Gegen Qinlian standard decoction alleviated irinotecan-induced diarrhea via PI3K/AKT/NF-κB axis by network pharmacology prediction and experimental validation combination

Chemicals and reagents

CPT-11 (Irinotecan HCl Trihydrate, Mw 677.19) was purchased from Melonepharma Co., Ltd. (Dalian, China). Weikeqi Biological Technology Co., Ltd (Chengdu, China) supplied the reference compounds Puerarin, Coptisine, Berberine, Palmatine, Daidzin, Liquiritin, Daidzein, Wogonoside, Wogonin, Baicalin and Baicalein (purity ≥ 98%). HPLC-grade reagent were obtained from Thermo Fisher Scientific (Thermo scientific, USA). The tumor Necrosis factor-α (TNF-α) kit, interleukin 1β (IL-1β) kit, and interleukin 6 (IL-6) kit were purchased from Multi Science (Lianke) Biotech Co., Ltd. (Hangzhou, China). PI3K antibody (60225-1-Ig), NF-κB p65 antibody (66535-1-Ig) were purchased from Proteintech Group, Inc (Wuhan, China). Abmart Shanghai Co., Ltd (Shanghai, China) provided the AKT (T55561) antibody and Servicebio Biotechnology Co., Ltd. (Wuhan, China) provided the β-actin antibody (GB12001). Professor Guihua Jiang (College of Pharmacy, Chengdu University of Traditional Chinese Medicine, China) identified all of the herbs procured from the Sichuan Neautus Traditional Chinese Medicine Co., Ltd. (Chengdu, China).

Cells and animals

The Human normal colonic epithelial cell (NCM 460) was purchased from Wuhan Fine Biological Technology Co., Ltd. (Wuhan, China). RPMI-1640 complete medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin was used to cultivate NCM 460 cells. The cells were kept at 37 °C in an environment that was humidified and added to 5% CO2.

Male ICR mice (weight 22 ± 2 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). (quality certification number: SCXK 2019-0010). The mice were housed in cages at 22 ± 2 ℃, 55% ± 5% relative humidity and with a 12 h light–dark cycle. The Institutional Animal Care and Use Committee (IACUC) of Chengdu University of Traditional Chinese Medicine provided the guidelines for all in vivo tests.

Preparation of GQD standard decoction

The GQD standard decoction extract was performed in accordance with ancient records [22]. The herbal material Pueraria lobata (Willd.) Ohwi (Ge-Gen, GG, 110.4 g) was firstly soaked in distilled water (1.6 L) for 30 min and then boiled to a solution volume of 1.2 L. Next, the other herbs, including Coptis chinensis Franch. (Huang-Lian HL, 41.4 g), Scutellaria baicalensis Georgi (Huang-Qin, HQ, 41.4 g) and Glycyrrhiza uralensis Fisch. (Gancao, GC, 27.6 g) were added in solution at the weight ratio of 8:3:3:2 and boiled for 3 h, When the volume of decoction is boiled to 0.4 L, the aqueous extract was filtered by gauze, then lyophilized. Ultrasonic extraction was used to dissolve 0.1 g of lyophilized GQD standard decoction powder in 10 mL of 70% methanol. The supernatant was filtered, and analyzed by LC–MS.

UPLC-Q-TOF–MS analysis

The Vanquish UHPLC system (Thermo Fisher Scientific) couppled with Q Exactive quadrupole-electrostatic field orbitrap mass spectrometer was used to evaluate GQD standard decoction powder. Samples were performed on a SunFire C18 column (3.0 × 150 mm, 3.5 μm, Waters, Massachusetts, USA). The column temperature was 30 ℃ and flow rate was 0.3 mL/min. The mobile phases A and B were deionized water with 0. 1% formic acid and acetonitrile with 0.1% formic acid, respectively. Installing the gradient elution program: 0–3 min, 10%–12% B; 3–5 min, 12%–13% B; 5–8 min, 13%–16% B; 8–16 min, 16%–20% B; 16–18 min, 20%–21% B; 18–20 min, 21% B; 20–38 min, 21%–40% B; 38–50 min, 40%–50% B; 50–54 min, 50%–51% B; 54–60 min, 51%–90% B.

