Materials
Cyclophosphamide was obtained from Jiangsu Shengdi Pharmaceutical Co., Ltd (Batch No. 18051125, Jiangsu, China). D2O was purchased from Norell (Landisville, USA). K2HPO4·3H2O and NaH2PO4·2H2O were obtained from Wuhan Yuancheng Technology Development Co., Ltd. (Wuhan, China). Lvjiao Buxue Granules was purchased from Jiuzhitang Co., Ltd. (Batch No. 201802029, Hunan, China), which production method conforms to the standard of Chinese Pharmacopoeia (2015 edition). Diyu Shengbai tablets were obtained from Chengdu Diao Co., Ltd. (Batch No. Z20026497, Chengdu, China). The enzyme-linked immunosorbent assay (ELISA) kits were obtained from MEIMIAN (Jiangsu, China).
Animals
Inbred strain female (6-8 weeks old) BALB/c mice, weighing 18-20 g, provided by Vital River Laboratory Animal Technology Co., Ltd. (Beijing) (License number SCXK-2016-0006). The animals were kept at room temperature (24±1) ℃, humidity (60±5)%, and freely eating under the natural rhythm of day and night before the experiments. All animal experiments were conducted in accordance with the NIH Guidelines for Care and Use of Laboratory Animals (U.S.A) and the Prevention of Cruelty to Animals Act (1986) of China, and these experiments were also approved by the Animal Ethics Committee of Shanxi University.
Development of leucopenia mice model and treatment protocols
Animals were randomly divided into a normal control group and five cyclophosphamide induced groups: control group (C), model group (M), low dose of Lvjiao Buxue Granules group (LBG-L), moderate dose of Lvjiao Buxue Granules group (LBG-M), high dose of Lvjiao Buxue Granules group (LBG-H), and Diyu Shengbai tablets group (DST), each group was consists of 8 mouse. The mice in LBG-L, LBG-M, or LBG-H groups were administered Lvjiao Buxue Granules (3 g/kg, 6 g/kg, 12 g/kg) suspension daily, and the mice in the DST group were administered Diyu Shengbai tablets (0.14 g/kg) suspension daily. Mice in the control and model groups received an equal volume of vehicle orally. The 4T1 breast cancer model was established in which the tumor grew to approximately 5*5 cm2. The five cyclophosphamide-induced groups would be injected with cyclophosphamide in the dose of 80 mg/kg by intraperitoneal to 4T1 breast cancer model on the 1st day, 3rd day, 5th day and 7th day individually. The treatment lasted for 7 days, and the state of the mice was observed and the weight was measured daily.
Sample collection and determination
After 1h of the last treatment on the 7th day, 0.4 mL blood samples were collected into 1.0 mL tube with EDTA within via the orbital blood. Animal blood analyzer (HEMAVET950) was applied to evaluate peripheral blood routine parameters of 400 μL whole blood: white blood cell count (WBC), neutrophil count (NE), lymphocyte count (LY), monocytes count (MO), red blood cell count (RBC), hemoglobin (HGB), red blood cell volume (HCT), platelet count (PLT), mean red cell volume (MCV) and mean corpuscular hemoglobin (MCH). On the 8th day, mice were sacrificed, and spleen, thymus and liver tissues were immediately weighed and collected. The calculation formula of the organ index is as follows: organ index = organ weight (mg)/bodyweight (g). Quickly transfer the spleen to a refrigerator at -80 ℃ and use it for metabolomics analysis.
Sample preparation for NMR measurements
After thawing the spleen tissue, take about 40 mg, cut it (on ice), add 650 μL of MeOH and H2O (v/v, 2:1) to a 2 mL centrifuge tube, and homogenize and extract twice on an ice bath. The homogenate was centrifuged at 4 ℃, 13 000 r·min-1 for 15 min. The supernatants were combined, transferred to a 2 mL centrifuge tube and blown with nitrogen. The dried sample was dissolved in 700 μL of phosphate buffer (pH 7.40, containing D2O, 0.1 mo/L, Na2HPO4/Na H2PO4, 0.01% TSP), centrifuge at 13 ℃, 13,000 r·min-1 for 20 min, 600 μL supernatant was transferred into a 5 mm NMR tube for 1H NMR analysis.
Metabolomics analysis
The 1H NMR spectral data were collected on a Bruker 600 MHz AVANCE III NMR spectrometer(Bruker, Germany). The sample was the Noesygppr1d sequence to suppress the water peak. The number of scans was 64 scans, and each scan required an acquisition time of 2.654s. The specific parameters were as follows: spectral width was 12 345.7 Hz; spectrum size was 65 536 data points; pulse width (PW) was 30° (12.7 μs); fourier transform LB was 0.3 Hz and relaxation delay time was 1.0 s. The 1H NMR spectrum of the spleen was corrected for chemical shifts using TSP (δ0.00) as the standard. The spectrum in the region of δ0.60 to 9.49 was divided into 0.01 equal widths and integrated. All resulting integration data are “mass” normalized to eliminate weight differences of spleen tissue.
Simca-P 14.1 (Umetrics, Sweden) was used to perform multivariate data analysis. Firstly, by principal component analysis (PCA) of the normalized data, to identify the degree of dispersion between the control group and the model group, and the outliers were eliminated. Next, partial least-squares discriminant analysis (PLS-DA) was used to distinguish the differences in metabolic profiles between the control, model and drug groups. Orthogonal-projection to latent structure-discriminant analysis (OPLS-DA) was used to find differential metabolites between the control group and the model group. Finally, ANOVA analysis was performed on the metabolites in SPSS 16.0 software, with VIP>1 and P<0.05 as differential metabolites.
Biological targets network analysis
All components of Lvjiao Buxue Granules were collected from the TCMSP database. For all ingredients, the initial structure formats (e.g., mol2 and SDF) were transformed into a unified SDF format using the Open Babel toolkit (version 2.4.1). The ingredients with suitable OB≥30% and DL≥0.18 were chosen as candidate ingredients for further research, which is used as a selection criterion for the ingredients in the traditional Chinese herbs. After ADME screening, some ingredients that did not meet the three screening criteria were also selected because of their high content and high biological activity. All databases and software mentioned above are public.
The PharmMapper server was used for potential target prediction analysis. Metabolite data were imported in Metascape, a plugin of Cytoscape 3.7.1 and subjected to metabolic enzyme analysis. Finally, Cytoscape 3.7.1 software was used to construct the “herb-chemical constituent-targets-pathway-metabolite” regulatory network of Lvjiao Buxue Granules for leucopenia treatment.
Determination of the levels of BCKDHA and ACADS
In order to further verify the results of biological targets network, the content of the key rate-limiting enzymes BCKDHA (branched chain keto acid dehydrogenase E1, alpha polypeptide) and ACADS (Acyl-CoA Dehydrogenase Short Chain) on the BCAAs degradation pathway were determined. The levels of BCAAs and the degradation rate of them could be affected by the content of these enzymes.
The levels of BCKDHA and ACADS in the liver tissue lysates were determined by ELISA kits according to manufacturer instructions.