Simultaneous Detection of Aldehyde Metabolites by Light-Assisted Ambient Ionization Mass Spectrometry.

In the clinic, small-molecule metabolites (SMMs) in blood are highly convincing indicators for disease diagnosis, such as cancer. However, challenges still exist for detection of SMMs due to their low concentration and complicated components in blood. In this work, we report the design of a novel "selenium signature" nanoprobe (Se nanoprobe) for efficient identification of multiple aldehyde metabolites in blood. This Se nanoprobe consists of magnetic nanoparticles that can enrich aldehyde metabolites from a complex environment, functionalized with photosensitive "selenium signature" hydrazide molecules that can react with aldehyde metabolites. Upon irradiation with UV, the aldehyde derivatives can be released from the Se nanoprobe and further sprayed by mass spectrometry through ambient ionization (AIMS). By quantifying the selenium isotope distribution (MS/MS) from the derivatization product, accurate detection of several aldehyde metabolites, including valeraldehyde (Val), heptaldehyde (Hep), 2-furaldehyde (2-Fur), 10-undecenal aldehyde (10-Und), and benzaldehyde (Ben), is realized. This strategy reveals a new solution for quick and accurate cancer diagnosis in the clinic.

Identification of sulfhydryl-containing proteins and further evaluation of the selenium-tagged redox homeostasis-regulating proteins.

Chemoproteomic profiling of sulfhydryl-containing proteins has consistently been an attractive research hotspot. However, there remains a dearth of probes that are specifically designed for sulfhydryl-containing proteins, possessing sufficient reactivity, specificity, distinctive isotopic signature, as well as efficient labeling and evaluation capabilities for proteins implicated in the regulation of redox homeostasis. Here, the specific selenium-containing probes (Se-probes) in this work displayed high specificity and reactivity toward cysteine thiols on small molecules, peptides and purified proteins and showed very good competitive effect of proteins labeling in gel-ABPP. We identified more than 6000 candidate proteins. In TOP-ABPP, we investigated the peptide labeled by Se-probes, which revealed a distinct isotopic envelope pattern of selenium in both the primary and secondary mass spectra. This unique pattern can provide compelling evidence for identifying redox regulatory proteins and other target peptides. Furthermore, our examiation of post-translational modification (PTMs) of the cysteine site residues showed that oxidation PTMs was predominantly observed. We anticipate that Se-probes will enable broader and deeper proteome-wide profiling of sulfhydryl-containing proteins, provide an ideal tool for focusing on proteins that regulate redox homeostasis and advance the development of innovative selenium-based pharmaceuticals.

Tanshinone â ¡A participates in the treatment of endometriosis by regulating adhesion, invasion, angiogenesis and inhibition of PI3K/Akt/mTOR signaling pathway.

Endometriosis (EMs) is a common gynecological disorder characterized by abnormal growth of the endometrial stroma and glands outside the uterus. Tanshinone IIA, the active component of Chinese medicine Danshen (Salvia miltiorrhiza Bge.), has a number of pharmacological effects such as anti­inflammation and anti­oxidation and serves a significant role in the treatment of EMs. In the present study, network pharmacology and experimental validation were used to elucidate the potential mechanism of tanshinone IIA for treating EMs. Several databases were used to collect information on EMs and tanshinone IIA and cross­targets for tanshinone IIA and EMs finally obtained. A total of 64 common targets were found between tanshinone IIA and EMs. Subsequently, a protein­protein interaction network was constructed, a total of 14 core targets were screened for enrichment analysis. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed. The network pharmacology showed that intercellular adhesion molecule (ICAM)­1, MMP­9 and VEGF are the core targets while PI3K/AKT pathway and mTOR pathway are the main signaling pathways through which tanshinone IIA regulates relevant biological processes to intervene in EMs. Finally, the therapeutic role and mechanism of tanshinone IIA on EMs was verified in vivo. Female Sprague­Dawley rats were treated by autologous transplantation to establish EMs. Serum inflammatory factors were detected by enzyme­linked immunosorbent assay (ELISA). The expression of ICAM­1, MMP­9 and VEGF in ectopic endometrial tissues of rats was determined by immunohistochemical. The expression of PI3K/Akt/mTOR pathway­related proteins and genes was detected by western blotting and quantitative PCR. It was found that tanshinone IIA treatment significantly decreased the formation of ectopic endometrium by reducing serum levels of TNF­α and IL­1ß, and down regulating the levels of ICAM­1, MMP­9 and VEGF in ectopic uterine tissue. In addition, tanshinone IIA can also block the activation of PI3K/Akt/mTOR signaling pathway by reducing the expression of related proteins and genes. In conclusion, tanshinone IIA can regulate adhesion, invasion and angiogenesis, thereby improving the pathological morphology of ectopic endometrium and inhibiting the formation of ectopic lesions. The PI3K/Akt/mTOR signaling pathway may play a key role in controlling this process.

