Abstract
Six 10-indolyl-cytochalasans, chaetoglobosin F (
Parkinson’s disease (PD) is a common long-term degenerative disorder of the central nervous system. It might be caused by a progressive selective degeneration and loss/death of dopaminergic neurons in the substantia nigra, as well as the decrease of dopamine content in the striatum. 1 There is still no effective drug to cure it radically. 2 It has been recognized that the causes of PD are genetic polymorphism and susceptibility combined with environmental factors. Oxidative stress, mitochondrial dysfunction, proteolysis stress, and immune abnormality eventually lead to the apoptosis of dopaminergic neurons, which increases the susceptibility of PD. 3 Researches showed that apoptosis was the main form of neuronal loss and oxidative stress was one of the major pathogenesis of PD. Natural products and natural product-derived drugs have many advantages in clinical trials. Therefore, neuroprotective natural product with antioxidative stress and antiapoptosis activity would be a potential leading compound for curing PD. 4
Endophytic fungi are an important source of natural products that exhibit a wide range of bioactivities. Cytochalasans are a class of fungal secondary metabolites containing a parent nucleus of hydrogenated isoindolone structure fused with a macrocyclic ring (a carbocycle, a lactone, or a cyclic carbonate). 5 Many cytochalasans have been found in fungi. For example, Buchanan et al have isolated 15 new 10-phenyl-[11]-cytochalasans, a 10-phenyl-22-oxa-[12]-cytochalasan, and 1 known 10-phenyl-[11]-cytochalasan from one Japanese fungus Daldinia sp.. 6 Initially, cytochalasans showed the distinctive effects on the cellular structure and movement in vitro.7,8 Gradually, cytochalasans were found to have various bioactivities, such as cytotoxic, immunomodulatory, anti-inflammatory, antimicrobial, antiparasitic, antiviral, and nematicidal properties.9 -11 More and more scientists realize the potential of cytochalasans as anticancer agents; however, the function of cytochalasans on neuroprotection effect is still neglected.
Our previous studies have reported six 10-indolyl-cytochalasans isolated from Chaetomiun globosum WQ, an endophytic fungus in Imperata cylindrical, 12 and two 10-phenyl-cytochalasans from the endophyte, Phomopsis sp. IFB-E060 in Vatica mangachapoi, respectively. 13 Among them, cytochalasin H was found to be able to inhibit N-methyl-4-phenylpyridinium iodide (MPP+)-induced apoptosis in pheochromocytoma cell line 12 (PC12) cells, which was closely relative to the reduction of the intracellular reactive oxygen species (ROS) level. 14 The protective effect of cytochalasin H to MPP+-damaged PC12 cells implied the potential application of cytochalasans in developing new anti-PD drugs. In order to investigate the antioxidative and neuroprotective effects of cytochalasans furtherly, radical-scavenging activity assay, hydrogen peroxide (H2O2) and MPP+-induced PC12 cell models were applied for these 8 natural cytochalasans in the present study.
Antioxidative capacity of the 8 cytochalasans (ie,
Structures of compounds 1-
Results of In Silico pkCSM Prediction of BBB Property.
BBB, blood–brain barrier.
Except compound
EC50 and Emax of the 8 Cytochalasans for Scavenging DPPH/ABTS Radical, and Their TEAC (
EC50, half maximal effective concentration; Emax, maximum effect; TEAC, trolox equivalent antioxidative capacity; SD, standard deviation; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.
H2O2 can cause oxidative damage and cell death in different types of cells, including PC12 cells.
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The 8 cytochalasans had no cytotoxicity to the PC12 cells (unpublished data) but mostly could protect the PC12 cells from the H2O2-induced damage. Similar to the radical scavenging assay in vitro,
Effects of the 8 Cytochalasans (0.005 μmol/L) for Inhibiting Hydrogen Peroxide-Induced PC12 Cell Damage (
LDH, lactate dehydrogenase; SD, standard deviation; EC50, half-maximal effective concentration; Emax, maximum effect.
ΔΔΔ indicate P < 0.001 compared to the control group; ** and *** indicate P < 0.01 and P < 0.001, respectively compared to the H2O2 model group.
