Efficacy of Poria cocos and Alismatis rhizoma against diet-induced … – Nature.com

Animals

Fifty 6-week-old male SpragueDawley (SD) rats were obtained from the Experimental Animal Science and Technology Development Co., Ltd., affiliated with Southern Medical University (license number: SCXK 2011-0015). The rats were housed in an SPF-grade barrier environment at an ambient temperature of 2125 with 5060% humidity and maintained on a 12-h light/12-h dark cycle in the Laboratory Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). All animals protocols followed the Regulations of Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine and were approved by the Animal Ethics Committee (ethical review number: 00157477). The entire research process including experimental procedures and data analysis, met the Essential 10 and Recommended Set of ARRIVE Guidelines 2.0 criteria25.

The following equipment was used: a multifunctional microplate reader (Enspire 2300, PerkinElmer, MA, USA), paraffin embedding machine (Histocore Arcadiac, Leica, Wetzlar, Germany), automatic dehydrator (Excelsior AS, Thermo Fisher Scientific, MA, USA), paraffin slicing machine (RM 2245, Leica, Wetzlar, Germany); optical microscope (BX46F, Olympus, Tokyo, Japan), vertical electrophoresis system (Mini-Protean Tetra, Bio-Rad, CA, USA), and automatic chemiluminescence imaging system (Tanon 4600, Tanon, Shanghai, China).

After one week of acclimatization, 40 rats were fed a high-fat diet (HFD) for four weeks. The HFD consisted of 52.2% maintenance feed, 0.4% premixed feed, 20% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 10% casein, 0.6% dicalcium phosphate, and 0.4% mountain flour. The rats in control group (n=10) were fed a chow diet. At the end of the fourth week, after the rats had fasted for 12 h, blood lipids indices, including TC, TG, LDL-C, and HDL-C, were analyzed to evaluate whether the model was successfully established. The model evaluation criteria were assessed as described previously26.

PC (batch number: 190301) and AR (batch number: 190701), were purchased from Zisun Chinese Pharmaceutical Co. Ltd. (Guangzhou, China). The Inspection Certificate Reports of PC and AR (details in the Supplementary Material 1) indicated that herbs traits, identification, check items, extracts, and content met the standards of Chinese Pharmacopoeia (2015) section IIV. A 15-g PC and 10-g AR mixture was pre-soaked in distilled water for 20 min and then extracted twice with ddH2O (1:10 and 1:8, w/v), producing a 0.454 g/mL crude drug extract. The extract was then diluted to 0.114 g/mL and 0.227 g/mL. All herbal solutions were stored at 4 until use.

The computational formula for determining the rat equivalent dose, according to body surface area based dosing27 (0.018, rat/human), is as follows:

$$Rat, dose=frac{human, equivalent, dose times, human, body, weight times 0.018}{rat, body, weight}$$

(1)

The daily dose of PCAR (25 g) for a person (70 kg) is 0.36 g/kg, which is equivalent to the 2.27 g/kg concentration used as the median dose for the rats (0.2 kg). The low and high doses of PCAR were 1.14 g/kg and 4.54 g/kg respectively. The volume of the intragastric liquid administered daily to the rats was 1mL/100g.

Before the intervention was implemented, 50 male SD rats were divided into five groups (10 rats per group): control, model, 1.14g/kg PCAR, 2.27g/kg PCAR and 4.54g/kg PCAR groups. In the control and model groups, PCAR was replaced with an equal volume of 0.9% sterile saline. After four weeks of treatment, all rats were sacrificed, and blood and livers were harvested for further analysis.

The rats from all five groups were fasted overnight at the end of the PCAR treatment period. The body weight (BW) of all rats was measured before anesthetization. The animals were anesthetized by an intraperitoneal injection (50 mg/kg) of 2% pentobarbital sodium (GuangzhouQiyunBiological Technology Co. Ltd., No.GH-40C0). Body length measurement and blood samples collection were performed under general anesthesia. Thereafter, all the animals were euthanized for liver sample collection. After standing for 12 h, the collected blood was centrifuged at 4 and 3,500 rpm for 10 min. The upper serum was collected and stored in a refrigerator at 20 . The excised livers were washed with cold normal saline, frozen in liquid nitrogen, and stored at 80C until use. The liver coefficient28 and Lees index28 were calculated after the experiment the following formulae:

$$mathrm{Liver, coefficient}=frac{mathrm{Liver, weight }(mathrm{g})}{mathrm{Body, weight }(mathrm{g})}$$

(2)

$${text{Lee}}prime {text{s}};{text{index}} = sqrt[3]{{frac{{{text{Body}};{text{weight}} left( {text{g}} right)*1000}}{{{text{Body}};{text{length}} left( {{text{cm}}} right)}}}}$$

(3)

TC, TG, LDL-C, and HDL-C concentrations were determined at the end of the fourth and eighth weeks using blood lipid detection kits provided by the Nanjing Jiancheng Biotechnology Company. Blood lipid detection was performed according to the manufacturers instructions.

Collected rat liver tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated in gradient grade ethanol, cleared with xylene, embedded in paraffin, and cut into 4m sections. The tissue slides were then dehydrated with xylene and gradient grade ethanol, incubated with hematoxylin for 5 min, stained with an eosin solution for 13 min, dehydrated again with gradient grade ethanol and xylene, and sealed with neutral gum. The liver cell nuclei (stained blue) and cytoplasm (stained red) were observed using a microscope.

