2.1 Chemicals, Materials and Reagents
HPLC grade acetonitrile and methanol were urchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Formic acid was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Distilled water was purchased from Guangzhou Watsons Food & Beverage Co., Ltd. (Guangzhou, China). The reference compounds Jatrorrhizine Hydrochloride, Quercetin and Notoginsenoside R1 were purchased from National Institutes for Food and Drug Control (Beijing, China). The reference compound Ononin was purchased from Shanghai Y ongheng Biotechnology Co., Ltd. (Shanghai, China). The reference compounds Ginsenoside Rg1, Ginsenoside Rb1, Coptisine, Epiberberine, Berberine hydrochloride and Palmatine were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China). The reference compounds Daidzin, Puerarin and Daidzein were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). The purity of each reference compound was determined to be over 98% by HPLC analysis. The Huangdi Anxiao Capsules were provided by the First Affiliated Hospital of Anhui University of traditional Chinese Medicine.
2.2 Ethics Statement
Spontaneous T2DM GK rats (200 ± 20 g, 5 months old, SPF grade) were purchased from Shanghai Slack Laboratory Animals Co., Ltd., license number: SCXK (Shanghai) 2017-0005. All animals were raised in accordance with the guidelines for the care and use of laboratory animals and approved by the Animal Ethics Committee of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.
2.3 Preparation of HDAXC Solution and Reference Solution
The accurately weighed 400 mg the content of HDAXC was dispersed in 50ml methanol, ultrasonically in water bath (power 300W, frequency 40KHZ) for 30min, filter it through a 0.22μm microporous filter membrane, and the filtrate was used for UPLC-Q-TOF-MSE analysis.13 reference standards were dissolved in methanol. Before the analysis, they were mixed to make the concentration between 3~5μg/L and filtered by 0.22μm 115 microporous filter membrane.
2.4 Collection and Treatment of Serum Samples
Random blood glucose of GK rats was higher than that of 11.1 mmol/L as the standard of successful T2DM model (Zhao et al. 2021). The T2DM GK rats were randomly divided into a GK control group (n=5) and a GK experimental group (n=5). The experimental group were given 15 g/kg HDAXC twice a day for 3 days. 12 hours before the last intragastric administration, no feeding, free drinking water, and the last intragastric administration were given a full day dose. The control group was given 20mL/kg distilled water by gavage. Blood samples were collected an after oral 4 hours administration, which were allowed to stand at room temperature for 30 min, centrifuged at 12,000 r/min for 5 min to obtain serum and store for use at -20 ℃. Taking 300 μl serum sample in EP tube add 1200 μl methanol to swirl for 30 s, centrifuge for 5 min at 12000 r/min, taking out the supernatant, drying it with nitrogen (N2) at 40 ℃, dissolving it again with 150 μl methanol, swirling for 30 s, centrifuging for 5min at 12,000 r/min, taking the supernatant was used for UPLC-Q-TOF-MSE analysis. Part for serum samples filtered through a 0.22 μm filter membrane, heated at 56 °C for 30 min, and collected to store at −80 °C. The serum samples were used for Quantitative PCR and Western bolt.
2.5 Cell culture and treatment
TNF-α-stimulated HUVEC cell model, which is often used for in vitro study of T2DM, has been mentioned in our previous study (Calandrelli R et al. 2020). Our experiment was separated into three groups: the control group, the model group, and the HDAXC treatment group. The cells were inoculated on a six-hole culture plate and cultured overnight. The storage solution of 20mol/L mannitol and 25mol/L glucose was configured respectively. The normal group added mannitol storage solution (culture medium itself contained 5mM glucose). The other groups were incubated in incubator for 48 hours after adding glucose storage solution and TNF-a to the final concentration of 25mM and 5ng/ml. In the drug-containing group, 20% volume of drug-containing serum was added after 24 hours, and cultured in incubator for 24 hours. Then, the cells were collected for subsequent experiments.
2.6 Chromatography and Mass Spectrometry Conditions
Chromatographic analysis using a Waters Acquity UPLC system (Waters Corporation, Milford, USA). The column used for the experiment was ACQUITY UPLCTM BEH C18 column (2.1 mm×100 mm, 1.7μm). Mobile phase A was 0.1% formic acid water. Mobile phase B was acetonitrile. The gradient was set as follows: 0-1.5 min, 5% B; 1.5-5 min, 5% - 15% B; 5-8 min, 15% -20% B; 8-9.5 min, 20% B; 9.5-12 min, 20% - 35% B; 12-15.5 min, 35% - 40% B; 15.5-17 min, 40%-50% B; 17-19 min, 50%-5% B; 19-21 min, 5% B. The column temperature was retained at 35 ℃ and the sample chamber temperature at 8 ℃. The flow rate was set at 0.2 mL/min. And sample injection volume was 1.0 μL.
The mass spectrometer used in the experiment was Waters Xevo G2 QTOF mass spectrometer (Waters Corporation, Milford, USA), and the Electrospray ionization source (ESI) included two modes, a negative ion mode and a positive ion mode. respectively, testing parameters: cone gas, 50 L/h; source temperature, 110 ℃ (-) and 120 ℃ (+); desolvation gas temperature, 350 ℃; desolvation gas flow, 600 L/h; the capillary voltage is 2.5 kV (ESI+) in positive ion mode and 2.0 kV (ESI-) in negative ion mode; low collision energy, 6 V; high collision energy, 20~40 V; mass range 50~1200.