MPP+ causes cellular damage mainly by promoting the formation of radical ROS and leading death of the dopaminergic neurons, which ultimately displays as symptoms of PD.
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Except compounds
Effects of the 8 Cytochalasans (0.025 μmol/L) for Inhibiting MPP+-Induced PC12 Cell Damage (
ND, not determined; EC50, half-maximal effective concentration; Emax, maximum effect; LDH, lactate dehydrogenase; ROS, reactive oxygen species; MPP + , N-methyl-4-phenylpyridinium iodide.
ΔΔΔ indicate P < 0.001 compared to the control group; ** and *** indicate P < 0.01 and P < 0.001, respectively compared to the MPP+ model group.
The previous review outlined some structure–activity relationship of cytochalasans: palladium-catalyzed hydrogenation reduced cytochalasin D skin-irritating effects while retained cytotoxicity; the hydroxy function at C-7 and the C-3 benzyl group was important to biological activity; derivatives with bromo, iodo, or azido functions significantly reduced the effectiveness.
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In this study, the 8 cytochalasans all had a similar parent nucleus of hydrogenated isoindolone structure with an indolyl or phenyl ring substituted at C-10. Various substituents in the parent nucleus and macrocycle should cause a difference in the function of cytochalasans markedly. First, compounds containing epoxy group sometimes had strong bioactivity, and the 6,7-epoxy group was also critical for cytochalasans. Chaetoglobosin F (
In conclusion, 7 among the 8 cytochalasin compounds, chaetoglobosin F (
Experimental
Materials and Chemicals
The rat PC12 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM) was purchased from GIBCO/Invitrogen (Gaithersburg, MD, USA). The standard VE with purify >98.8% was obtained from the Chinese National Institutes For Food and Drug Control (Beijing, China). MPP+ and DPPH were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were bought from AMROSCO LLC (Solon, OH, USA). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co., Ltd (Hangzhou, Zhejiang, China). Penicillin, streptomycin, and ABTS radical cation scavenging activity assay kit were bought from Beyotime Institute of Biotechnology (Shanghai, China). LDH and ROS assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). All the chemicals and reagents were of the analytical grade.
Sample Preparation
Eight cytochalasan compounds (ie,
Assay of DPPH Radical Scavenging Activity
The DPPH radical scavenging activity of 8 cytochalasan compounds was examined via spectrophotometric analysis. 24 Different concentrations (10−6, 10−5, 10−4, 10−3, 10−2, 10−1, and 5 × 10−1 mmol/L) of samples were prepared and standard VE was assayed as the positive control. The solution 0.01% DMSO without the tested sample was used as the blank control. The absorbance was then recorded at 517 nm, and the scavenging activity was expressed as the percentage of scavenged DPPH radical in the assay system. The EC50 (expressed as mmol/L) value, denoting the concentration of the sample required to scavenge 50% DPPH free radicals, was calculated by graphical regression analysis, and Emax (expressed as %) was calculated by Scott Law. 25
Assay of ABTS Radical Cation Scavenging Activity
The ABTS radical cation (ABTS+) scavenging activity of the sample was performed using the method of Cai et al. 26 Different levels of cytochalasan compound samples (10−6, 10−5, 10−4, 10−3, and 10−2 mmol/L) and trolox, an analog of VE, standard solution (6, 3, 2, 1, 0.2, and 0.1 mmol/L) were prepared and assayed under the same conditions. The absorbance of 734 nm was recorded to get the percentage of scavenged ABTS radical curve. Results were expressed in terms of trolox equivalent antioxidative capacity (TEAC), that is, mol trolox/mol. Similar to DPPH assay, EC50 and Emax of ABTS assay were also calculated.