Total RNA was extracted from the livers using TRIzol solution (Thermo Fisher Scientific) following the manufacturers protocol. RNA purity was assessed by determining the absorbance at 260 and 280 nm using a spectrophotometer (IMPLEN, CA, USA). The RNA concentration was measured using the Qubit RNA Assay Kit along with the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies). A cDNA library was constructed using PCR amplification. The quality of the cDNA library was confirmed by quantitation, using a Qubit 2.0. The insert size was detected using Agilent 2100 istrument, and the library concentration was confirmed by quantitative PCR. All the samples were sequenced on an Illumina HiSeq 2500 platform. Raw data with a Q30 base percentage of over 86.09% were obtained. The obtained clean reads were blasted and mapped against the reference genome database (Species: Rattus norvegicus; Version: Rnor_6.0) using the TopHat229 software. The mapping rates of the different samples against the reference database were between 73.76% and 83.81%. Fragments per kilobase per million fragments mapped values were calculated and used to normalize gene expression levels.

The R studio software (v 1.1.463)30 was used for data processing. All gene expression values were transformed into fold change (FC for group comparisons, using the DESeq2 package (v 1.26.0)31. DEGs were defined according to the criteria |log2(FC)|>0.585 and false discovery rate (FDR) 0.05. A volcano plot, processed using the ggplot2 package (v 3.4.1)32 was used to display the DEGs in both comparison sets based on the relationship between log10(FDR) and log2(FC). In addition, hierarchical clustering analysis was performed using the pheatmap package (v 1.0.12)33 to compare the DEGs among the groups. Raw sequencing data and processed data were uploaded to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE212771.

Gene ontology (GO) enrichment analysis was performed to determine the main mechanisms involved in the occurrence and treatment of HLP. First, all DEGs were mapped to GO terms in the GO database (http://www.geneontology.org/), falling under three categories, molecular function (MF), cellular component (CC), and biological process (BP). Gene numbers were calculated for every term, and significantly enriched GO terms were defined using a hypergeometric test. The calculated P-value were subjected to false discovery rate (FDR) correction, with Q0.05 used, as a threshold. GO terms that met this criterion were defined as significantly enriched.

GSEA was performed to verify which biologically significant gene sets were significantly associated with the different groups of samples. The annotated gene set for rattus norvergicus was acquired from the Kyoto Encyclopedia of Genes and Genomes (KEGG)34 database and processed as a GMT file using R software (v 4.0.2)30. Other files containing the gene expression data in .txt format and the phenotype labels in .cls format for the model vs. control and the model vs. PCAR groups were loaded into the GSEA (v 4.3.2)35. Finally, following standard protocols, 1,000 permutations were performed for each analysis to identify enriched pathways based on gene expression.

The RT-qPCR was used to detect the expressional levels of candidate targets. Total RNA was extracted from rat liver tissues using a lysis buffer with a Tissue RNA Purification Kit (#RN001A, EZBioscience) according to the manufacturers instructions. After determining the concentration and purity of the RNA using a nucleic acid quantitative analyzer (NanoDrop One, ThermoFisher Scientific Inc.), reverse transcription was conduceted using a Color Reverse Transcription Kit (#A0010CGQ, EZBioscience). RT-qPCR was then performed with (ROX2 Plus) 2xColor SYBR Green qPCR Master Mix (#A0012R2, EZBioscience) on an fluorescence quantitation PCR system (CFX96, Bio-Rad). Relative mRNA expression levels were normalized to -actin (ACTB). The measurement results were expressed as 2CT. Primer sequences used in this study are listed in Supplementary Material 2.

Compounds contained in PC and AR were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; http://www.tcmsp-e.com) 36according to oral bioavailability (OB)30% and drug-like properties (DL)0.18 criteria. Three-dimensional (3D) structure diagrams of the compounds were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). If no 3D structural diagram was available, two-dimensional (2D) structural diagram were used. The 2D structure diagram was converted to 3D using Chem3D software37. All structure diagrams were saved in PDB format. Next, the PDB structure files were imported into AutoDock4 (v 4.2.6)38 to add charging information and display rotatable keys. The files were saved in the required pdbqt format. The protein crystal structures corresponding to the core target genes were downloaded from the PDB database and imported into PyMOL39 (v 2.4.0 open-source; https://github.com/schrodinger/pymol-open-source) to remove water molecules and impurities, following the addition of hydrogen atoms in the AutoDock4 and pdbqt formats. These compounds were used as ligands and proteins as receptors for molecular docking analysis. AutoDock4 was used to estimate the binding capacities of the molecules and targets. The results were visualized using PyMOL software (v 2.4.0 open source).

Statistical analysis was performed using SPSS statistical software (v22.0, SPSS, Inc, Chicago, IL, USA). All data were analyzed using one-way analysis of variance (ANOVA) after the normality test. If the data met the condition of homogeneity of variance condition, the least significant difference (LSD) and Tukeys post-hoc tests were used for three and four comparison groups, respectively. Welchs analysis of variance combined with the GamesHowell post hoc test was applied under conditions of heterogeneity of variance when there was a significant difference between the groups. Statistical data are presented as the meanstandard deviation. A P-value of<0.05 was defined as indicative of a significant difference.

Following the animal ethics principle of minimizing harm to animals, the control and model groups of rats used in this study were shared with the two groups previously described in published literature26. Permissions was obtained from the previous publisher. The medicine used in this study, Poria cocos and Alismatis rhizoma, differs from that used in the published literature. Additionally, the efficacy results, transcriptome data, comprehensive targets screening process and RT-qPCR validation results of PCAR have not been published before, thus supporting for the originality of the study.

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Efficacy of Poria cocos and Alismatis rhizoma against diet-induced ... - Nature.com