Metabolomic analysis of Lobophytum sp.
Lobophytum sp. collection and identification
Samples of the soft coral Lobophytum sp. samples were gathered from the Egyptian Red Sea off the coast of Hurghada in January 2023. The collected samples were cleaned with seawater, tap water, and distilled water to get rid of any impurities, sand, or salts. The samples were brought to the lab in an ice box, stored in sterile plastic bottles, and kept cold. A voucher specimen (2023-DUPD-12) was kept at the Department of Pharmacognosy, Faculty of Pharmacy, Deraya University, Egypt, where the specimen was identified using common taxonomic keys.
Extraction of Lobophytum sp.About 350 g of a Lobophytum sp. sample were air dried for a month in the shade. The dried sample was subsequently processed with an OC-60B/60B grinder (60–120 mesh, Henan, Mainland China) to give 285 g fine powder. The powder was extracted using 6 L of 70% ethanol, which were macerated for 5 d at room temperature. A rotary evaporator (Buchi Rotavapor R-300, Cole-Parmer, Vernon Hills, IL, USA) was used to concentrate the mixture under vacuum at 45 °C to provide 50 g of crude extract.Metabolomic analysisAn Acquity Ultra Performance Liquid Chromatography system connected to a Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer (Waters, Milford, CT, USA) was used to perform high-resolution liquid chromatography-mass (HR-LC–MS) metabolic tentative identification for 1 mg of the Lobophytum sp. extract, which had been weighted using a delicate electric balance (Sartorius, type 1712, Germany) and dissolved in 1 mL of HPLC-grade methanol.An ACE C18 column, measuring 75 mm by 3.0 mm and 5 µm, was used as the HPLC column (ACE, Mainz, Germany). The mobile phase consisted of HPLC grade water (A), which was obtained in-house, from a direct Q-3 water purification system (Millipore, Watford, UK), and acetonitrile (B) with 0.1% formic acid in each solvent. All reagents were purchased from Fisher Scientific, Hemel Hempstead, UK, and were of analytical grade. The gradient program was 10% B at first, and after 30 min of linear growth to 100% B at a flow rate of 300 µL/min, it was isocratic for 5 min before linearly falling back to 10% B in 1 min. Before the injection, the column was re-equilibrated with 10% B for 9 min. Every sample was analyzed for a total of 45 min. The tray temperature was kept at 12 °C, and the injection volume was 10 µL. Both positive and negative ESI ionization modes of high-resolution mass spectrometry were used to include the highest number of metabolites from the investigated methanol extract, with a capillary temperature of 320 °C and a spray voltage of 4.5 kV. The mass range of 150–1500 m/z was chosen. The dereplication was achieved for each m/z ion peak with metabolites recorded in the customized databases such as METLIN and the Dictionary of Natural Products (DNP) database52 based on established parameters (m/z threshold of ± 3 ppm and retention time), which provided a high level of confidence in metabolites identity. As a result, 20 compounds were dereplicated from Lobophytum sp. methanol extract, and the number of the remaining unknown metabolites were refined. The raw data were processed, aligned, and merged into one dataset according to the method previously developed in our lab53,54.Data analysis for HR-ESI–MSThe MassConvert tool from ProteoWizard was first used to split the raw data into two data sets based on the ionization mode (positive and negative modes). In MZmine 2.1055, the sliced data sets were imported and processed utilizing predefined settings to extract features from raw data. With MZmine, the following data processing steps were carried out: peak detection (using chromatographic builder and mass detection), deconvolution, deisotoping, filtering, alignment, and gap filling. Adducts and complexes were identified, and formula prediction procedures were performed to minimize feature misassignment by removing adducts and complexes and to forecast potential chemical formulae for each feature (see Supplementary Information for full details of all settings and procedures utilized to process data in MZmine). After that, the data was exported as a CSV file for additional cleanup. Antibase® (February 2013) and Marinlit® (September 2013) molecular formula data sets were used, with an algorithm being used. The provided molecular weights do not distinguish between monoisotopic, average, and most abundant masses in these versions of the manually curated databases. Next, exact monoisotopic masses of each metabolite were computed and used to create the customised library. Peak identification and dereplication were performed using the integrated Excel macro in the customized library, which integrated the processed data from MZmine. Unidentified peaks and ―Hits‖ were cross-checked with the MS raw data in Xcalibur 2.2. Excel macros were created to enable the combination of positive and negative ionisation mode data files produced by MZmine, as well as the elimination of background peaks. Ion peaks from the medium were removed whereas features with peak intensities 20 times higher in the samples than in the medium were kept. This was done by using an algorithm to determine the intensity of each m/z in both