Cubosomes-assisted transdermal delivery of doxorubicin and indocyanine green for chemo-photothermal combination therapy of melanoma.

Melanoma is a highly aggressive form of skin cancer with limited therapeutic options. Chemo-photothermal combination therapy has demonstrated potential for effectively treating melanoma, and transdermal administration is considered the optimal route for treating skin diseases due to its ability to bypass first-pass metabolism and enhance drug concentration. However, the stratum corneum presents a formidable challenge as a significant barrier to drug penetration in transdermal drug delivery. Lipid-nanocarriers, particularly cubosomes, have been demonstrated to possess significant potential in augmenting drug permeation across the stratum corneum. Herein, cubosomes co-loaded with doxorubicin (DOX, a chemotherapeutic drug) and indocyanine green (ICG, a photothermal agent) (DOX-ICG-cubo) transdermal drug delivery system was developed to enhance the therapeutic efficiency of melanoma by improving drug permeation. The DOX-ICG-cubo showed high encapsulation efficiency of both DOX and ICG, and exhibited good stability under physiological conditions. In addition, the unique cubic structure of the DOX-ICG-cubo was confirmed through transmission electron microscopy (TEM) images, polarizing microscopy, and small angle X-ray scattering (SAXS). The DOX-ICG-cubo presented high photothermal conversion efficiency, as well as pH and thermo-responsive DOX release. Notably, the DOX-ICG-cubo exhibited enhanced drug permeation efficiency, good biocompatibility, and improved in vivo anti-melanoma efficacy through the synergistic effects of chemo-photothermal therapy. In conclusion, DOX-ICG-cubo presented a promising strategy for melanoma treatment.

Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability.

Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1−30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to −100 °C, and stored at −135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability (p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

Photothermal MnO2 nanoparticles boost chemo-photothermal therapy-induced immunogenic cell death in tumor immunotherapy.

The induction of immunogenic cell death (ICD) is an attractive strategy for generating in situ autologous tumor cell-based vaccines and thus has great potential in cancer prevention and personalized immunotherapy. However, the effectiveness of ICD in tumor immunotherapy has been greatly limited, mainly by low induction efficiency and the immunosuppressive tumor microenvironment (TME). Herein, we report a new strategy for chemo-photothermal therapy-induced ICD by employing photothermal MnO2 nanoparticles loaded with doxorubicin (DOX) in tumor immunotherapy to overcome the low efficiency of traditional ICD inducers and the immunosuppressive TME. Specifically, we prepared photothermal bovine serum albumin (BSA)-templated MnO2 NPs (BSA/MnO2 NPs) with good aqueous dispersibility and high biocompatibility through the direct reduction of KMnO4 with BSA, and we then efficiently loaded DOX, an ICD inducer, onto the MnO2 NPs through coordination (DOX-BSA/MnO2 NPs). The DOX-BSA/MnO2 NPs achieved high photothermal conversion efficiency, highly efficient tumor targeting, TME-responsive DOX release and modulation of the hypoxic TME. Notably, a marked in vivo synergistic therapeutic effect was achieved in a triple-negative breast carcinoma-bearing mouse model by combining chemo-photothermal therapy-induced ICD with amelioration of the immunosuppressive TME. Our research highlights the great promise of modulating the TME with photothermal MnO2 nanosystems to enhance ICD-induced antitumor immunotherapy.

Ferroptosis due to Cystathionine γ Lyase/Hydrogen Sulfide Downregulation Under High Hydrostatic Pressure Exacerbates VSMC Dysfunction.

