Animals and agents
C57BL/6 mice were purchased from Shanghai Super-B&K Laboratory Animal Corp., Ltd. (Shanghai, China) and kept at 22°C under a 12-h light/dark cycle with unlimited access to water and standard rodent diet. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of Second Military Medical University. Anisodamine (ANI) was obtained from Shanghai First Biochemical Phamaceutical Company (Shanghai, China). Neostigmine (NEO) was obtained from San-Wei Pharmaceutical Company (Shanghai, China). 3-methyladenine (3-MA, Cat.No. M9281) and methyllycaconitine (MLA, Cat.No. M168) were obtained from Sigma-Aldrich (Saint Louis, MO, USA).
Caco-2 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured with DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) at 37°C in a humidified incubator with 5% CO2. Caco-2 cells were primed with 10 ng/ml LPS (Sigma, Louis, MO, USA) for 1 h, and then stimulated with 3% DSS in the presence or absence of 10, 30, 100 μM ANI/NEO compound for 24 h.
Induction of Colitis
Colitis was induced with 3% (w/v) DSS (mol.wt. 36,000 to 50,000 kDa, MP Biomedicals, Solon, OH, USA) dissolved in drinking water for 7 days as previously described. Briefly, C57BL/6 mice were provided with a solution of filtered water containing 3% DSS ad libitum. Every other day, the DSS solution was replenished. Control mice received only normal drinking water.
Clinical score and histological analysis
Body weight, the disease activity index (DAI) were determined by two investigators blinded to the treatment groups. The DAI combines scores for diarrhoea and the presence of occult or overt blood in the stool. A scoring system was used to assess diarrhea and the presence of occult or overt blood in the stool . Changes of body weight are indicated as loss of baseline body weight as a percentage. Postmortem, the colon was removed and pieces of colonic tissue were used for ex vivo analysis. For histology, rings of the transverse part of the colon were fixed in 4% buffered formalin and embedded in paraffin. Sections were stained with H&E according to standard protocols. Histological scoring was performed in a blinded way by a pathologist as previously described . Briefly, no evidence of inflammation was scored as 0, low level of inflammation with scattered infiltrating mononuclear cells as 1, moderate inflammation with multiple focim as 2, high level of inflammation with increased vascular density and marked wall thickening as 3, maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet cells as 4.
Reverse Transcription and Real-time PCR
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for the extraction of total RNA from colon tissuess and Caco-2 cells. Reverse transcription was conducted for the extracted RNA to obtain the cDNA with PrimeScript™ RT Master Mix (Takara, Otsu, Shiga, Japan). Real-time PCR was then conducted in the LightCycler quantitative PCR apparatus (Stratagene, Santa Clara, CA, USA) using the FastStart Unitversal SYBR Green Master (Roche, Konzern-Hauptsitz, Grenzacherstrasse, Switzerland). In order to eliminate the effect of DSS on PCR, 1.25 mM spermine was added into PCR reactions. The expression values of INF-γ, TNF-α, IL-6 and IL-22 were normalized to GAPDH in the same sample and then normalized to the control. After transfection with control siRNA or ATG5 siRNA, Caco-2 cells were primed with 10 ng/ml LPS for 1 h, and then stimulated with 3% DSS in the presence or absence of 100 μM ANI/NEO compound for 24 h. The expression values of TNF-α, IL-1β and IL-6 were were normalized to β-actin in the same sample and then normalized to the control. The primer sequences are listed in Table 1.
Enzyme-linked Immunosorbent Assay
Levels of INF-γ and TNF-α in colonic tissue were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Westang Biotechnology, Shanghai, China).
