SU5416 directly inhibits nNOS enzyme activity in a concentration-dependent manner.

<p>The inhibitory effects of SU5416 on rat cerebellum nNOS were shown in the graph. The IC₅₀ value was also indicated. Each individual point was an average from three independent experiments.</p

SU5416 prevents MPP⁺-induced apoptosis in a concentration-dependent manner.

<p>(A) SU5416, but not VRI, prevented MPP⁺-induced cell death in a concentration-dependent manner. CGNs were treated with SU5416, VRI, EPTU, 7-nitroindazole (7-NI), 1400 W or DMSO (vehicle control) at the indicated concentrations for 2 hours and then exposed to 35 µM MPP⁺. Cell viability was measured by MTT assay at 24 hours after MPP⁺ challenge. (B) SU5416 blocked neuronal loss induced by MPP⁺. CGNs were pre-incubated with or without 20 µM SU5416 and exposed to 35 µM MPP⁺2 hours later. At 24 hour after MPP⁺ challenge, CGNs were assayed with FDA/PI double staining. (C) SU5416 reversed the morphological alteration induced by MPP⁺. CGNs were pre-incubated with or without 20 µM SU5416 and exposed to 35 µM MPP⁺2 hours later. At 24 hour after MPP⁺ challenge, CGNs were assayed with nNOS and Hoechst double staining. (D) The number of apoptotic nuclei with condensed chromatin was counted from representative Hoechst staining photomicrographs and represented as a percentage of the total number of nuclei counted. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *<i>p</i><0.05 and **<i>p</i><0.01 <i>versus</i> MPP⁺ group in (A) or <i>versus</i> control in (D); ^##<i>p</i><0.01 <i>versus</i> MPP⁺ group in (D) (Turkey’s test).</p

SU5416 attenuates the deficit of locomotion behavior on zebrafish larval induced by MPTP.

<p>One dpf zebrafish embryos were treated with 200 µM MPTP for 2 days, and then co-incubated with 10 µM MPTP and SU5416 or VRI at the indicated concentrations for 72 hours, and zebrafish larval co-treated with MPTP and 150 µM L-dopa or 20 µM L-deprenyl were used as positive controls. After treatment, zebrafish were collected to perform locomotion behavior test using Viewpoint Zebrabox system and total distances travelled in 10 min were calculated. Data, expressed as percentage of control, were the mean ± SEM of 12 fish larvae per group from 3-time independent experiments. ^##<i>p</i><0.01 <i>versus</i> control group; **<i>p</i><0.01 <i>versus</i> MPTP group (Turkey’s test).</p

nNOS depletion abolishes the neuroprotective effects of SU5416 against MPP+-induced neuronal death in PC12 cells.

<p>(A) PC12 cells were transfected with pG418-GFP plasmid (vector), pG418-GFP plasmid encoding nNOS ShRNA (ShnNOS) and pG418-GFP plasmid encoding negative control ShRNA (ShNC). The levels of nNOS and β-actin in the cell lysates were analyzed by Western blotting assay by using specific antibodies. (B, C) nNOS depletion abolished the neuroprotective effects of SU5416 against MPP⁺-induced neuronal death in PC12 cells. PC12 cells transfected with vector, ShnNOS, or ShNC were treated with 20 µM SU5416 for 2 hours and then exposed to 1 mM MPP⁺. Cell viability (B) and cytotoxicity (C) were measured at 24 hours after MPP⁺ challenge by MTT and LDH assays, respectively. Data were the mean ± SEM of three separate experiments; **<i>p</i><0.01 <i>versus</i> control; ^##<i>p</i><0.01 <i>versus</i> MPP⁺ group; ^&<i>p</i><0.05 and ^&&<i>p</i><0.01 <i>versus</i> MPP⁺ vector group (Turkey’s test).</p

SU5416 protects against MPTP-induced TH+ region area decrease in zebrafish.