This MS system operated in positive ionization modes with m/z 100–1500. Positive and negative ion spray modes were 3.2 and 2.5 kV. 350 °C for the ion source heating. Fragment voltage was 50 V, and the scan mass ratio was within m/z 50–1500.

The mass detection was carried out with a m/z tolerance of 10 ppm and an RT tolerance of 0.01 min, and the noise level set to 1.5. Finally, the mgf data file and its associated. csv metadata file, which included Peaks height and areas integration, were exported.

Quantification 11 ingredients identified with the reference standard of GQD standard decoctionHPLC instrumentation and conditions

The HPLC determination conditions were exhibited in Additional file 1: Material S1.

Methodological investigation

According to the recommendations of Chinese Pharmacopoeia, the specificity, linearity, instrument precision, method repeatability, sample stability and method recovery of the high performance liquid phase content determination method were verified.

Content determination of 11 components

The above method was used to prepare 15 batches of sample solutions of 11 ingredients identified with the reference standard in GQD standard decoction. Among them, 10 μL was injected under high performance liquid phase conditions, the peak area of each compound was measured, and the content of 11 ingredients of multiple batches of GQD standard decoction was calculated.

Network pharmacology analysisScreening the components of GQD standard decoction

The GQD standard decoction’s candidate compounds were recognized by LC–MS. And the TCMSP (https://old.tcmsp-e.com/tcmsp.php) was used to obtain compounds' pharmacological information. For the efficacy prediction, based on the body’s drugs ADME, compounds with OB ≥ 30% and DL ≥ 0.18 were chosen. And the active compounds information was queried and standardized by the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/).

Prediction of potential proteins

The proteins of active component was obtained from SWISS Target Prediction database (http://swisstargetprediction.ch/), and collected the top 15 proteins for docking scores or the proteins with a docking score of 1. The related disease proteins were searched from two databases using “chemotherapy induced diarrhea” as the keywords: the GeneCards database (http://www.genecards.org) OMIM database (http://www.omim.org). The GQD standard decoction absorbable component proteins and CID-related proteins were intersected to yield possible GQD treatments CID proteins, which were then displayed in VENNY2.1.

GO and KEGG enrichment analysis

The clusterProfiler R package was used to evaluate the biological consequences of GQD standard decoction for CID treatment. And GO and KEGG pathways with P < 0.05 was identified.

Construction of compounds-targets-diseases network

Cytoscape 3.7.2 showed the following network construction. The PPI of GQD standard decoction in treating CID were constructed by STRING database (https://string-db.org) and Cytoscape, in which the network proteins were screened with a combined score > 0.7. The compounds-targets-diseases (C-T-D) network was visualized with Cytoscape software and analyzed with Network Analyzer.

Cell viability analysis

NCM 460 cells (6000 cells/well) were planted in 96-well plates overnight for adherence. After 12 h, cells were cultured with a fresh culture medium, SN-38, GQD standard decoction @SN-38. After 24 h of treatment, CCK8 (Beyotime, Shanghai, China) was given to cells for 4 h at 37 °C, and the optical density was measured at 450 nm with a microplate plate reader (Thermo Fisher Scientific, Inc.).

Prevention effects of GQD standard decoction on NCM460 cell damage

NCM 460 cells were planted in 24-well plates (2 × 105 cells/well). Cells were cultured with a fresh culture medium, SN-38, GQD standard decoction @SN-38 (500 nmol/L) after overnight culture. The equivalent amounts of GQD standard decoction in various groups were 18.75, 37.5 and 75 µg/ml. Untreated cells were utilized as control. After 24 h, each well’s cell supernatant was collected. ELISA kits were used to quantify TNF-α, IL-1β, IL-6 in the cell supernatants.