Proteins were extracted from colonic tissue and Caco-2 cells using a standard extraction reagent supplemented with protease inhibitors (Kangchen, Shanghai, China). Protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). The proteins were separated using SDS-PAGE and electro-transferred to nitrocellulose membranes and then incubated with the rabbit anti-Beclin-1 monoclonal antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-LC3 polyclonal antibody (1:500; Novus Biologicals, Littleton, CO, USA), rabbit anti-p62 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA) and mouse anti-beta-actin antibody (1:1000, Beyotime Biotechnology, Shanghai, China). After that, the membranes were incubated with a Donkey anti-rabbit or Donkey anti-mouse secondary antibody (1:5000, LI-COR Biosciences, Lincoln, NE, USA) accordingly. The image was acquired with an Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA).
Autophagy Flux Assessment
Caco-2 cells were seeded on the cultural slides and transfected with tandem fluorescent AdPlus-mCherry-GFP-LC3B plasmid (Beyotime Biotechnology, Shanghai, China) when the confluence reached to 50–70% . In brief, cultured in DMEM supplemented with 10% FBS for 24 h, cells were incubated with plasmids for 8 h and then changed into DMEM supplemented with 10% FBS for another 48 h to ensure the expression of the genes. After transfection, cells were primed with 10 ng/ml LPS for 1 h, and then stimulated with 3% DSS in the presence or absence of 10, 30, 100 μM ANI/NEO compound for 24 h. Cellular autophagosomes (G+C+) and autolysosomes (G−C+) were detected by confocal microscopy (Leica TCS SP8, Leica, Biberach, Germany). Total number of puncta (>1 μm) per cell was counted.
Transient transfection and siRNA
The following siRNAs against ATG5 (Gene ID: 11793) were synthesized by Genepharm Biotech (Shanghai, China): siRNA1, 5’-GCCUGUAUGUACUGCUUUATT-3’, 5’-UAAAGCAGUACAUACAGGCTT-3’; siRNA2, 5’-GAACCAUACUAUGCAUUAUTT-3’, 5’-AUAAUGCAUAGUAUGGUUCTT-3’; siRNA3, 5’-GGGAAGAAGAGAUUGUUUATT-3’, 5’-UAAACAAUCUCUUCUUCCCTT-3’. All siRNAs consisted of 21 nucleotides and contained symmetric 3’ overhangs of two deoxythymidines. Caco-2 cells were transfected with siRNAs as previously reported .
Animals and treatment
For the detection of ANI/NEO compound effection on symptoms of DSS-induced colitis, C57BL/6 mice were received 3% DSS for 7 days and treated with vehicle, ANI (20 mg/kg, i.p.), NEO (40 μg/kg, i.p.) or ANI and NEO compound (ANI/NEO, 5 mg/kg and 10 μg/kg, 10 mg/kg and 20 μg/kg, 20 mg/kg and 40 μg/kg, i.p.) twice a day from day 3 to day 7. Body weight, the disease activity index, colon length and histologic score were examined as mentioned above. Protein levels of LC3-II/LC3-I ratio, Beclin-1, P62 and mRNA levels of INF-γ, TNF-α, IL-6, IL-22 in colonic tissue were measured as mentioned above. To test the influence of autophagy and 7nAChR on the protective effect of ANI/NEO combination in DSS-induced colitis, C57BL/6 mice were received 3% DSS for 7 days and treated with vehicle, 3-MA (10 mg/kg, i.p.), MLA (10 mg/kg, i.p.), ANI/NEO compound (20 mg/kg and 40 μg/kg, i.p.), 3-MA+ANI/NEO compound or MLA+ANI/NEO compound respectively. 3-MA or MLA was given daily and ANI/NEO compound twice a day from day 3 to day 7. Body weight, the disease activity index, colon length and histologic score were examined and the expression of INF-γ, TNF-α, IL-6, IL-22 in colonic tissue were tested with RT-PCR as mentioned above.
Data are expressed as means ± SEM. For nonparametric data, a Kruskal-Wallis test followed by a Dunn’s post-test was used. For continuous variables, the statistical differences between groups were determined by one-way analysis of variance followed by a Student-Newman-Keuls test. Statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.