Hydrostatic pressure, stretch, and shear are major biomechanical forces of vessels and play critical roles in genesis and development of hypertension. Our previous work demonstrated that high hydrostatic pressure (HHP) promoted vascular smooth muscle cells (VSMCs) two novel subsets: inflammatory and endothelial function inhibitory VSMCs and then exacerbated VSMC dysfunction. However, the underlying mechanism remains unknown. Here, we first identified that aortic GPX4 (a core regulator of ferroptosis) significantly downregulated association with VSMC novel phenotype elevation in SHR rats and hypertension patients. In primary VSMCs, HHP (200 mmHg) increased iron accumulation, ROS production, and lipid peroxidation compared with normal pressure (100 mmHg). Consistently, the ferroptosis-related gene (COX-2, TFRC, ACSL4, and NOX-1) expression was also upregulated. The ferroptosis inhibitor ferrostatin-1 (Fer-1) administration blocked HHP-induced VSMC inflammatory (CXCL2 expression) and endothelial function inhibitory (AKR1C2 expression) phenotyping switch association with elevation in the GPX4 expression, reduction in the reactive oxygen species (ROS), and lipid peroxidation production. In contrast, the ferroptosis inducer RLS3 increased HHP-induced CXCL2 and AKR1C2 expressions. These data indicate HHP-triggering ferroptosis contributes to VSMC inflammatory and endothelial function inhibitory phenotyping switch. In mechanism, HHP reduced the VSMC GSH content and cystathionine gamma-lyase (CSE)/hydrogen sulfide (H2S)-an essential system for GSH generation. Supplementation of the H2S donor-NaHS increased the VSMC GSH level, alleviated iron deposit, ROS and lipid peroxidation production. NaHS administration rescues both HHP- and RLS3-induced ferroptosis. Collectively, HHP downregulated VSMC CSE/H2S triggering GSH level reduction, resulting in ferroptosis, which contributed to the genesis of VSMC inflammation and endothelial function inhibitory phenotypes.

Fabrication of a "Selenium Signature" Chemical Probe-Modified Paper Substrate for Simultaneous and Efficient Determination of Biothiols by Paper Spray Mass Spectrometry.

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.

Shikonin overcomes drug resistance and induces necroptosis by regulating the miR-92a-1-5p/MLKL axis in chronic myeloid leukemia.

Development of resistance to tyrosine kinase inhibitors (TKIs) targeting the BCR/ABL fusion protein represents a major challenge in the treatment of chronic myeloid leukemia (CML). Since apoptosis resistance is the fundamental mechanism impeding TKIs' therapeutic effects, alternative approaches that induce nonapoptotic cell death are being pursued to treat TKI-resistant CML. Induction of necroptosis, a distinct, caspase-independent form of programmed cell death, may be a valuable strategy in this respect. The present study shows that shikonin, an herbal compound used in traditional Chinese medicine, overcomes TKI resistance in BCR/ABL-positive CML cells by inducing necroptosis via activation of RIPK1/RIPK3/MLKL signaling. This effect occurs both in vitro and in vivo and involves downregulation of miR-92a-1-5p, a poor-prognosis marker frequently overexpressed in leukemia patients. Based on gene expression experiments, we conclude that miR-92a-1-5p promotes CML progression by inhibiting MLKL expression. Accordingly, we show that antagomiR-mediated in vivo inhibition of miR-92a-1-5p reduces the growth of CML tumors in mice through necroptosis induction. Our research suggests that therapies that relieve MLKL suppression by targeting miR-92a-1-5p may represent a useful strategy to treat TKI-refractory CML.

Acaricidal activity of plant-derived essential oil components against Psoroptes ovis in vitro and in vivo.

