogenesis and Ameliorates Ovariectomy-Induced Osteoporosis by Inhibiting Reactive Oxygen Species Production and Activating Nrf2 Signaling.

Osteoporosis is characterized by a decrease in bone mass and destruction of the bone microarchitecture, and it commonly occurs in postmenopausal women and the elderly. Overactivation of osteoclasts caused by the inflammatory response or oxidative stress leads to osteoporosis. An increasing number of studies have suggested that intracellular reactive oxygen species (ROS) are strongly associated with osteoclastogenesis. As a novel angiotensin (Ang) II receptor blocker (ARB), azilsartan was reported to be associated with the inhibition of intracellular oxidative stress processes. However, the relationship between azilsartan and osteoclastogenesis is still unknown. In this study, we explored the effect of azilsartan on ovariectomy-induced osteoporosis in mice. Azilsartan significantly inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and downregulated the expression of osteoclast-associated markers (Nfatc1, c-Fos, and Ctsk) in vitro. Furthermore, azilsartan reduced RANKL-induced ROS production by increasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Mechanistically, azilsartan inhibited the activation of MAPK/NF-κB signaling pathways, while Nrf2 silencing reversed the inhibitory effect of azilsartan on MAPK/NF-κB signaling pathways. Consistent with the in vitro data, azilsartan administration ameliorated ovariectomy (OVX)-induced osteoporosis, and decreased ROS levels in vivo. In conclusion, azilsartan inhibited oxidative stress and may be a novel treatment strategy for osteoporosis caused by osteoclast overactivation.

TGF­ß inhibitor RepSox suppresses osteosarcoma via the JNK/Smad3 signaling pathway.

Osteosarcoma (OS) is the most common malignant bone tumor and the long­term survival rates remain unsatisfactory. Transforming growth factor­ß (TGF­ß) has been revealed to play a crucial role in OS progression, and RepSox is an effective TGF­ß inhibitor. In the present study, the effect of RepSox on the proliferation of the OS cell lines (HOS and 143B) was detected. The results revealed that RepSox effectively inhibited the proliferation of OS cells by inducing S­phase arrest and apoptosis. Moreover, the inhibitory effect of RepSox on cell migration and invasion was confirmed by wound­healing and Transwell assays. Furthermore, western blotting revealed that the protein levels of molecules associated with the epithelial­mesenchymal transition (EMT) phenotype, including E­cadherin, N­cadherin, Vimentin, matrix metalloproteinase (MMP)­2 and MMP­9, were reduced by RepSox treatment. Concurrently, it was also revealed that the JNK and Smad3 signaling pathway was inhibited. Our in vivo findings using a xenograft model also revealed that RepSox markedly inhibited the growth of tumors. In general, our data demonstrated that RepSox suppressed OS proliferation, EMT and promoted apoptosis by inhibiting the JNK/Smad3 signaling pathway. Thus, RepSox may be a potential anti­OS drug.

Chicago sky blue 6B (CSB6B), an allosteric inhibitor of macrophage migration inhibitory factor (MIF), suppresses osteoclastogenesis and promotes osteogenesis through the inhibition of the NF-κB signaling pathway.

Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator involved in various pathophysiological and inflammatory states. Accumulating line of evidence suggests a role for MIF in regulating bone metabolism and therefore a prime candidate for therapeutic targeting. In this study, we showed that Chicago sky blue 6B (CSB6B) suppresses RANKL-induced osteoclast and bone resorption in vitro via the inhibition of NF-κB signaling activation and promoting proteasome-mediated degradation of MIF. Consequently, the induction of NFATc1 was impaired resulting in downregulation of NFATc1-responsive osteoclast genes. We also demonstrated that CSB6B treatment enhanced primary calvarial osteoblast differentiation and bone mineralization in vitro via the suppression of NF-κB activation and upregulation of Runx expression. Using two murine models of osteolytic bone disorders, we further showed that administration of CSB6B protected mice against pathological inflammatoryc calvarial bone destruction induced by titanium particles mice as well as estrogen-deficiency induced bone loss as a result of ovariectomy. Together, as an MIF inhibitor, CSB6B can inhibit osteoclast differentiation and bone resorption function and enhance the mineralization of osteoblasts through the inhibition of NF-κB pathway. MIF is a prime target for therapeutic targeting for the treatment of osteolytic bone disorders and the MIF inhibitor CSB6B could be potential anti-osteoporosis drug.

[Expression and clinical significance of plasma small RNA in patients with pancreatic cancer].