Real-time PCR analysis

Animal Total RNA Isolation Kit (Foregene, Chengdu, China) was used to extract and purify Total RNA from NCM 460 cells. The 5*All-In-One MasterMix (abm, USA) was used to synthesize complementary DNA (cDNA) Then, water that was devoid of nucleases diluted cDNA. Real-time PCR was performed on an Applied Biosystems 7500 machine (Life Technologies) using 2 × SYBR Green qPCR Master Mix (Servicebio, Wuhan, China). Each gene’s e relative mRNA expression was determined in relation to β-Actin mRNA, and all genes are shown as fold changes from the negative control group. Each sample's mean was determined by six replicate experiments. The results are expressed using the relative quantitative analysis of 2(− ΔΔCT). All PCR primers are summarized in Table 1.

Table 1 Primers and probes for real-time PCREffects on CPT-11-induced diarrheal mice model

After one week of adaptation, the mice were divided into five groups at random (n = 5): Control, CPT-11, GQD-L (0.7 g/kg), GQD-M (1.4 g/kg), GQD-H (2.8 g/kg). The CPT-11 was dissolved in water plus 1% Tween and the GQD standard decoction powder were dissolved in water. In the control group, the mice were only given water plus 1% Tween (10 ml/kg, i.p.) from days 7 to 10 and water (10 ml/kg, i.g) from days 1 to 14. In the CPT-11 and GQD standard decoction-treated groups, the mice received injection of CPT-11 suspensions (50 mg/kg, i.p.) from days 7 to 10, and GQD standard decoction extract were intra-gastric administration to mice in GQD standard decoction-treated groups from days 1 to 14. The GQD standard decoction’s dose was based on preliminary experiment and the group’s previous study [13]. Body weight, stool consistency, and fecal hemorrhage were monitored daily throughout the treatment period. The disease activity index (DAI) provides a complete evaluation of body weight loss (0−4), stool consistency state (0−4), and fecal bleeding (0−4) [23]. At day 15, all mice were sacrificed by euthanasia with isoflurane, then, the duodenum and colon were collected.

Histopathological analysis

Each group's colon tissues were photographed and sized. And 1.0 cm samples of duodenum and colon will be collected and preserved in 4% paraformaldehyde for 24 h and embedded in paraffin. And samples will be processed for hematoxylin–eosin (H&E) staining for general morphological observations. And mucin content was quantified using Periodic acid–Schiff staining (PAS) staining.

ELISA determination

Weighing and homogenizing the distal colon tissues in PBS. After centrifugation at 3000 rpm for 30 min, then, supernatant was collected for IL-6, TNF-α, IL-1β concentration measurement.

Immunohistochemical analysis

Immunohistochemical staining of paraffin slices revealed Occludin, ZO-1. Paraffin sections were blocked in 10% normal rabbit serum + 5%nonfat dry milk + 3% BSA + 0.1% Triton X-100 for 30 min, and then treated overnight with anti-Occludin, ZO-1 at 4 ℃. The slices were rinsed three times with PBS and HRP-goat anti-rabbit IgG antibody (1:200) was incubated for 50 min. The sections were treated with diamino-benzidine and counterstained with hematoxylin for 3 min, and visualized using the Nikon DS-U3 microscope system (Tokyo, Japan).

Western blot assays

Colon tissue was extracted with RIPA buffer with protease inhibitor. After high-speed centrifugation, BCA protein assay kit (Biosharp, China) was use to determine protein content. Protein suspension (30 µg) combined with 5× loading buffer (Biosharp, China) was electrophoresed on sodium dodecyl sulfate-polyacrylamidegel (SDS-PAGE, 7.5–10%) and transferred to 0.22 µm PVDF membranes (Millipore, USA). Membranes were occluded with 5% nonfat milk for 2 h and kept at 4 °C overnight. After washing with TBST (0.1% Tween), the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The ChemiDocTM XRS + system (Bio-Rad) was used to observe immunoreactive bands with HRP substrate (Luminata, Millipore). ImageJ 1.48 (National Institutes of Health, Bethesda, MD, USA) software examined protein expression levels. The results are representative Mouse anti-PI3K (1:5000), rabbit anti-AKT (1:1000), Mouse anti-NF-κB p65 (1:2000), and three independent experiments used β-Actin levels as controls.

Statistical analysis

All data were expressed as mean ± SEM. Comparing several groups with one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. Statistical analysis and graphing were performed using GraphPad Prism 8.0 software (San Diego, CA, USA).

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