BACKGROUND: Treatment of Psoroptes ovis in cattle is limited to topical acaricides or systemic treatment with macrocyclic lactones. Treatment failure of macrocyclic lactones has been reported. The aim of this study was to evaluate a potential alternative treatment against P. ovis. METHODS: The acaricidal activity against P. ovis of four plant-derived essential oil components, i.e. geraniol, eugenol, 1,8-cineol and carvacrol, was assessed in vitro and in vivo. In vitro contact, fumigation and residual bioassays were performed. In addition, 12 Belgium Blue cattle were artificially infested and treated topically once a week for three successive weeks with carvacrol in Tween-80 (treatment group) or with Tween-80 alone (control). The efficacy of carvacrol was determined by the reduction in lesion size and mite counts. Six additional animals were topically treated with carvacrol to assess local adverse reactions. RESULTS: Three components showed a concentration-dependent acaricidal activity in a contact assay, with LC50 of 0.56, 0.38 and 0.26% at 24 h for geraniol, eugenol, and carvacrol, respectively. However, 1,8-cineol showed no activity at any of the tested concentrations in a contact bioassay. In a fumigation bioassay, carvacrol killed all mites within 50 min after treatment, whereas geraniol, eugenol and 1,8-cineol needed 90 to 150 min. Following a 72 h incubation period in a residual bioassay, carvacrol killed all mites after 4 h of exposure to LC90, while geraniol and eugenol killed all mites only after 8 h exposure. Based on these results, carvacrol was further assessed in vivo. Mite counts in the treatment group were reduced by 98.5 ± 2.4% at 6 weeks post-treatment, while in the control group the mite population had increased. Topical application of carvacrol only caused mild and transient erythema 20 min after treatment. No other side effects were observed. CONCLUSIONS: Considering the strong acaricidal activity of carvacrol in vitro and in vivo and the mild and transient local side effects, carvacrol shows potential as an acaricidal agent in the treatment of P. ovis in cattle.

Bu Yang Huan Wu decoction prevents reperfusion injury following ischemic stroke in rats via inhibition of HIF-1 α, VEGF and promotion ß-ENaC expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu Yang Huan Wu Decoction (BYHW) is a famous traditional Chinese medicine (TCM) formula used in China for the treatment of cerebral ischemic stroke. But the protective effects and underlining mechanisms of BYHW remain unclear. AIM OF THE STUDY: This study was designed to investigate the protective effects and underlining signaling mechanisms of BYHW on brain tissues in a rat model of cerebral ischemic reperfusion (I/R) injury. MATERIALS AND METHODS: Liquid chromatography was used to verify the composition of BYHW. The cerebral edema and infarct volume were measured by magnetic resonance imaging (MRI). The morphology and ultrastructure of ischemic penumbra brain tissues were observed by hematoxylin-eosin (HE) and transmission electron microscopy (TEM). The expression levels of HIF-1 α, VEGF and ß-ENaC were tested using immunohistochemistry technique, western blot and quantitative PCR analysis, respectively. RESULTS: Administration of BYHW significantly decreased cerebral edema, rat neurological function scores, reduced brain infarct volume. At the same time, BYHW had protective effect on the blood-brain barrier (BBB), which improved the morphology and ultrastructure of ischemic penumbra brain tissues. BYHW treatment significantly decreased the protein and mRNA levels of HIF-1 α and VEGF compared with the model treatment. In addition, BYHW treatment significantly up-regulated the protein and mRNA levels of ß-ENaC. CONCLUSIONS: BYHW protected against cerebral I/R injury in MCAO rats through inhibiting the activation of the HIF-1 α /VEGF pathway and stabilizing ion channel of ß-ENaC in brain, indicating that BYHW shows potential for stroke treatment in acute stage.

Shikonin exerts antitumor activity in Burkitt's lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR pathway and acts synergistically with doxorubicin.

Burkitt's lymphoma (BL) is a highly aggressive malignancy molecularly characterized by deregulation of the C-MYC proto-oncogene. Recently, it has been confirmed that phosphatidylinositol-3-kinase (PI3K) pathway activation is a crucial element in the malignant transformation of the B cells in BL. Despite the better outcome of adults with BL treated with high-intensity chemotherapy regimens, the overall survival rate for patients older than 60 years remains dismal. Shikonin, a natural naphthoquinone derived from Chinese herbal medicine plant, has the potential to induce cell death in a series of human cancer. In the present study, we investigated the effect and molecular mechanisms of Shikonin in treatment with BL. Shikonin suppressed cellular proliferation and induced caspase-dependent apoptosis in BL cells. Inhibition of C-MYC and suppression of PI3K/AKT/mTOR pathway played critical roles in SHK-induced apoptosis in BL both in vitro and in vivo. Besides, Shikonin potentiated doxorubicin-induced growth inhibition and apoptosis in vitro. Furthermore, the growth of a subcutaneous xenograft tumor model of BL was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice. In summary, these data suggest that shikonin may be an encouraging chemotherapeutic agent in the clinical treatment of BL.