OBJECTIVE: The aim of this study was to identify six miRNAs expressed in plasma of patients with pancreatic cancer (PCa) and analyze their value as a diagnostic index of pancreatic cancer. METHODS: Plasma total RNAs were extracted from 30 PCa patients and 26 normal controls, and the abundance of six microRNAs was measured using real-time PCR. The possibility to combine them with CA19-9 as diagnostic biomarkers was analyzed. RESULTS: The expression level of miR-21, miR-210, miR-155, miR-20a, miR-25 and miR-196a in plasma of patients with pancreatic cancer were 1.65×10(6), 5.98×10(4), 2.83×10(3), 3.47×10(6), 2.76×10(6), and 1.03×10(3) (copies/µl), while the normal controls were 4.08×10(5), 2.54×10(4), 8.55×10(2), 1.79×10(6), 9.32×10(5), and 4.67×10(2) (copies/µl), respectively, with a significant difference between the two groups (P < 0.05). The areas under the ROC curve of miR-21, miR-210, miR-155, miR-20a, miR-25 and miR-196a were 0.893, 0.810, 0.820, 0.766, 0.816 and 0.729, respectively. MiR-21 had the highest diagnostic value when it was used as diagnostic marker alone. The combination of miR-155 and miR-25 was more effective to distinguish PCa from normal than to be used alone, and the area under the ROC curve was 0.913 (95%CI 0.838-0.988) .When CA199 associated with miR -210 and miR-25, respectively, the areas under the ROC curves were 0.96 (95%CI was 0-1.0) and 0.942 (95% CI was 0.876-1.0), which were higher than CA199 alone (0.862, 95%CI was 0.748-0.975). There was a high improvement in diagnostic sensitivity and accuracy when miR-210 and miR-25 were combined with CA19-9, respectively. CONCLUSIONS: Plasma miR-21, miR-155, miR-25, miR-210 have diagnostic value for pancreatic cancer, and deserve further study.

Isolation of talathermophilins from the thermophilic fungus Talaromyces thermophilus YM3-4.

Six indole alkaloids with various levels of prenylation were isolated from the thermophilic fungus Talaromyces thermophilus strain YM3-4. Their structures were identified by NMR and MS spectroscopic analyses. Compounds 1 and 2 are new analogues of the key versatile precursor notoamide E. Compound 3 is a novel analogue of preechinulin, and compound 4 was reported as a natural occurring cyclo(glycyltryptophyl) for the first time. The metabolite profile of this thermophilic organism displayed a biosynthetic pathway for talathermophilins.

Anoxybacillus tengchongensis sp. nov. and Anoxybacillus eryuanensis sp. nov., facultatively anaerobic, alkalitolerant bacteria from hot springs.

Two novel thermophilic, spore-forming bacterial strains, T-11(T) and E-112(T), were isolated from hot springs in Tengchong and Eryuan counties of Yunnan province in south-west China. The strains were Gram-stain-positive rods, occurring singly or in chains. Growth of strain T-11(T) was observed between 30 and 75 °C (optimum 50 °C) and at pH 7-11 (optimum pH 8.5), while the temperature range for strain E-112(T) was 35-70 °C (optimum 55 °C) and the pH range was 7.0-11.0 (optimum pH 8.0). The DNA G+C contents of strains T-11(T) and E-112(T) were 41.1 and 42.6 mol%, respectively. On the basis of 16S rRNA gene sequence similarity, the two strains were shown to be related most closely to Anoxybacillus species. The chemotaxonomic characteristics [predominant isoprenoid quinone menaquinone 7 (MK-7); major fatty acids iso-C(15 : 0) and iso-C(17 : 0)] also supported the affiliation of strains T-11(T) and E-112(T) to the genus Anoxybacillus. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains T-11(T) and E-112(T) from Anoxybacillus species with validly published names. Strains T-11(T) and E-112(T) therefore represent two novel species, for which the names Anoxybacillus tengchongensis sp. nov. (type strain T-11(T) =CCTCC AB209237(T) =KCTC 13721(T)) and Anoxybacillus eryuanensis sp. nov. (type strain E-112(T) =CCTCC AB209236(T) =KCTC 13720(T)) are proposed.

Isolation of putative biosynthetic intermediates of prenylated indole alkaloids from a thermophilic fungus Talaromyces thermophilus.

The putative key biosynthetic intermediates of prenylated indole alkaloids have long been proposed but never isolated. Two such alkaloids, named talathermophilins A and B (1 and 2), were isolated from a thermophilic fungus Talaromyces thermophilus strain YM1-3 and were identified by NMR and MS spectroscopic analyses. The ratio of 1 and 2 in the culture broths was unexpectedly rather constant (about 2:3), which even remained unchanged despite the addition of exogenous 1 or 2, suggesting that talathermophilins might be of special function for the extremophilic fungus.

Diversity of thermophilic fungi in Tengchong Rehai National Park revealed by ITS nucleotide sequence analyses.

The geothermal sites near neutral and alkalescent thermal springs in Tengchong Rehai National Park were examined through cultivation-dependent approach to determine the diversity of thermophilic fungi in these environments. Here, we collected soils samples in this area, plated on agar media conducive for fungal growth, obtained pure cultures, and then employed the method of internal transcribed spacer (ITS) sequencing combined with morphological analysis for identification of thermophilic fungi to the species level. In total, 102 strains were isolated and identified as Rhizomucor miehei, Chaetomium sp., Talaromyces thermophilus, Talaromyces byssochlamydoides, Thermoascus aurantiacus Miehe var. levisporus, Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea flava, Myceliophthora sp. 1, Myceliophthora sp. 2, Myceliophthora sp. 3, and Coprinopsis sp. Two species, T. lanuginosus and S. thermophilum were the dominant species, representing 34.78% and 28.26% of the sample, respectively. Our results indicated a greater diversity of thermophilic fungi in neutral and alkaline geothermal sites than acidic sites around hot springs reported in previous studies. Most of our strains thrived at alkaline growth conditions.