2.7 Network Pharmacology Analysis
2.7.1 Screening of Prototype Component Targets and T2DM Targets
The prototype components identified by UPLC-Q-TOF-MSE were converted to simplified molecular input line entry specification (SMILES) in chem 2014 software. Inputing the conversion results into Swiss target prediction (http://www.swisstargetprediction.ch/), and set organization to “Homo sapiens” to search and filter the target of the components (Gfeller et al. 2014).
In the GeneCards (http://www.genecards.org/) and DrugBack (https//www.drugbank.com/) enter the key word “type 2 diabetes mellitus”, and search the corresponding target of the disease (Stelzer et al. 2016; Wishart et al. 2018). By combining prototype component targets with T2DM targets, the common targets of serum drug components and disease were selected. These common targets are the prediction target of HDAXC acting on T2DM.
2.7.2 Construction and Analysis of “Component-Target” Network
The prototype components and common targets are introduced into the software of Cytoscape3.7.2 to construct the component-target network. The network analyzer function in the software of Cytoscape3.7.2 is used to analyze the network. The node represents the absorbed prototype components and target of HDAXC. The edge shows the relationship between the components of TCM and targets. According to the results of topological analysis, the key compounds of HDAXC acting on T2DM were selected.
2.7.3 The Protein-Protein Interaction Network Analysis of Common Targets
The common targets were uploaded to STRING (https://string-db.org/) database, construct a protein-protein interaction (PPI) network, and set the protein type to “Homo sapiens”, the minimum required interaction score is set to 0.400, and the display hides the free point to obtain the PPI network. The data of PPI network was downloaded and imported into the software of Cytoscape3.7.2. The topological analysis of PPI network is carried out with the help of the “network analyzer” function of the software of Cytoscape3.7.2. The targets with degree of freedom, betweenness centrality and closeness centrality greater than the median are selected as the key targets.
2.7.4 GO and KEGG Pathway Enrichment Analysis
The common targets were imported into the OmicShare tools(https://www.omicsshare.com/)for Gene Ontology enrichment(GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG)( Xiang L et al. 2021). The enrichment results of GO function and KEGG pathway were obtained by setting p-value. Go enrichment refers to the description of genes and proteins, mainly including three aspects: biological process (BP), molecular function (MF), and cellular component (CC).
2.7.5. Construction of “Component-Target-Pathway” Network
According to the enrichment results of KEGG pathway and literature search, the signal pathways related to T2DM were screened, the relevant targets of HDAXC in the treatment of T2DM were found, and the relevant information of HDAXC was combined to construct “component-target-pathway” network.
2.8. In in-vivo experiments
2.8.1 Quantitative PCR Verifies the Effect of HDAXC on Gene Expression
Total RNA from serum samples was isolated by TRIzol reagent (Thermo Fisher). Real-Time PCR System (PTC-200; Bio-Rad, Hercules, CA, USA). Removal of genomic DNA reaction: total RNA (mass 1 μ g), 5 × gDNA Eraser Buffer 2.0 μ L RNA Eraser 1.0 μ L were added into 0.2 mL tube, supplemented with DEPC water to 10 μ L, gently mixed and centrifuged. PCR was heated at 42 ℃ for 2 min and immediately incubated with ice for 1 min. PrimeScript RT Enzyme Mix I 1.0 μ L, RT Primer Mix 1.0 μ L, RNase Free dH2O 4.0 μ L and RevertAidTM M-MuLV Reverse Transcniptase 4.0 μ L are added to the EP tube. On the PCR instrument, 37 ℃, 15 min,85 ℃, 5s. The above reaction solution was taken out and stored at -20 ℃, and quantitative PCR (qPCR) was performed using the ABI Real-Time PCR System (StepOne Plus; ABI). The primers are shown in Table 1. The relative expression of each target gene was quantified and normalized by 2−ΔΔCT.
2.8.2 Western bolt Verifies the Effect of HDAXC on Gene Expression
Each group included an equal mass of HUVEC cell samples and was subjected to western blot assay to detect the levels of AKT1, VEGFA, SRC, EGFR. Total proteins in HUVEC cell were lysed using RIPA lysis buffer. Lysates were centrifuged at 12,000 rpm for 10 min to obtain the supernatant, and the protein concentration was detected by ECL luminescence kit (UA276616, Thermo). Samples were mixed with the loading buffer and boiled for 10 min in a boiling water. Then, the protein was loaded on 10% SDS-PAGE and transferred to PVDF membranes. Subsequently, the membranes were sealed with 5% skim milk powder at room temperature for 2 h with incubated primary antibody. Next, the membrane was washed with TBST and added to the secondary antibody to incubate at room temperature for 1 h. Last, the color was developed after washing the membrane with TBST.
2.9 Data Analysis
Importing the mass spectrum data of drug-containing serum and blank serum into UNIFI software deduct the endogenous components in the serum, and compare the retention time, mass-to-charge ratio and secondary mass spectrum of the remaining ion peak with the ion peak of the HDAXC solution. If they are consistent, they are confirmed as the prototype components in HDAXC. SPSS20.0 software was used for data analysis and processing, and the results are expressed as mean±standard deviation (SD)(x̄±s). One-way analysis of variance (ANOVA) designed by randomized block design was used to analyze the differences in multiple groups. The result was considered statistically significant when P<0.05.