The “whole ingredients extract” of Astragali Radix improves the symptoms of dextran sulfate sodium-induced ulcerative colitis in mice through systemic immunomodulation

Chemicals and reagents

Astragalosides (I, II, III and IV), calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pufeide Biotech Co., Ltd. (Chengdu, China). Acetonitrile (ACN) as HPLC grade was purchased from Merck (Darmstadt, Germany). All chemicals used were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q water prepared from Sigma-Aldrich Co. (Millipore, MA, USA).

Anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8a-PE were provided by eBioscience (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits IL-2, IL-6 and MCP-1 were supplied by Neobioscience Technology Co., Ltd. (Shenzhen, China). Primary antibody against COX-2 and the secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, United States), all antibodies were diluted with 1:1000. Astragaloside I, Astragaloside II, Astragaloside III, Astragaloside IV (the purities of all standards were higher than 98% by HPLC analysis) were purchased from Chengdu Pefeide Biotech Co., Ltd. (Chengdu, China). Acetonitrile (ACN) purchased from Merck (Darmstadt Germany) is HPLC grade. And other chemicals used were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Herbs and herbal extracts

Astragali Radix (Astragalus, Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao) was purchased from Guangjitang CSPC Pharm Group (Guizhou, China). The herb sample was authenticated by the Dr. YU Hua (Macao, China) and the voucher specimen (HQ-2017001) was deposited in Institute of Chinese Medical Sciences, University of Macau.

The WIE and WAE were prepared with the methods as reported previously. For WIE, 400 g of powered AR was gradient-extracted with tenfold volume of 95% ethanol, 50% ethanol and water at 60 °C for 1 h for each, the filtered extracts were concentrated under reduced pressure, and then lyophilized using a Virtis freeze dryer. On the other hand, the WAE was prepared with the same protocol but using water instead of ethanol.

HPLC–MS analysis

The contents of total polysaccharides in WIE and WAE were determined using phenol–sulfuric acid method [11].

The contents of Saponins (Astragaloside I~ IV) and flavonoids (calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin) were determined by a Waters Alliance HPLC system coupled with a Water ACQUITY QDa Mass Detector (Waters Corp., Milford, USA). Samples were eluted on a Waters Atlantis T3 column (150 mm × 2.1 mm, 3.0 µm) maintained at 25 °C. Elution was performed with a mobile phase of A (0.1% formic acid in water) and B (0.1% formic acid in ACN) under a gradient program: 0–2 min, 21% B; 2–12 min, 22–25% B; 12–20 min, 25–50% B; 20–30 min, 50–70% B. The flow rate was 0.4 mL/min, and the injection volume was 5 μL. The analytes were monitored by mass spectrometry with an electrospray ion source operating in the positive ion mode (ESI +) using single ion recording (SIR). The monitored ions were m/z 269.09 ([M + H]+, formononetin), 285.05 ([M + H]+, calycosin), 431.21 ([M + H]+, ononin), 447.13 ([M + H]+, calycosin-7-O-β-d-glucoside), 891.39 ([M + Na]+, astragaloside I), 849.44 ([M + Na]+, astragaloside II) and 807.30 ([M + Na]+, astragaloside III and IV), respectively. Between two injections, the column was washed with 100% B for 3 min and equilibrated with the initial mobile phase for 5 min.

Experimental animals and treatments

Male C57BL/6 mice (22–24 g) supplied by the Faculty of Healthy Science Animal Centre of University of Macau were fed on a standard laboratory diet with free access to water at a controlled temperature of 22 ± 1 °C and relative humidity of 50% with a 12 h light/dark cycle. After one week of acclimatization, mice were randomly divided into six groups, control (CTRL), model (DSS, 3%), high dose of WIE (WIE-H, 3 g/kg), medium dose of WIE (WIE-M, 1.5 g/kg), low dose of WIE (WIE-L, 0.3 g/kg) and WAE group (WAE-H, 3 g/kg). Each group has 6 mice by random allocation. The control group was free to intake water for 14 days. Other groups were free to intake 3% dextran sulfate sodium (DSS) solution for 7 days, then changed to water for another 7 days. Extracts were administrated orally to the mice for 14 days in WIE and WAE groups. The body weight of each mouse was measured and recorded every day. All the experimental protocols were in accordance with the National Institutes of Health guidelines for the Care of Use of Laboratory Animals, and approved (reference No: UMARE-004–2020) by the Animal Research Ethics Committee, University of Macau, Macao SAR, China.

Colon index and spleen index

In the end of experiment, mice were sacrificed by CO2 inhalation, the colon was dissected, and the length of the colon was measured using a ruler. Colon index (cm/g) = length of colon (cm)/body weight (g). The spleen was immediately excised and weighed. Spleen index (mg/g) = weight of spleen (mg)/body weight (g).

White blood cell counting

Mice tail was wiped with warm water and disinfected with 75% alcohol. 5 μL of blood was collected from the tail vein and mixed with 95 μL of 0.2% acetic acid. Then the white blood cells were recorded with cell counting plate.

ELISA assay for serum and colon

At the end of the experiment, blood samples were collected from the orbits of mice and centrifuged at 4,000 rpm for 10 min at 4 °C to obtain the serum sample. Additionally, the colon tissues in different groups were lysed by cell lysis buffer (Beyotime, Jiangsu, China), and the protein concentration was detected using a BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Cytokines (IL-2, MCP-1, and IL-6) in the serum and lysate were measured using mouse ELISA kits according to the manufacturer’s protocol.

Flow cytometry analysis

At the end of the experiment, spleens were isolated from mice and gently grinded and filtrated through a 40 μm strainer. Red blood cells were removed by red cell lysis buffer. Subsequently, 5–10 × 105 splenocytes was incubated with FITC-CD3, APC-CD4 and PE-CD8a for 30 min at 4 °C in darkness and washed with PBS twice. The numbers of CD3+, CD4+ and CD8+ T lymphocytes was measured by LSRFortessa™ Flow Cytometer (BD, USA) and signified as percentage of total number of lymphocytes.

Histopathology and immunohistochemistry

The colon tissue was fixed with 10% neutral phosphate buffered formalin for 7 days. After washing with tap water, the tissues were sequentially placed in gradient ethanol by standard methods [12], and processed in xylene-ethanol mixture (45 min)-xylene (20–60 min)-melted paraffin at 70 °C (2 h). The embedded colon tissue were cut into 6 μm sections on Paraffin Microtome (Thermo, UK).

Slices were deparaffinized and rehydrated with standard methods, then processed with Hematoxylin staining for 5–10 min, differentiated with 0.5% hydrochloric acid ethanol for 30 s, and stained with 0.5% Eosin for 10 s, following by rinsing with tap water for 25 min. Finally, the slices were dehydrated with standard protocol and sealed.

For immunohistochemistry assay, slices were deparaffinized and rehydrated by standard methods [12], and subjected to antigen retrieval by microwave (320 W, 11 min). The cyclooxygenase-2 was stained according to the commercial kit (Solarbio, Shanghai, China).

Statistical analysis

All data from a minimum three experiments were presented as mean ± SD. Data were analyzed on GraphPad Prism 6.0 software based on a one-way ANOVA with Dunnet’s multiple comparisons test; P < 0.05 was considered difference significantly.

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