Reagents and antibodies
Baicalin (98%), with batch number YS121121, was purchased from the Xi'an Yuansen Biotechnology Company. 3-MA, LC3-II, LC3-I, Beclin-1, P62, p-AMPK, AMPK, mTOR, and Tubulin monoclonal antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA). The CCK-8 kit, RIPA lysis buffer, polycarbonate Lucifer Yellow (dextran) and the reverse Transcription Kit were purchased from Sparkjade (Jinan, China). The Caco2 cell line was obtained from Chinese Academy of Sciences (Beijing, China). The Millicell system and fluorescence plate reader were provided by Sparkjade (Jinan, China).
Animals and experimental protocol
Mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and the animal production license number was SCXK (Beijing) 2012-0001. 10 healthy SPF-grade Balb/c H-2d male mice were used as donors (weighing 18–22 g and aged 8–10 weeks); 56 SPF-grade CB6F1 female mice were used as transplant recipients(weighing 18–22 g and aged 8–10 weeks).
Inbred Balb/c H-2d mice were sacrificed by cervical dislocation, and the bone marrow and spleen mononuclear cells were obtained aseptically. Next, CB6F1 mice were irradiated with 60co X rays, and then the transplant was completed by infusing the mononuclear cell suspension (1×107 bone marrow cells + 1×107 spleen cells), obtained from donor Balb/c mice (above), via tail vein[7].
Mice were randomly divided into the model control, low dose, medium dose, and high dose groups. Model control group mice were fed with normal saline and treated group mice were fed with 15, 30 or 60 mg/(kg·d) baicalin for 15 days immediately after modeling.
In order to examine the influecing autophagy effect of baicalin, we also compared treated with 30mg baicalin alone group, to a group treated with 30mg baicalin combined with the autophagy inhibitor 3-MA[8].All animal procedures were performed with the approval of the Animal Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. All experiments were performed in accordance with relevant guidelines and regulations.
Cell lines culture, tested by transepithelial electrical resistance
The human colon carcinoma cell line Caco-2 was grown in Dulbecco's modified Eagle's minimum essential medium (DMEM, pH 7.4) (Invitrogen) supplemented with 25 mM glucose, 10% inactivated fetal bovine serum (FBS) (Lonza), 1% penicillin streptomycin (PS) and 1% non-essential amino acid solution (Invitrogen). After the cells were stimulated by TNF-α (100 ng/ml) for 21 h at 37°C, transepithelial electrical resistance (TER) was tested to verified if the intestinal barrier dysfunction cell model was established. For the measurement of TER, 1×105 cells were seeded on polycarbonate transwell inserts with a diameter of 12 mm and a pore size of 0.4 µm. TERs were recorded using the Millicell system, and resistances obtained over a blank filter were subtracted from the obtained values. For assessment of Lucifer Yellow, or dextran, permeable transwell inserts were washed with HBSS followed by apical application of the tracer compounds. After 2 h of gentle agitation at 37°C, basal tracer concentration was measured using a fluorescence plate reader, and then used to determine relative sodium-to-chloride permeability according to the Goldmann-Hodgkin-Katz equation.
After cell model was established, cells were treated with 10, 20,40µg/ml baicalin, respectively. To confirm the autophagy effect 20µg/ml baicalin was chosen to be combined with 3-MA.
Clinical aGVHD classification criteria
Clinical score for aGVHD was determined by observing weight loss, posture, activity, hair, skin integrity, and diarrhea on the 15th day after transplantation (Table 1). The criteria for determining aGVHD was taken from work by Cooke et al[9].
Table 1
criteria | Grade 0 | Grade 1 | Grade 2 |
Weight loss | < 10% | 10–25% | > 25% |
Posture | Normal | Hunching noted only at rest | Severe hunching impairs movement |
Activity | Normal | Mile to moderately decreased | Stationary unless stimulated |
Fur texture | Normal | Mile to moderate ruffling | Severe ruffling/poor grooming |
Skin integrity | Normal | Scaling of paws/tail | Obvious areas of denuded skin |
Diarrhea | ≤ 1 time | 2–3 times | ≥ 4times |
Individual mice from coded cages received a score of 0 to 2 for each criteria (maximum score of 12), as described above.
