S1PR1 regulates ovarian cancer cell senescence through the PDK1-LATS1/2-YAP pathway.

Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

ABSTRACT: Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

[Acupuncture regulates PI3K/Akt/mTOR signaling and up-regulates cell autophagy to protect sy-novial tissue of adjuvant arthritis rats].

OBJECTIVE: To observe the effect of acupuncture of "Yinlingquan"(SP9) and "Sanyinjiao"(SP6) on expression of phosphatidylinositol-3 kinase/protein kinase B/mammalian target protein of rapamycin (PI3K/Akt/mTOR) signaling in adjuvant arthritis (AA) rats, so as to explore its mechanism underlying improvement of AA. METHODS: Forty-eight male Wistar rats were randomly divided into normal control, AA model, acupuncture and medication (tripterygium wilfordii) groups, with 12 rats in each group. The AA model was established by putting the rats in a windy, cold and wet environment for 12 h, once every day for 21 days and injection of Freund's complete adjuvant (CFA) into the sole of the right hindlimb on the 21st day. Manual acupuncture stimulation was applied at SP9 and SP6 for 30 min/time, once a day for 21 days. Rats of the medication group received gavage of tripterygium wilfordii tablets solution (8 mg/kg), once a day for 21 days, and those of the normal control group and model group received gavage of the same amount of normal saline, once a day for 21 days. The degree of joint swelling and arthritis index (AI) were detected 1 day before modeling, 3 days after modeling, and 21 days after the treatment. Twenty-four hours after the last treatment, the contents of serum cytokines interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA); and changes of synovial ultrastructure were observed under electron microscope. Western blot was used to detect the expression levels of PI3K, Akt, phosphorylated protein kinase B (p-Akt), phosphorylated mammalian rapamycin target protein (p-mTOR), mTOR, microtubule associated protein 1 light chain 3B (LC3-Ⅱ) and mammalian atg6 homologous protein (Beclin-1) in the synovial membrane tissue. RESULTS: Compared with the normal control group, the joint swel-ling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in the synovium were increased in the model group (P<0.05), while the expression levels of LC3-Ⅱ and Beclin-1 proteins in the synovium were significantly decreased (P<0.05). Compared with the model group, the joint swelling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR were significantly decreased in the acupuncture and medication groups (P<0.05), while the expresion levels of LC3-Ⅱ and Beclin-1 proteins were significantly increased （P<0.05）. Comparison between the two treatment groups showed that the therapeutic effects of acupuncture were obviously weaker than those of medication in down-regulating TNF-α and IL-6, Akt, p-Akt and mTOR levels (P<0.05) and in up-regulating Beclin-1 expression (P<0.05). Outcomes of electron microscope displayed widened nuclear membrane space, some fractured mitochondrial cristae with vacuoles, expanded rough endoplasmic reticulum, reduction of autophagosomes in the cytoplasm and ruptured synovial cell membrane in the model group, and increase of autophagosomes, deformed organelles in the acupuncture and medication groups. CONCLUSION: Acupuncture can relieve the inflammatory reactions and joint synovial injury of the affected joint in AA rats which may be associated with its effects in inhibiting PI3K/Akt/mTOR signaling and increasing the auto-phagy level of synovial cells.

Identification of a Strong Anthocyanin Activator, VbMYBA, From Berries of Vaccinium bracteatum Thunb.

Wufanshu (Vaccinium bracteatum Thunb.), which is a wild member of the genus Vaccinium, accumulates high concentration of anthocyanin in its berries. In this study, the accumulated anthocyanins and their derivatives in Wufanshu berries were identified through UHPLC-MS/MS analysis. Candidate anthocyanin biosynthetic genes were identified from the transcriptome of Wufanshu berries. qRT-PCR analyses showed that the expression of anthocyanin structural genes correlated with anthocyanin accumulation in berries. The R2R3-MYB, VbMYBA, which is a homolog of anthocyanin promoting R2R3-MYBs from other Vaccinium species, was also identified. Transient expression of VbMYBA in Nicotiana tabacum leaves confirmed its role as an anthocyanin regulator, and produced a higher anthocyanin concentration when compared with blueberry VcMYBA expression. Dual-luciferase assays further showed that VbMYBA can activate the DFR and UFGT promoters from other Vaccinium species. VbMYBA has an additional 23 aa at the N terminus compared with blueberry VcMYBA, but this was shown not to affect the ability to regulate anthocyanins. Taken together, our results provide important information on the molecular mechanisms responsible for the high anthocyanin content in Wufanshu berries.