Effect of Hua Yu Xiao Zheng decoction on the expression levels of vascular endothelial growth factor and angiopoietin-2 in rats with endometriosis.

The aims of the present study were to investigate the effects of a traditional Chinese medicine, Hua Yu Xiao Zheng (HYXZ) decoction, on surgically induced endometriosis in a rat model and to determine the possible underlying regulatory mechanisms. A total of 108 female Sprague-Dawley rats were divided into the control group (n=12) and endometriosis group (EM group; n=96), in which endometriosis was surgically induced in model rats by autotransplantation of endometrial tissues and 72 rats survived. After 3 weeks, the EM model rats were randomly divided into four subgroups (n=18), including the untreated model group, and three groups administered 7, 14 or 21 g/kg HYXZ decoction. Following 28 days of treatment, the associated proteins and genes of ectopic endometrial tissues were analyzed using immunohistochemistry, western blotting and quantitative polymerase chain reaction to investigate the underlying mechanisms. Compared with the model group, the size of the endometriotic implants decreased significantly in the HYXZ-treated groups. Furthermore, the expression levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were significantly decreased in HYXZ-treated groups compared with the model group. These results indicate that HYXZ affected the inhibition of angiogenesis and decreased the endometriotic implant volumes and histopathological scores. The effectiveness of HYXZ may be partially attributed to the decrease of VEGF and Ang-2 expression levels in the ectopic endometrium.

Naringenin reduces oxidative stress and improves mitochondrial dysfunction via activation of the Nrf2/ARE signaling pathway in neurons.

Oxidative stress and mitochondrial dysfunction are considered to be major contributing factors in the development and progression of many neurodegenerative diseases. Naringenin (NAR) is an abundant flavanone in the Citrus genus and has been found to exert antioxidant, anticarcinogenic and antimutagenic effects. However, the potential underlying mechanism of its antioxidant effects remains unclear. In the present study, the authors investigated the antioxidant effect of NAR on neurons in vitro. Neurons isolated from the brains of Sprague-Dawley rats were randomly divided into a control group, model group, NAR-L group, NAR-M group and NAR-H group. The model group received hypoxia and re-oxygenation treatment, and the NAR-L, NAR-M and NAR-H groups received 20, 40 and 80 µM NAR, respectively. The levels of reactive oxygen species (ROS) in each group were detected by chloromethyl-2',7'dichlorodihydro fluorescein diacetate staining, and differences in mitochondrial dysfunction were analyzed through measurement of mitochondrial membrane potential (∆ψm), adenine nucleotide translocase transport activity and adenine nucleotide levels. MTT and flow cytometry assays were also used to analyze cell proliferation and apoptosis, and the effects of NAR on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway were investigated using small interfering RNA methods. The authors detected an increased accumulation of ROS in the model group, and high-dose NAR could significantly reduce the levels of ROS. Furthermore, NAR could improve mitochondrial dysfunction, as indicated by increased levels of high-energy phosphates, enhanced mitochondrial ANT transport activity and increased mitochondrial membrane potential. Moreover, NAR increased cell viability and decreased the rate of cell apoptosis. NAR also increased the expression of Nrf2 and its downstream target genes. These findings demonstrated that NAR could reduce oxidative stress and improve mitochondrial dysfunction via activation of the Nrf2/ARE signaling pathway in neurons.

Effects of Bu Shen Yi sui capsule on NogoA/NgR and its signaling pathways RhoA/ROCK in mice with experimental autoimmune encephalomyelitis.