<p>One dpf zebrafish embryos were co-incubated with 200 µM MPTP and SU5416, VRI or 0.3% DMSO (vehicle control) at the indicated concentrations for 2 days. After treatment, zebrafish were collected to perform whole-mount immunohistochemistry. (A) Representative pictures of whole-mount immunostaining of zebrafish brain from different treatment groups. (B) Magnification of diencephalic area of zebrafish larval (indicated by red bracket in Fig. 2A). (C) Statistical analysis of TH⁺ region area in each treatment group (20 fish embryos per group). Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ^##<i>p</i><0.01 <i>versus</i> control; *<i>p</i><0.05 and **<i>p</i><0.01 versus MPTP group (Turkey’s test).</p

Legislative Documents

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Image_1_Identification of Steroidogenic Components Derived From Gardenia jasminoides Ellis Potentially Useful for Treating Postmenopausal Syndrome.jpg

Estrogen-stimulating principles have been demonstrated to relieve postmenopausal syndrome effectively. Gardenia jasminoides Ellis (GJE) is an herbal medicine possessing multiple pharmacological effects on human health with low toxicity. However, the therapeutic effects of GJE on the management of postmenopausal syndrome and its mechanism of action have not been fully elucidated. In this study, network pharmacology-based approaches were employed to examine steroidogenesis under the influence of GJE. In addition, the possibility of toxicity of GJE was ruled out and four probable active compounds were predicted. In parallel, a chromatographic fraction of GJE with estrogen-stimulating effect was identified and nine major compounds were isolated from this active fraction. Among the nine compounds, four of them were identified by network pharmacology, validating the use of network pharmacology to predict active compounds. Then the phenotypic approaches were utilized to verify that rutin, chlorogenic acid (CGA) and geniposidic acid (GA) exerted an estrogen-stimulating effect on ovarian granulosa cells. Furthermore, the results of target-based approaches indicated that rutin, CGA, and GA could up-regulate the FSHR-aromatase pathway in ovarian granulosa cells. The stimulation of estrogen production by rat ovarian granulosa cells under the influence of the three compounds underwent a decline when the follicle-stimulating hormone receptor (FSHR) was blocked by antibodies against the receptor, indicating the involvement of FSHR in the estradiol-stimulating activity of the three compounds. The effects of the three compounds on estrogen biosynthesis- related gene expression level were further confirmed by Western blot assay. Importantly, the MTT results showed that exposure of breast cancer cells to the three compounds resulted in reduction of cell viability, demonstrating the cytotoxicity of the three compounds. Collectively, rutin, chlorogenic acid and geniposidic acid may contribute to the therapeutic potential of GJE for the treatment of postmenopausal syndrome.</p

SU5416 reverses the elevated intracellular NO induced by MPP+ in CGNs.

<p>CGNs were pre-incubated with EPTU, 7-NI or SU5416 at the indicated concentrations for 2 hours, and exposed to 35 µM MPP⁺. Intracellular NO level was measured using DAF-FM diacetate as a probe at 8 hour after MPP⁺ challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **<i>p</i><0.01 <i>versus</i> MPP⁺ group (ANOVA and Dunnett’s test).</p

Anti-angiogenic effects of SU5416 and VRI in zebrafish.

<p>One dpf Tg<i>(fli-1:EGFP)</i> transgenic zebrafish embryos were treated with SU5416, VRI or DMSO (vehicle control) at the indicated concentrations for 2 days. After treatment, intersegmental-vessel formations were observed under fluorescence microscopy. Deficit of blood vessels was indicated by yellow asterisks.</p

SU5416 reduces the expression of nNOS protein elevated by MPP+ in CGNs.

<p>(A) CGNs were pre-treated with 20 µM SU5416 or DMSO (vehicle control) for 2 hours, and then treated with 35 µM MPP⁺ for various durations as indicated. The total proteins were extracted for Western blot analysis with specific iNOS, nNOS and β-actin antibodies. (B) Statistical analysis of nNOS expression in each treatment group. Data are expressed as the ratio to OD values of the corresponding controls. Data, expressed as percentage of control, were the mean ± SEM of five separate experiments; *<i>p</i><0.05 <i>versus</i> MPP⁺ group at the same time (Turkey’s test).</p