Effect and Related Mechanism of Platelet-Rich Plasma on the Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

Objective: Human adipose-derived stem cells (hADSCs) are ideal seed cells for the regeneration of alveolar bone defects. Platelet-rich plasma (PRP), which is rich in growth factors, promotes tissue repair. The purpose of the present study was to investigate whether PRP promotes the osteogenic differentiation of hADSCs and to perform high-throughput sequencing to explore the possible mechanism. Methods: hADSCs were divided into the three following groups: CON group, OM group, and PRP group. Osteogenesis was detected by Alizarin Red staining on day 14. Total RNA was extracted from the OM and PRP groups for high-throughput sequencing. The target genes of the differentially expressed osteogenic-related miRNAs were predicted, and combined miRNA/mRNA analysis was then performed. The mRNA and protein expression levels of hsa-miR-212-5p, type 1 cannabinoid receptor (CNR1), alkaline phosphatase (ALP), Runx2, osteocalcin (OCN), and collagen 1 A1 (COL1A1) in the OM and PRP groups were detected by qRT-PCR and Western blot analyses. The binding between hsa-miR-212-5p and CNR1 was detected by a dual-luciferase reporter assay. Results: Both the OM and PRP groups exhibited enhanced proliferation of hADSCs, and the differences at 48 h and 72 h were statistically significant (P < 0.05). The PRP group had significantly more calcium nodules than the CON group (P < 0.05). Through high-throughput sequencing analysis, differential miRNA and mRNA expression profiles were obtained. During hADSC osteogenesis, the expression of hsa-miR-212-5p was downregulated, and the expression of CNR1 was upregulated. hsa-miR-212-5p was found to bind directly to the 3' UTR of CNR1. Conclusions: The present findings indicated that downregulation of hsa-miR-212-5p and upregulation of CNR1 may be involved in the process by which PRP promotes the osteogenic differentiation of hADSCs.

Discovery of a Novel Small-Molecule Inhibitor Disrupting TRBP-Dicer Interaction against Hepatocellular Carcinoma via the Modulation of microRNA Biogenesis.

MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.

Discovery of diarylheptanoids that activate α7 nAchR-JAK2-STAT3 signaling in macrophages with anti-inflammatory activity in vitro and in vivo.

Acute inflammatory diseases, such as sepsis, are life-threatening illnesses. Regulating the α7 nicotinic acetylcholine receptor (α7 nAchR)-mediated signaling may be a promising strategy to treat sepsis. Diarylheptanoids have long been found to exhibit anti-inflammatory properties. However, the possible mechanism of diarylheptanoids has rarely been investigated. In this study, we isolated and synthesized 49 diarylheptanoids and analogues and evaluated their anti-inflammatory activities. Among them, compounds 28 and 40 markedly blocked lipopolysaccharide (LPS)-induced production of nitric oxide (NO), interleukin-1ß (IL-1ß) and interleukin-6 in murine RAW264.7 cells. Furthermore, compounds 28 and 40 also effectively attenuated LPS-induced sepsis, acute lung injury, and cytokines release in vivo. Mechanistically, compounds 28 and 40 significantly induced phosphorylation of janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling and suppression of nuclear factor-κB (NF-κB) pathway. Furthermore, blocking α7 nAchR could effectively abolish compounds 28 and 40-mediated activation of JAK2-STAT3 signaling as well as inhibition of NF-κB activation and NO production in LPS-exposed RAW264.7 cells. Collectively, our findings have identified a new diarylheptanoid, compound 28, as an agonist of α7 nAchR-JAK2-STAT3 signaling, which can be potentially developed as a valuable candidate for the treatment of sepsis, and provide a new lead structure for the development of anti-inflammatory agents targeting α7 nAchR-JAK2-STAT3 signaling.

Identification of a Novel TAR RNA-Binding Protein 2 Modulator with Potential Therapeutic Activity against Hepatocellular Carcinoma.

Imbalance miRNAs contribute to tumor formation; therefore, the development of small-molecule compounds that regulate miRNA biogenesis is an important strategy in oncotherapy. Here, (-)-Gomisin M1 (GM) was found to modulate miRNA biogenesis to inhibit the proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells. GM modulated expression profiles of miRNA and protein in HCC cells and suppressed tumor growth in a mouse model. Mechanistically, GM affected miRNA maturation by targeting TAR RNA-binding protein 2 (TRBP), with an efficacy higher than that of enoxacin, and promoted the binding of TRBP with Dicer. Structural simplification and a preliminary structure-activity relationship study via the synthesis of 20 GM derivatives showed that compound 9 exhibited more potent inhibitory activity in HCC cell proliferation and affinity for TRBP than did GM. These results suggest that TRBP may be a novel potential therapeutic target in HCC and compound 9 may be a potential drug candidate for the treatment of HCC.

Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway.

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3', 5'-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Salinomycin promotes T-cell proliferation by inhibiting the expression and enzymatic activity of immunosuppressive indoleamine-2,3-dioxygenase in human breast cancer cells.

Indoleamine 2,3 dioxygenase (IDO) is upregulated in many tumor types, including breast cancer, and plays a reputable role in promoting tumor immune tolerance. The importance of the immunosuppressive mechanism of IDO by suppressing T-cell function has garnered profound interest in the development of clinical IDO inhibitors. Herein, we established a screening method with cervical HeLa cells to induce IDO expression using interferon-γ (IFN-γ). After screening our chemical library, we found that salinomycin potently inhibited IFN-γ-stimulated kynurenine synthesis with IC50 values of 3.36-4.66 µM in both human cervical and breast cancer cells. Salinomycin lowered the IDO1 and IDO2 expression with no impact on the expression of tryptophan-2,3-dioxygenase. Interestingly, salinomycin potently repressed the IDO1 enzymatic activity by directly targeting the proteins in cells. Molecular docking revealed an alignment that favors nucleophilic attack of salinomycin in the catalytic domain of IDO1. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway by IFN-γ was significantly suppressed by salinomycin, via inhibiting the Jak1, Jak2, and STAT1/3 phosphorylation. Moreover, it inhibited IFN-γ-induced activation of the nuclear factor (NF)-κB pathway by inhibiting IκB degradation and NF-κB phosphorylation without affecting BIN1 expression. Furthermore, salinomycin significantly restored the proliferation of T cells co-cultured with IFN-γ-treated breast cancer cells and potentiated antitumor activity of cisplatin in vivo. These findings suggest that salinomycin suppresses kynurenine synthesis by inhibiting the catalytic activity of IDO1 and its expression by inhibiting the JAK/STAT and NF-κB pathways. Salinomycin warrants further investigation as a novel dual-functional IDO inhibitor for cancer immunotherapy.

Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus Penicillium sclerotiorum cib-411.

Nine new azaphilone alkaloids, penazaphilones A-I (1-9), were isolated from the solid fermented rice culture of Penicillium sclerotiorum cib-411. The structures of compounds 1-9 were elucidated based on HRESIMS, NMR, and CD spectroscopic data. The structures of 5 and 8 were confirmed by X-ray crystallographic analyses. Biological evaluation showed that compounds 1, 5, 6, and 8 inhibited the production of nitric oxide (NO) on RAW 264.7 cells stimulated by lipopolysaccharide with IC50 values of 15.29, 9.34, 9.50, and 7.05 µM, respectively. Meanwhile, they did not exhibit obvious cytotoxicity at a concentration of 50.0 µM.

Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D.

BACKGROUND: Drugs that inhibit the MEK/ERK pathway have therapeutic benefit in bladder cancer treatment but responses vary with patients, for reasons that are still not very clear. Interferon-α (IFN-α) is also used as a therapeutic agent for bladder cancer treatment but the response rate is low. It was found that IFN-α could enhance the cytotoxic effect of MEK inhibition. However, the potential mechanisms of that are still unclear. Understanding of the cross-talk between the IFN-α and MEK/ERK pathway will help enhance the efficacy of IFN-α or MEK inhibitors on bladder cancer. METHODS: Immunoprecipitation and pull-down assay were used to reveal the formation of signaling complex. The protein expressions were detected by western blot and immunohistochemistry. The cAMP level, Phosphodiesterase 4D (PDE4D) activity and Prostaglandin E2 (PGE2) concentration in cells, serum and tissues were detected by enzyme-linked immunosorbent assay. The role of PDE4D in bladder tumorigenesis in vivo was examined by the xenograft model. Tissue microarray chips were used to investigate the prognostic roles of PDE4D and tumor progression locus 2 (TPL2) in bladder cancer patients. RESULTS: IFN-α down-regulated the cyclooxygenase-2 (COX-2) expression in bladder cancer cells through the inhibition of TPL2/NF-κB pathway; IFN-α also inhibited COX-2 expression by suppressing cAMP signaling through TPL2-ERK mediated PDE4D activity. Reduction of the intracellular cAMP level by PDE4D potentiated the antitumor effect of IFN-α against bladder cancer in vitro and in vivo. Further analysis of clinical samples indicated that low PDE4D expression and high level of TPL2 phosphorylation were correlated to the development and poor prognosis in bladder cancer patients. CONCLUSIONS: Our data reveal that IFN-α can exert its antitumor effect through a non-canonical JAK-STAT pathway in the bladder cancer cells with low activity of IFN pathway, and the TPL2 inhibition is another function of IFN-α in the context of bladder cancer therapy. The antitumor effects of IFN-α and MEK inhibition also depend on the PDE4D-mediated cAMP level in bladder cancer cells. Suppression of the TPL2 phosphorylation and intracellular cAMP level may be possible therapeutic strategies for enhancing the effectiveness of IFN-α and MEK inhibitors in bladder cancer treatment.