the bacterial extracts and the media extracts. Through the use of RT and an m/z threshold of ± 3 ppm, the Excel macro was able to dereplicate each m/z ion peak with compounds in the customized database, providing information on the putative identities of all the metabolites in the Lobophytum sp. extract and sequentially sorting the number of unknowns that remained for the extract. Hits from the database were accessed using ChemBioFinder version 13 (PerkinElmer Informatics, Cambridge, UK). After that, the data was converted into a CSV file and exported to SIMCA-P V 13.0 Umetrics, Umeå, Sweden. As a result, each feature in the extract was given a feature ID number, ionization mode, m/z, retention time, potential molecular formulas, and peak intensity54.Biological studyAnimalsA total of 32 male Sprague Dawley rats, at the age of 8 weeks, with a weight range of 200–250 g, were selected as subjects for this research investigation. The subjects were confined within polystyrene enclosures maintained at a consistent temperature of 25 ± 2 °C, while being subjected to a regular 12-h cycle of alternating light and darkness. The animals were provided with a diet consisting of standard chow pellets and unrestricted access to water. One week prior to the experiment, the animals were acclimated to the laboratory environment.Extract preparationThe Lobophytum sp. extract was dried and dissolved in 0.5% carboxymethylcellulose (CMC). It was administered orally by gavage at a dose of 40 mg/kg daily throughout the entire duration of the experiment. A solution containing rotenone was prepared, with a concentration of 2 mg/mL in sunflower oil. The rats in the group receiving rotenone were given subcutaneous injections of 2 mg/kg/d.Ethics approvalAll animal treatments adhered rigorously to the institutional and international ethical guidelines for the utilization and welfare of laboratory animals, and all experiments were conducted in accordance with the ARRIVE guidelines. The experimental protocols underwent approval by the Experimental Animal Center and Research Ethical Committee, Deraya University, Minia, Egypt.Acute toxicity studyAn acute toxicity study was performed according to the method outlined by Lorke17. During the initial phase, a group of rats was divided into three smaller groups, each consisting of three rats. The groups were administered the extract at a dosage of 10, 100, or 1000 mg/kg body weight. The animals were closely monitored for a period of 24 h to detect any indications of toxicity or mortality.In the second phase, the rats were divided into three groups. The extract was then administered to all three groups at a dose of 1600, 2900, and 5000 mg/kg. The LD50 was determined based on the findings of the final phase, using the following formula:$$LD50=\sqrt{(D0\times D100)}$$D0 is the highest dose that resulted in no mortality, D100 is the lowest dose that resulted in mortality.Experimental designA population of rats was subjected to random allocation into four distinct groups, each containing eight individuals. Group 1 was administered a solution containing 0.5% CMC orally and sunflower oil subcutaneously for a duration of 4 weeks. Group 2 was administered the oral extract dissolved in 0.5% CMC via gavage on a daily basis, along with subcutaneous administration of sunflower oil, over a period of 4 weeks. Group 3 was administered 0.5% CMC via the oral route, while also receiving rotenone (Sigma Aldrich) (2 mg/kg) per day, administered subcutaneously. This treatment regimen continued for a duration of 4 weeks. Group 4 was administered the oral extract via gavage on a daily basis, along with rotenone (2 mg/kg/d, s.c.), for a duration of 4 weeks24. After a period of 24 h following the final injection, the rats were anesthetized using thiopental sodium at a dose of 50 mg/kg56 and scarified by decapitation. The entire brains were then carefully dissected, and the midbrains and striata from one hemisphere were removed. These brain regions were subsequently stored at a temperature of − 80 °C for preservation purposes. The stored samples were later utilized for neurochemical analysis, while the second brain was immersed in a 10% formalin solution (pH 4.0) for the purpose of histological assessment.Histological examinationHematoxylin and eosin (H&E) stainingThe paraffin beeswax tissue blocks underwent sectioning at a thickness of 4 μm using a sliding microtome. The specimens were obtained and mounted onto glass slides, followed by the removal of paraffin. Subsequently, a staining technique involving hematoxylin and eosin was employed. Finally, an impartial researcher scrutinized the samples using a light microscope, which was equipped with a camera57.ImmunohistochemistryParaffin tissue sections with a thickness of 5 μm were meticulously prepared following the established protocol outlined by Johansson et al. in their seminal work published in 200258. Immunohistochemical staining was performed in accordance with the manufacturer’s protocols utilizing a TH rabbit monoclonal antibody (1:200 dilution, Abcam, Cambridge, UK; catalog number ab75875).Assessment of SOD and MDA levels in the brainThe estimation of the activities of the endogenous antioxidant enzyme superoxide dismutase (SOD) was conducted in accordance with the protocols provided by the manufacturer’s kits (Bio-Diagnostic, Giza, Egypt). The expression of SOD activities is quantified in units per gram tissue59,60. Cerebral MDA content assessment was conducted utilizing commercially available kits in accordance with the protocols specified by the manufacturer (Bio-Diagnostic, Giza, Egypt).Estimation of cerebral acetylcholinesterase activityThe colorimetric assay involved the evaluation of the hydrolysis process of butyrylthiocholine into butyrate and thiocholine, which occurred in the presence of cholinesterase. The formation of thiocholine was subsequently subjected to reaction with DTNB, resulting in the acquisition of a pigmented product. This product was subsequently quantified by measuring its absorbance at a wavelength of 405 nm. The magnitude of absorbance augmentation is contingent upon the level of cholinesterase activity present within the homogenate61.Gene expression analysis by real-time PCRIn order to ascertain the mRNA expression levels of the target genes, the total RNA was extracted utilizing a TRIzol reagent (Invitrogen, Waltham, MA, USA) in accordance with the guidelines provided by the manufacturer. The quantification of the isolated RNA was performed utilizing the Nanodrop 1000 instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA) to determine its concentration. The process of cDNA synthesis was carried out utilizing a High-Capacity cDNA Reverse Transcription Kit. To conduct a quantitative analysis, we employed real-time PCR to identify the overall mRNA transcripts of the target genes. This was done using the step-one real-time PCR platform and the maxima SYBR green master mix from (Thermo Fisher Scientific Inc., Waltham, MA, USA). The primer sequences of the target genes were acquired from the National Center for Biotechnology Information (NCBI) and are presented in Table 2. The assessment of target gene expression was conducted utilizing the 2-ΔΔCt method, which involves normalizing the results to the housekeeping gene GAPDH.
Table 2 Primers list for real-time PCR.Enzyme-linked immunosorbent assay (ELISA)Using an ELISA plate reader and a commercially available ELISA kit (Cusabio), the quantity of α-synuclein in rat brain tissues was assessed. The results are represented as ng/mg protein.Statistical analysisThe data were reported in the form of the mean ± standard deviation (SD) and subjected to statistical analysis using a one-way ANOVA followed by the Tukey’s test as a post-hoc analysis. The statistical analyses were performed using GraphPad Prism software 9 (GraphPad Software, Inc. La Jolla, CA, USA). The statistical significance of the results was determined based on probability values below 0.05.Network pharmacologySoft coral-metabolite networkThe metabolites identification, carried out by the LC–HR–ESI–MS technique, tentatively identified 20 metabolites, and a basic network, linking the soft coral (Lobophytum sp.) to the identified metabolites was constructed.Metabolite-genes networkA network (metabolite-genes) was constructed, based on chemical data extracted for each compound from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)62 (last accessed on 10-08-2023) and SwissTargetPrediction database http://www.swisstargetprediction.ch/result.php?job=215444691&organism=Homo_sapiens63 (last accessed on 11–8-2023) was used to find out the targets of each identified compound related to the human species (Homosapien). The top genes were selected in the SwissTargetPrediction database with a probability score > 0.Gene-Parkinsonism networkDisGenet (https://www.disgenet.org/)64 (last accessed on 13-8-2023) online database was used to find out the target genes related to parkinsonism. A filter option was chosen in the database, and a word filter (Parkinson) was applied.Metabolites–Parkinsonism genesA network was formed based on extracted from the DisGenet. The determined genes related to parkinsonism were selected and in a backward step, each identified gene related to parkinsonism was referred to its corresponding metabolite.Protein–protein interaction (PPI) networkInteractions between proteins of the identified genes related to parkinsonism were obtained from the STRING database62. The graphical diagram was obtained from the same database.Complete pharmacology networkA complete pharmacology network was formed by combining the Soft Coral-Metabolite Network (Gene-Parkinsonism Network) and (Metabolites–Parkinsonism Genes Network).Networks construction visualization and analysisThe networks were constructed, visualized, and analyzed using the software Cytoscape 3.9.0. (https://cytoscape.org/download.html)65.Gene ontology (GO) and enrichment analysisThe gene ontology and enrichment analysis were performed on the genes related to parkinsonism among the gene set identified by metabolites. The determination of the gene ontology in terms of the cellular components, biological processes, molecular function, and biological pathways that are related to this set of genes was performed using ShinyGO 0.7566.
Unveiling Lobophytum sp. the neuroprotective potential of Parkinson’s disease through multifaceted mechanisms, supported by metabolomic analysis and network pharmacology