Tanshinone IIA alleviates brain damage in a mouse model of neuromyelitis optica spectrum disorder by inducing neutrophil apoptosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD), an autoimmune astrocytopathic disease associated with the anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions primarily located on the optic nerves and spinal cord. Tanshinone IIA (TSA), an active natural compound extracted from Salvia miltiorrhiza Bunge, has profound immunosuppressive effects on neutrophils. OBJECTIVE: The present study aimed to evaluate the effect of TSA on NMOSD mice and explore the underlying mechanisms. Mice were initially administered TSA (pre-TSA group, n = 20) or vehicle (vehicle group, n = 20) every 8 h for 3 days, and then NMOSD model was induced by intracerebral injection of NMOSD-immunoglobulin G (NMO-IgG) and human complement (hC). In addition, post-TSA mice (n = 10) were administered equal dose of TSA at 8 h and 16 h after model induction. At 24 h after intracerebral injection, histological analysis was performed to assess the inhibitory effects of TSA on astrocyte damage, demyelination, and neuroinflammation in NMOSD mice, and western blotting was conducted to clarify the effect of TSA on the NF-κB and MAPK signaling pathways. Furthermore, flow cytometry and western blotting were conducted to verify the proapoptotic effects of TSA on neutrophils in vitro. RESULTS: There was a profound reduction in astrocyte damage and demyelination in the pre-TSA group and post-TSA group. However, prophylactic administration of TSA induced a better effect than therapeutic treatment. The number of infiltrated neutrophils was also decreased in the lesions of NMOSD mice that were pretreated with TSA. We confirmed that prophylactic administration of TSA significantly promoted neutrophil apoptosis in NMOSD lesions in vivo, and this proapoptotic effect was mediated by modulating the caspase pathway in the presence of inflammatory stimuli in vitro. In addition, TSA restricted activation of the NF-κB signaling pathway in vivo. CONCLUSION: Our data provide evidence that TSA can act as a prophylactic agent that reduces NMO-IgG-induced damage in the mouse brain by enhancing the resolution of inflammation by inducing neutrophil apoptosis, and TSA may serve as a promising therapeutic agent for neutrophil-associated inflammatory disorders, such as NMOSD.

P2X receptors and trigeminal neuralgia.

There is currently no effective cure for trigeminal neuralgia (TN) - a relatively common disease that causes long-term pain in patients. Previous research has shown that ionotropic ATP signaling through excitatory and calcium-permeable P2X receptor channels plays a critical role in pathological pain generation and maintenance. In this paper, we review several hypotheses on the pathogenic mechanisms underlying TN. We further discuss pathways or agents that can target P2X expression in TN, thereby affecting pain induction and maintenance.

Effects of 1,8-cineole on neuropathic pain mediated by P2X2 receptor in the spinal cord dorsal horn.

As an intractable health threat, neuropathic pain is now a key problem in clinical therapy, which can be caused by lesions affecting the peripheral nervous systems. 1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. Research has shown that 1,8-cineole inhibits P2X3 receptor-mediated neuropathic pains in dorsal root ganglion. The P2X2 and P2X3 receptors participate in the transmission of algesia and nociception information by primary sensory neurons. In the present study, We thus investigated in the spinal cord dorsal horn whether 1,8-cineole inhibits the expression of P2X2 receptor-mediated neuropathic pain. This study used rats in five random groups: group of chronic constriction injury(CCI) with dimethysulfoxide control (CCI + DMSO); group of CCI; sham group(Sham); group of CCI treated with a low dose 1,8-cineole (CCI + 50 mg/kg); group of CCI with a high dose (CCI + 100 mg/kg). We observed the effects of 1,8-cineole on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). We examined P2X2 receptors mRNA change in rat spinal cord dorsal horn by In situ nucleic acid hybridization(ISH) and Quantitative realtime polymerase chain reaction (qRT-PCR) methods. Western Blotting and Immunohistochemical staining methods were used to observe P2X2 receptor protein expressions in the rat spinal cord dorsal horn. It demonstrated that oral administration of 1,8-cineole inhibits over-expression of P2X2 receptor protein and mRNA in the spinal cord and dorsal horn in the CCI rats. And the study explored new methods for the prevention and treatment of neuropathic pain.

Microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in dorsal root ganglia and neuropathic pain.

Schwann cell transplantation is a promising method to promote neural repair, and can be used for peripheral nerve protection and myelination. Microcapsule technology largely mitigates immune rejection of transplanted cells. We previously showed that microencapsulated olfactory ensheathing cells can reduce neuropathic pain and we hypothesized that microencapsulated Schwann cells can also inhibit neuropathic pain. Rat Schwann cells were cultured by subculture and then microencapsulated and were tested using a rat chronic constriction injury (CCI) neuropathic pain model. CCI rats were treated with Schwann cells or microencapsulated Schwann cells and were compared with sham and CCI groups. Mechanical withdrawal threshold and thermal withdrawal latency were assessed preoperatively and at 1, 3, 5, 7, 9, 11 and 14 days postoperatively. The expression of P2X3 receptors in L4-5 dorsal root ganglia of the different groups was detected by double-label immunofluorescence on day 14 after surgery. Compared with the chronic constriction injury group, mechanical withdrawal threshold and thermal withdrawal latency were higher, but the expression of P2X3 receptors was remarkably decreased in rats treated with Schwann cells and microencapsulated Schwann cells, especially in the rats transplanted with microencapsulated Schwann cells. The above data show that microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in L4-5 dorsal root ganglia and neuropathic pain.

1,8-cineole decreases neuropathic pain probably via a mechanism mediating P2X3 receptor in the dorsal root ganglion.

1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. The therapeutic effects of 1,8-cineole on neuropathic pain and the molecular mechanisms of its pharmacological actions remain largely unknown. In the present study, we investigated the analgesic mechanisms of orally administered 1,8-cineole in a rat model of chronic constriction injury (CCI) and examined the drug-induced modulation of P2X3 receptor expression in dorsal root ganglia. The mechanical withdrawal threshold and thermal withdrawal latency were measured in rats to assess behavioural changes 7 and 14 days after CCI surgery. Changes in P2X3 receptor mRNA expression of L4-5 dorsal root ganglia were analysed using quantitative real-time polymerase chain reaction at the 7th and 14th postoperative day. Additionally, we examined the expression of P2X3 receptor protein in L4-5 dorsal root ganglia 7 and 14 days after surgery using immunohistochemistry and western blots. We found that 1,8-cineole can alleviate pathological pain caused by P2X3 receptor stimulation and explored new methods for the prevention and treatment of neuropathic pain.

Identification of candidate genes involved in anthocyanin accumulation in the peel of jaboticaba (Myrciaria cauliflora) fruits by transcriptomic analysis.

Jaboticaba is a grape-like fruit that accumulates high levels of anthocyanins in the peel and is proposed as a good source of functional pigments. However, the molecular mechanisms underlying anthocyanin accumulation in jaboticaba peel remains to be elucidated. In this study, we employed RNA-seq technique to compare the transcriptomic differences between green-colored and black-colored jaboticaba peels. Over 5 million high-quality reads were assembled into 62,190 unigenes with an average length of 737 bp, 29,320 (47.15%) of them were annotated by public databases. 2152 unigenes were found to be differentially expressed (830 upregulated and 1322 downregulated). Gene ontology analysis and pathway enrichment annotation revealed that 18 differentially expressed genes encode phenylalanine ammonialyase, 4-coumaroyl:CoA-ligase, chalcone synthase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, anthocyanidin synthase, UDP-glucose: flavonoid 3-O-glucosyltransferase, glutathione S-transferase, Cytochrome b5 were associated with anthocyanin biosynthesis. Additionally, 54 differentially expressed transcription factors were identified. Furthermore, the expression of genes involved in biosynthesis and signal transduction of ethylene and abscisic acid were negatively and positively correlated with that of anthocyanin pathway genes and anthocyanin accumulation, respectively. Quantitative reverse transcription PCR analysis of candidate genes showed trends similar to those in the RNA-seq analysis. McMYB, a homolog of AtMYB113, induced anthocyanin accumulation in tobacco leaves when co-infiltrated PsbHLH3. These results will contribute to further understanding of the molecular mechanisms regulating anthocyanin accumulation in jaboticaba peel.