Cell Culture and Treatment
PC12 cells with cell composition and function close to the mesencephalic dopamine neuron have become a common cell model of PD in vitro. 27 PC12 cells were cultured in DMEM containing 10% FBS, 1% streptomycin and penicillin at 37°C in a 5% carbon dioxide humidified NAPCO 5400 incubator (JOUAN, France). The cells in the period of logarithmic phase were used for all experiments. H2O2 and/or MPP+-induced PC12 cell models were established based on our previous work (unpublished data) and other reports. 28
For H2O2-induced model, the cells in 96-well or 24-well plates (1 × 105 cells/mL) were pretreated with the different concentration of test compounds (0.001, 0.0025, 0.005, 0.01, 0.05, 0.1, and 0.2 μmol/L) for 0.5 hours, respectively. Then the sample cells were stimulated with H2O2 solution (final concentration 200 μmol/L) for another 4 hours. DMEM without cells was used as the blank. The solution 0.01% DMSO in DMEM with H2O2 stimulation was used as the H2O2-induced model, while without H2O2 was the control. The positive control chose 1057.6 μmol/L VE.
Similarly, for the MPP+-induced model, the cells were pretreated for 24 hours with a series concentration of cytochalasan compounds (0.005, 0.01, 0.025, 0.05, 0.1, 0.15, and 0.2 μmol/L) as the test groups. The MPP+-damaged experiment was accomplished by stimulating the PC12 cells with 500 μmol/L MPP+ for 48 hours. The cell morphology was observed using an XDS-1B Inverted Biomicroscope (Chongqing Optical Instrument Co., Ltd., Chongqing, China).
The PC12 cells in H2O2 and/or MPP+-induced models were collected and further analyzed the viability by MTT method, while the supernatants were collected for enzyme-linked immunosorbent assay (ELISA) analysis of LDH activity. The MPP+-induced PC12 cells were also determined the ROS levels.
Cell Viability Detection
Toxicity of the tested compounds to PC12 cells, as well as the influence on H2O2 and/or MPP+-induced PC12 cells, was analyzed by the determination of the cell viability using MTT method. 29 PC12 cells with different treatments were inoculated in 96-well culture plates (1 × 105 cells/mL) for 48 hours. Each well was added with 10 µL MTT (5 mg/mL) and then continued to culture 4 hours. After removal of the MTT solution, 150 µL DMSO was added to solubilize the purple formazan crystals produced. The absorbance of each well was measured at 490 nm on a Bio-Tek ELx800 Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). The reader was controlled via Hyper Terminal Applet ELISA software. The cell viability was calculated with the following formula and expressed as a percentage. EC50 and Emax of the inhibition on cell damage were also calculated.
LDH Activity Assay
The tested cytochalasan compounds at the optimal concentration were chosen with LDH assay, 0.005 μmol/L for H2O2-induced PC12 cell model and 0.025 μmol/L for MPP+-induced PC12 cell model. LDH activity in the supernatant was assayed using the ELISA kit. The assay procedure was employed according to the kit protocol booklet instructions. The absorbance was measured at 450 nm on a Bio-Tek ELx800 Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). The reader was controlled via Hyper Terminal Applet ELISA software.
ROS Assay
Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detection kit was used to assess the ROS level in MPP+-induced PC-12 cells. Briefly, the cells were seeded in 96-well plates (2 × 105 cells/mL), treated with different samples to incubate for 24 hours and cultured with 500 mmol/L MPP+ for 48 hours. After washing cells with phosphate buffer saline (PBS), 2 mL PBS and 2 µL DCFH-DA (final concentration 10 μmol/L) were added to each well and reacted for 30 minutes at 37°C. The cells were collected after dissociated, and fluorescence was recorded by a Becton Dickinson (BD) FACS Calibur Flow Cytometry (BD Biosciences, San Jose, CA, USA) with 488 P excitation and 525 P emission filters. The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as fold of the control.
Statistical Analysis
All data presented are mean value ± standard deviation of 3 independent experiments. EC50 and Emax were calculated by SPSS 17.0 and Scott Law, respectively. Figures were obtained using Microsoft Office Excel 2016. SPSS 17.0 was also applied in a one-way analysis of variance. Dunnett test was used to compare with the control group, and the Student–Newman–Keuls test was used to compare among the groups. Differences were considered significant with a P-value <0.05.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Major Program for The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJA350005), Jiangsu Provincial Key Research and Development Project (BE2017339 & BE2019338), The Key Research and Development Project of Yangzhou (YZ2019043 & YZ2018063), and the Science and Technology Innovation Training Fund of Yangzhou University (2015CXJ074).