BACKGROUND: Axon growth inhibitory factors NogoA/Nogo receptor (NgR) and its signaling pathways RhoA/Rho kinase (ROCK) play a critical role in the repair of nerve damage in multiple sclerosis (MS). Bu Shen Yi Sui Capsule (BSYSC) is an effective Chinese formula utilized to treat MS in clinical setting and noted for its potent neuroprotective effects. In this study, we focus on the effects of BSYSC on promoting nerve repair and the underlying mechanisms in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: The EAE mouse model was induced by injecting subcutaneously with myelin oligodendrocyte glycoprotein (MOG) 35-55 supplemented with pertussis toxin. BSYSC was orally administrated at dose of 3.0 g/kg once a day for 40 days. The levels of protein gene product (PGP) 9.5, p-Tau, growth associated protein (GAP) -43, KI67 and Nestin in the brain or spinal cord on 20 and 40 day post-induction (dpi) were detected via immunofluorescence and Western blot analysis. Furthermore, NogoA/NgR and RhoA/ROCK signaling molecules were studied by qRT-PCR and Western blot analysis. RESULTS: Twenty or 40 days of treatment with BSYSC increased markedly PGP9.5 and GAP-43 levels, reduced p-Tau in the brain or spinal cord of mice with EAE. In addition, BSYSC elevated significantly the expression of KI67 and Nestin in the spinal cord 40 dpi. Further study showed that the activation of NogoA/NgR and RhoA/ROCK were suppressed by the presence of BSYSC. CONCLUSIONS: BSYSC could attenuate axonal injury and promote repair of axonal damage in EAE mice in part through the down-regulation of NogoA/NgR and RhoA/ROCK signaling pathways.

You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui pills (YGPs) are an effective traditional Chinese formula being used clinically for the treatment of multiple sclerosis (MS). Previous studies demonstrated that YGPs exerted the potent neuroprotective effects in murine models of experimental autoimmune encephalomyelitis (EAE), which is an equivalent animal model for multiple sclerosis (MS). However, the mechanism of YGPs functions remained unclear. AIM OF THIS STUDY: The aim of this study was to evaluate the therapeutic effect of YGPs in MOG35-55-induced EAE mice and to further elucidate the underlying molecular mechanism. METHODS: Female C57BL/6 mice were divided into six groups, including the non-treated EAE model, prednisone acetate- and 1.2, 2.4 or 4.8g/kg YGPs-treated EAE groups, and a normal control group. The EAE model was established by injecting the mice subcutaneously with MOG35-55 antigen. The body weights were measured and the neurological functions were scored in each group. The pathology and morphology of the brain and spinal cord was examined. The expression of MAP-2 was detected by immunofluorescent staining. The levels of netrin1, DCC, RhoA, Rac1, and Cdc42 were assayed by immunohistochemistry, qRT-PCR and Western blot on day 40 post-immunization (PI). RESULTS: YGPs treatments significantly reduced neurological function scores in EAE mice, where the inflammatory infiltration was reduced and the axon and myelin damage in both brain and spinal cord was alleviated. In the brain and spinal cord tissues, YGPs increased the expression of neuronal factors MAP-2, netrin1 and DCC. The expression of Rac1 and Cdc42 were increased, while RhoA was reduced following YGPs treatments. CONCLUSION: Our results demonstrated that YGPs exhibited a neuroprotective effect on promoting nerve regeneration at the brain and spinal cord in EAE mice induced by MOG35-55. Netrin1, DCC and the Rho family GTPases of RhoA, Racl, Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.

Acute and subchronic toxicity as well as evaluation of safety pharmacology of traditional Chinese medicine "Huhezi".

The study was conducted to evaluate the toxicity and safety pharmacology of the traditional Chinese medicine, "Huhezi" granules. The results of acute toxicity test showed that the granules' LD50 was more than 5000 mg/kg, which indicated that the "Huhezi" belonged to actually non-toxic drug. Subchronic toxicity study showed that non-toxic reaction were detected in high (1000 mg/kg), medium (500 mg/kg) and low dose (250 mg/kg) of "Huhezi" groups by measuring rat body weight, organ coefficient, blood physiological indexes and blood biochemical indexes. Pathological examination showed that no tissue lesions were observed in test organs except liver (mild granular degenerationand reversible vesicular degeneration), spleen (Langerhans cells infiltrating) and kidney (homogeneous red staining of renal tubule). Safety pharmacology study found that "Huhezi" had no effects on the central nervous system, respiratory system and cardiovascular system. These results suggested that the dose of "Huhezi" at or below 1000 mg/kg through oral administration is considered safe.

Pure total flavonoids from Citrus paradisi Macfadyen act synergistically with arsenic trioxide in inducing apoptosis of Kasumi-1 leukemia cells in vitro.