Microencapsulated Schwann cell transplantation inhibits P2X2/3 receptors overexpression in a sciatic nerve injury rat model with neuropathic pain.

Transplantation of Schwann cells (SCs) can promote axonal regeneration and formation of the myelin sheath, reduce inflammation, and promote repair to the damaged nerve. Our previous studies have shown that transplantation of free or micro-encapsulated olfactory ensheathing cells can relieve neuropathic pain. There are no related reports regarding whether the transplantation of micro-encapsulated SCs can alleviate neuropathic pain mediated by P2X2/3 receptors. In the present study, we micro-encapsulated SCs in alginic acid and transplanted them into the region surrounding the injured sciatic nerve in the rat model of chronic constriction injury (CCI). The mechanical withdrawal threshold and thermal withdrawal latency were measured to assess changes in behavior 14 days after the surgery in CCI model rats. Ultrastructural changes in the injured sciatic nerve were assessed using transmission electron microscopy. Co-expression of P2X2/3 receptors with other markers in neurons in the L4-5 dorsal root ganglia (DRG) were assessed using double-label immunofluorescence 14 days after surgery. We determined P2X2/3 mRNA expression and protein level changes in the DRG using quantitative real-time polymerase change reaction technology and Western blotting analysis. We have investigated that the transplantation of micro-encapsulated SCs can alleviate pathological pain caused by P2X2/3 receptor stimulation and explored new methods for the prevention and treatment of neuropathic pain.

Design, synthesis and antifungal activity evaluation of coumarin-3-carboxamide derivatives.

A series of coumarin-3-carboxamides/hydrazides have been designed and synthesized, all the target compounds were evaluated in vitro for their antifungal activity against Botrytis cinerea, Alternaria solani, Gibberella zeae, Rhizoctorzia solani, Cucumber anthrax and Alternaria leaf spot, some of the designed compounds 4a-4g exhibited potential activity in the primary assays, this highlighted by the compounds 4a, 4d, 4e and 4f, EC50 values of which against Rhizoctorzia solani were as low as 1.80 µg/mL, 2.50 µg/mL, 2.25 µg/mL and 2.10 µg/mL, respectively, exhibiting more effective control with that of the positive control than Boscalid. Furthermore, compounds 4a and 4e represented equivalent antifungal activity with Boscalid against Botrytis cinerea.

Biosensor of alkaline phosphatase based on non-fluorescent FRET of Eu3+-doped oxide nanoparticles and phosphorylated peptide labeled with cyanine dye.

Dephosphorylation of biomolecules under the catalysis of alkaline phosphatase (ALP) is a critical physiological process. Abnormal levels of ALP activity have been associated with a number of diseases; thus, a simple and sensitive assay of ALP activity is highly demanded. Herein, to simulate biological conditions, we labeled a hydrosoluble phosphorylated heptapeptide Gly-Pro-Gly-Asn-p-Tyr-Gly-Ala (pGA) with aminated heptamethine cyanine dye (Cy) to give a low fluorescent labeled peptide Cy-pGA. The synthesized Cy-pGA and Eu3+-doped oxide Y0.6Eu0.4VO4 nanoparticles (NPs) were employed respectively as acceptor and donor to in situ form a non-fluorescent Fluorescence Resonance Energy Transfer (FRET) Cy-pGA-NP system, with the help of the strong interaction between Eu3+ ions in the NPs and phosphate group in Cy-pGA. The breaking of the FRET system of Cy-pGA-NP was triggered by the removal of phosphate group in Cy-pGA catalyzed by ALP and resulting in the release of fluorescent Y0.6Eu0.4VO4 NPs. Thus, the formed Cy-pGA-NP as a sensitive sensor can very well respond to the activity of ALP by measuring the time-resolved fluorescent intensity at near-infrared 617 nm (λ ex = 320 nm, delay time 400 µs). This sensor can not only accurately measure the activity of ALP (1-5 mU/mL) in the designed solutions, but it can also be applied to detect the activity of ALP in biological samples, such as cell lysate and human serum, without the interference of autofluorescent background of biosamples and screen ALP inhibitor by a simple mix-and-measure manner. Graphical abstract A biosensor of alkaline phosphatase (ALP) based on non-fluorescent FRET of Eu3+-doped oxide Y0.6Eu0.4VO4 nanoparticles and the phosphorylated heptapeptide labeled with cyanine dye (Cy-pGA).