Lambertellin from Pycnoporus sanguineus MUCL 51321 and its anti-inflammatory effect via modulation of MAPK and NF-κB signaling pathways.

Lambertellin (1) and ergosta-5,7,22-trien-3-ol (2) were isolated from the solid rice fermentation of the plant pathogenic fungus Pycnoporus sanguineus MUCL 51321. Their structures were elucidated using comprehensive spectroscopic methods. The isolated compounds were tested on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Lambertellin (1) exhibited promising inhibitory activity against nitric oxide (NO) production with IC50 value of 3.19 µM, and it significantly inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Lambertellin (1) also decreased the expression of pro-inflammatory cytokines IL-6 and IL-1ß. The study of the mechanistic pathways revealed that lambertellin (1) exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophage cells by modulating the activation of the mitogen activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. Therefore, lambertellin (1) could be a promising lead compound for the development of new anti-inflammatory drugs.

Pretreatment of 2,4-dinitroanisole (DNAN) producing wastewater using a combined zero-valent iron (ZVI) reduction and Fenton oxidation process.

A combined zero-valent iron (ZVI) reduction and Fenton oxidation process was tested for the pretreatment of 2,4-dinitroanisole (DNAN) producing wastewater. Operating conditions were optimized and overall performance of the combined process was evaluated. For ZVI process, almost complete reduction of nitroaromatic compounds was observed at empty bed contact time (EBCT) of 8h. For Fenton process, the optimal pH, H2O2 to Fe(II) molar ratio, H2O2 dosage and hydraulic retention time (HRT) were found to be 3.0, 15, 0.216 mol/L and 5h, respectively. After pretreatment by the combined ZVI-Fenton process under the optimal conditions, aromatic organic compound removal was as high as 77.2%, while the majority of COD remained to be further treated by sequent biological process. The combined anaerobic-aerobic process consisted of an anaerobic baffled reactor (ABR) and a moving-bed biofilm reactor (MBBR) was operated for 3 months, fed with ZVI-Fenton effluent. The results revealed that the coupled ZVI-Fenton-ABR-MBBR system was significantly efficient in terms of correcting the effluent's main parameters of relevance, mainly aromatic compounds concentration, COD concentration, color and acute toxicity. These results indicate that the combined ZVI-Fenton process offers bright prospects for the pretreatment of wastewater containing nitroaromatic compounds.

Reductive transformation and detoxification mechanism of 2,4-dinitrochlorobenzene in combined zero valent iron and anaerobic-aerobic process.

A combined zero valent iron (ZVI) and anaerobic-aerobic process was adopted for the treatment of 2,4-dinitrochlorobenzene (DNCB)-containing wastewater. The transformation pathway, reduction of acute toxicity and enhancement of biodegradability were investigated. After pretreatment by ZVI, DNCB in wastewater could be completely converted into 2,4-diaminochlorobenzene (DACB). The ratio of BOD5/COD increased from 0.005 +/- 0.001 to 0.168 +/- 0.007, while EC(50, 48 hr) (V/V) increased from 0.65% to 5.20%, indicating the enhancement of biodegradability and reduction of acute toxicity with the pretreatment by ZVI. DACB was further dechlorinated to m-phenylenediamine during the anaerobic process using methanol as electron donor, with EC(50, 48 hr) increasing from 5.20% to 48.2%. After the subsequent anaerobic-aerobic process, m-phenylenediamine was degraded completely, with effluent COD of 67.5 +/- 10.8 mg/L. This effluent of the subsequent anaerobic-aerobic process was not toxic to zebrafish. The combined ZVI and anaerobic-aerobic process offers bright prospects for the treatment of chlorinated nitroaromatic compound-containing wastewater.