To investigate the potential effects of pure total flavonoid compounds (PTFCs) from Citrus paradisi Macfadyen separately or combined with arsenic trioxide on the proliferation of human myeloid leukemia cells and the mechanisms underlying the action of PTFCs. The effects of PTFCs separately or combined with arsenic trioxide on the proliferation and apoptosis of leukemia cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescence microscopy, and flow cytometry. Their effects on the expression levels of apoptosis-related regulators were determined by Western blot assay. PTFCs combined with arsenic trioxide significantly inhibited the growth of Kasumi-1 cells, and apoptosis was confirmed by flow cytometry analysis. Hoechst 33258 staining showed more significant morphological changes and more apoptosis following the combined treatment. Western blots showed changes in the expression of genes for poly ADP-ribose polymerase (PARP), caspase 3/9, and P65. The results indicated that PTFCs separately or combined with arsenic trioxide inhibited proliferation of leukemia cells in vitro and induced their apoptosis by modulating the expression of apoptosis-related regulator genes.

In vitro acaricidal activity of 1,8-cineole against Sarcoptes scabiei var. cuniculi and regulating effects on enzyme activity.

1,8-Cineole found in many essential oils is a monoterpene and acts as a repellent against Sarcoptes scabiei var. cuniculi. In the present study, the acaricidal activity of 1,8-cineole against S. scabiei var. cuniculi was evaluated and the acaricidal mechanism was also investigated by assaying enzyme activities. The results showed that the lethal concentration of 50% (LC50) value (95% confidence limit (CL)) and the lethal time of 50% (LT50) value (95% CL) of 1,8-cineole were 2.77 mg/mL and 3.606 h, respectively. The pathological changes under transmission electron microscopy showed that the morphology of the mitochondria was abnormal, the cell nuclear membrane was damaged, and the nuclear chromatin was dissoluted. The activities of superoxide dismutase (SOD), glutathione-s-transferases (GSTs), monoamine oxidase (MAO), nitric oxide synthase (NOS), and acetylcholinesterase (AChE) were significantly changed after treatment with 1,8-cineole for 4, 8, 12, and 24 h. SOD and GSTs are associated with the protection mechanism of scabies mites. And, the activities of SOD and GSTs were increased as compared with the control group. MAO, AChE, and NOS are associated with the nervous system of scabies mites. The activity of MAO was increased whereas the AChE was suppressed. The activity of NOS was suppressed in the high-dose group whereas increased in the middle-dose group and low-dose group. These results indicated that the mechanism of 1,8-cineole mainly attributed to the changes of these enzyme activities related to the nervous system of scabies mites.

Microvascular density and vascular endothelial growth factor and osteopontin expression during the implantation window in a controlled ovarian hyperstimulation rat model.

Vascular endothelial growth factor (VEGF) and osteopontin (OPN) are suggested to facilitate angiogenesis and vascular remodeling in endometrial receptivity. Determination of the endometrial microvascular density (MVD) is the commonest method used to indirectly assess the levels of vasculogenesis and angiogenesis; however, the associations among VEGF, OPN and MVD remain unclear. Controlled ovarian hyperstimulation (COH) with the gonadotrophin-releasing hormone agonist-long protocol may impair endometrial receptivity, and Traditional Chinese Medicine (TCM) may exert therapeutic effects to relieve this impairment. The aim of the present study was to investigate the effects of COH on implantation biology and pregnancy outcome, and to explore the potential therapeutic role of the TCM Zi Dan Yin (ZDY). Female Sprague Dawley rats were divided into four groups: Control, COH, ZDY and COH + ZDY. On days 3, 4 and 5 of pregnancy (D3, D4 and D5, respectively), endometrial MVD was measured with cluster of differentiation 34 immunohistochemical detection, and VEGF and OPN protein and mRNA expression was detected through western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. On D10, the average number of implantation sites was observed. Subsequent to conceiving and bearing newborn rats, the number of live births from each group was recorded. COH was shown to have adverse effects on implantation and pregnancy outcome. The MVD was found to be significantly increased in the COH group compared with that in the control, ZDY and COH+ZDY groups. The results of the protein and RT-qPCR analysis of VEGF and OPN revealed the same trend. Conversely, ZDY reversed the changes in endometrial MVD, VEGF and OPN, and was indicated to improve uterine receptivity and pregnancy outcome. No significant difference was observed among the control, ZDY and COH + ZDY groups. In conclusion, since the results for MVD and VEGF and OPN expression were consistent, MVD could be used as an alternative approach to identify the period of receptivity in rats.