Microwave-assisted synthesis and antifungal activity of novel fused Osthole derivatives.

Based on the microwave-assisted synthetic protocol developed in our previous work, we have synthesized a series of novel furo[3,2-c]coumarins as fused Osthole derivatives, via the reaction of 4-hydroxycoumarins and ß-ketoesters catalyzed by DMAP. All the target compounds were evaluated in vitro for their antifungal activity against six phytopathogenic fungi, some compounds exhibited potential activity in the primary assays. Especially compounds 6c, 7b, 8b and 8c (shown in Fig. 1) were the most active ones, EC50 values of these four compounds against Colletotrichum capsica, Botrytis cinerea and Rhizoctonia solani were further investigated. 6c was identified as the most promising candidate with the EC50 value at 0.110 µM against Botrytis cinerea and 0.040 µM against Colletotrichum capsica, respectively, representing better antifungal activity than that of the commonly used fungicide Azoxystrobin.

Microwave-assisted Synthesis and antifungal activity of coumarin[8,7-e][1,3]oxazine derivatives.

The synthesis of novel coumarin[8,7-e][1,3]oxazine derivatives through a microwave-assisted three-component one-pot Mannich reaction is described in this study. All the target compounds were evaluated in vitro for their antifungal activity against Botrytis cinerea, Colletotrichum capsici, Alternaria solani, Gibberella zeae, Rhizoctonia solani, and Alternaria mali. The preliminary bioassays showed that 5e, 5m, and 5s exhibited good antifungal activity and the most active compound was 5m with an [Formula: see text] value as low as 2.1 nM against Botrytis cinerea.

Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan.

RAN (Ras-related nuclear protein) plays crucial roles in multiple cellular processes in yeast, animals and plants. Here we present a DlRan gene and its alternative splicing transcripts containing premature terminator codons (PTCs), identified from embryogenic cultures in longan. Multiple alignment and splicing pattern analyses indicated that DlRan-1 transcript harboring PTC was the consequence of alternative splicing. The accumulation of DlRan PTC-containing transcripts increased significantly when the embryogenic calli were treated with the translation inhibitor, cycloheximide, indicating that DlRan-1 may be targeted by NMD. The analysis of expression profiles of DlRan transcripts revealed that differential expression levels of the alternative spliced DlRan transcripts occurred during the development of embryogenic callus, globular-shaped embryos, and cotyledon-shaped embryos, respectively, in the longan somatic embryogenesis, and were in consistent with the embryo development in corresponding wild-type transcripts. The present work offers evidence to speculate that the alternatively spliced PTC-containing transcripts can be functional and may shed light on expression regulation of DlRan during development of the longan somatic embryos.

Chemotherapeutic effects of different paclitaxel plus poldine combination methods for treatment of ovarian carcinoma.

The study aimed to evaluate the curative effects and toxicity of different paclitaxel (PTX) plus poldine chemotherapeutic combination methods for treatment of advanced ovarian carcinoma. A total of 27 patients with ovarian epithelial carcinoma were divided into four groups: A1, taxotere plus poldine intravenous chemotherapy (n=5); A2, taxotere intravenous chemotherapy combined with poldine intraperitoneal chemotherapy (n=7); B1, paclitaxel plus poldine intravenous chemotherapy (n=6); B2, paclitaxel intravenous chemotherapy combined with poldine intraperitoneal chemotherapy (n=9). Toxic side effects were observed after chemotherapy, and the short-term effects were assessed. Some 25 (25/27) cases completed a four-course treatment, the remaining two stopping halfway due to anaphylactic shock. The total effective rate for the A1 Group was 60% (3/5) and that of A2 group was 71.4% (5/7), Figuires for the B1 and B2 groups were 50% (3/6) and 66.7% (6/9), respectively. In comparisons of toxic side reactions, there were significant differences between taxotere groups and paclitaxel groups, and between intravenous chemotherapy alone groups and intravenous plus intraperitoneal combination chemotherapy groups (p<0.05). Chemotherapy of toxol plus poldine was effective in treatment of advanced ovarian cancer, the toxicities of intravenous plus intraperitoneal combination chemotherapy was lower than that of intravenous chemotherapy alone, and the heart toxicity with taxoere was lower than with paclitexal.