Drugs and reagents.
Ginger and pinellia were bought in September 2019. The roots of Ginger were produced in Laiwu City, Shandong Province, China, voucher specimen no. J7201. The tubers of pinellia were produced in Xihe County, Gansu Province, China, voucher specimen no. J7654. Both of them were authenticated by Professor Jizhu Liu (Guangdong Pharmaceutical University, Guangzhou, China). Pure standards of -gingerol and -shogaol were from Manst Biotechnology Co., Ltd., Chengdu, China. Pure standards of ephedrine and succinic acid were from China Institute of Food and Drug Verification. Ondansetron and cisplatin were from Qilu Pharmaceutical Company, China. Kaolin powder was from China Pharmaceutical Chemical Reagents Company, China. 1-PBG was from Sigma-Aldrich Company, USA.
Preparation of kaolin pellets and XBXT formula.
Kaolin pellets were prepared according to our previously reported method (25). The roots of ginger 200 g and the tubers of pinellia 400 g were soaked in 4800 mL distilled water overnight and boiled for 1.5 h at 100℃. After filtration, the dregs were boiled in distilled water one more time following the same procedure. The extract from two soaking and filtering was lyophilized to form a dried powder.
UPLC-Q-Orbitrap MS analysis of XBXT.
XBXT samples were analyzed by UPLC-Q-Orbitrap MS using ThermoFisher Scientific (TF, Dreieich, Germany) Dionex UltiMate 3000. The separation was performed on a RP-C18 column (150 mm × 2.1 mm, 1.8 µm, Welch), operating at a flow rate of 0.3 mL/ min throughout the gradient. The column temperature was 35°C, and the injection volume was 10 μL, Eluent A was 0.1% formic acid in water, and eluent B was 0.1% formic acid in acetonitrile methanol. Separation was conducted under the following conditions: 0 - 1 min, 2% B; 1 - 5 min, 20% B; 5 - 10 min, 50% B; 10 - 15 min, 80% B; 15 - 20 min, 95% B; 20 - 25 min, 95% B; 25 - 26 min, 2% B. 26 - 30 min, 2% B. For the MS analysis, ESI source conditions were set as following: sheath gas flow rate as 40 Arb, aux gas flow rate as 15 Arb, capillary temperature 300℃, full mass resolution as 70000, MS/MS resolution as 17500, spray voltage as 3.8 kV (positive). The auxiliary gas heater temperature and capillary temperature were both controlled to 350°C. Nitrogen was used for spraying stabilization and damping gas in the Ctrap. The analysis was performed in the full scan mode with a positive ion swing. The resolution was 70,000 (full mass). The full MS scan ranges were set from 150 to 2000 m/z. All of the collected data in the profile were acquired and processed using Thermo Xcalibur 3.0 software (Thermo Scientific, USA). Results of UPLC-Q-Orbitrap MS analysis of XBXT formula were shown in Figure S1.
Animals and treatment.
56 male Wistar rats (200 - 230 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd, China) and housed in environmental conditions (temperature: 23 ± 2℃, the relative humidity: 40% ~ 60%, the alternating time of day and night: 12 h/12 h). The rats were free to access standard diet and water. All the animal experiments were approved by the Committee on Laboratory Animal Care and Use of Guangdong Pharmaceutical University (Guangzhou, China). After adaptive feeding for 3 days, the rats were randomly divided into 7 groups (n = 8): group 1 (normal control group (Control)); group 2 (cisplatin model group (C-Model)); group 3 (ondansetron + cisplatin model group (C-Ondansetron); group 4 (XBXT + cisplatin (C-XBXT)); group 5 (1-PBG model group (P-Model); group 6 (ondansetron + 1-PBG model group (P-Ondansetron); group 7 (XBXT + 1-PBG model group (P-XBXT).
Kaolin pellets were added before drug administration in 3 days to make the rats familiar with the presence of kaolin pellets. Those rats that still took in large amounts of kaolin pellets on the third day were excluded. On the day of drug administration, the rats of C-XBXT and P-XBXT were given XBXT (1.6 g/kg [body weight (B.W.)]) by gavage respectively. The rats of C-Ondansetron and P-Ondansetron were given ondansetron (1.3 mg/kg [B.W.]) by gavage respectively. The rats of Control, C-Model and P-Model were given distilled water (10 g/kg [B.W.]) by gavage respectively. The above dosage was given by gavage every 12 h (8: 00 AM, 20: 00 PM) until the end of the experiment for a total of 7 times. After 1 h, the rats of C-Model, C-Ondansetron and C-XBXT were injected with cisplatin intraperitoneally (i.p.) at the concentration of 6 mg/kg (B.W.) and the rats of P-Model, P-Ondansetron and P-XBXT were injected with 1-PBG intraperitoneally at the concentration of 25 mg/kg (B.W.), while the rats of Control were injected with saline of equal volume intraperitoneally.
During the experiment, the spirit, activity, fur, appetite and stool of the rats were observed every day. The kaolin intake of rats was measured every 24 h. All animals were anesthetized by intraperitoneal injection of 2% nembutal (45 mg/kg B.W.) before sacrifice.
The co-localization of CaM and 5-HT3R in small intestine and brain were detected by immunofluorescence. The brain and small intestine tissues were embedded in paraffin and sliced into 4 μm sections. The slides were blocked with goat serum for 30 min at room temperature and incubated overnight at 4℃ with the following antibodies: rabbit anti-CaM (1: 2000, Abcam, #ab45689); rabbit anti-5HT3R (1: 2000, Abcam, #ab13897). After that, the slices were incubated with corresponding Fluor-conjugated secondary antibody (1: 600, Servicebio, #GB21303) at room temperature for 1 h. Nuclei of cells were stained with DAPI. The expression of protein in cells was observed by fluorescence microscope (Nikon, Japan).
Western blot analysis.
Total proteins of the brain and intestinal tissue were extracted using RIPA lysis buffer (P1003B, Beyotime, China), which containing protease and phosphatase inhibitor. The protein concentration was detected with the BCA protein quantitative kit (P0011, Beyotime, China). Proteins were electrophoresed by SDS-PAGE gel and transferred to a PVDF membrane. After blocked in 5% non-fat dry milk for 1h at room temperature, the membrane were incubated separately at 4℃ overnight with the following antibodies: CaMKII(1: 2000, Abcam, #ab22609), p-CaMKII (1: 2000, Abcam, #ab171095), ERK (1: 2000, CST, #9107S), p-ERK (1: 2000, CST, #4370S). Then membranes were washed and incubated for 1h at room temperature with an anti-rabbit or anti-mouse secondary antibody. Immunoreactive complexes were incubated in ECL kit and exposed for film using BioSpectrum Imaging System (UVP, Upland, CA). The gray value of the protein was measured with Image-J software.
Graphpad Prism 6 statistical software (San Diego, CA, USA) was used for data analysis. All data were expressed as mean ± SEM. Two-way analysis of variance (ANOVA) was used to compare differences between multiple groups, and the results were analyzed by Tukey’s multiple comparisons test. P value less than 0.05 was considered to be significant.