Small intestinal mucosa histologic and pathological inflammation score analysis
Small intestinal tissue was collected from mice. The tissue was fixed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining following standard protocols. HE images were observed under a light microscope. Histological damage of the tissues was calculated using a pathological inflammation score(Table 2) which was determined by examining damage of the small intestine tissue, crypt destruction, and the degree of mucosal ulcer and the extent of cell infiltration (The scores for each criteria were added to get the pathological score of small intestine[10]). Motic Images Advanced 3.0 software was used to measure intestinal villi height and crypt depth.
Table 2
Pathological inflammation score in small intestine
Small intestine | 0 | 0.5 | 1 | 2 | 3 | 4 |
villous blunting crypt | normal | focal and rare | focal and mild | diffuse and mild | diffuse and moderate | diffuse and severe |
crypt regeneration |
crypt loss |
luminal sloughing of cellular debris |
lamina propria inflammatory cell infiltrate |
mucosal ulceration |
Detection of TNF-α and IL-10 level
The expression of tumor necrosis factor (TNF)-α and interleukin (IL)-10 was measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Small intestine tissue was collected and quickly frozen in liquid nitrogen for subsequent mRNA extraction. Total RNA was extracted using the guanidine isothiocyanate-phenol-chloroform method. Extracted RNA was reverse-transcribed using the Sparkjade reverse transcription kit and the resulting complementary DNA was analyzed for the expression of the target molecules using the LightCycler 480 real-time PCR system. The primer sequences are provided in Table 3. The resulting gene expression levels of the target molecules were normalized based on GAPDH expression.
Table 3
Gene | forward | reverse |
GAPDH | 5’-CAA CTT TGT CAA GCT CAT TTC C-3’ | 5’-GGT CCA GGG TTT CTT ACT CC-3’ |
TNF-α | 5’-CAT GCA CCA CCA TCA AGG AC-3’ | 3’-GGC CTG AGA TCT TAT CCA GCC-3’ |
IL-10 | 5’-CTA TGC TGC CTG CTC TTA CTG-3’ | 5’-AGC AGT ATG TTG TCC AGC TG-3’ |
Western Blot Analysis
Cells were lysed using RIPA lysis buffer and total protein was collected. Next 20µg of each protein sample was loaded on to 10% SDS-PAGE gels, and the protein was concentrated at 80V for 20 min and separated at 120V for ~ 1h. After the electrophoresis was stopped, the samples were transferred to PVDF at 110V at 4℃ based on the wet blotting method protocol. Each immunoblot was blocked in 5% nonfat milk TBST for 2h,then incubated with primary antibodies like LC3, P62, Beclin-1, AMPK, mTOR or β-tubulin at 4℃ overnight. The membrane was washed 3 times, then incubated with diluted secondary antibodies for 2h. After the secondary antibody solution was fully washed away, the PTG ECL chemiluminescence detection kit was used to develop the blot. Then the blot was transferred to an imaging machine for exposure and analysis.
Immunohistochemistry (IHC) analysis
The tissues of small intestine (2–3 cm) were fixed with 10% formaldehyde, decolorized, cleared, paraffin embedded and sectioned. Antigen retrieval was performed for 20 min in a pressure cooker. The sections were cooled, rinsed with PBS, incubated in a 3% H2O2 solution for 10 min and rinsed with PBS again. The tissue sections were incubated in goat serum for 20 min to remove the serum, incubated with primary antibodies against AMPK and LC3 at 37°C for 1 h, rinsed with PBS, incubated with biotin-labeled secondary antibodies at room temperature for 30 min and again rinsed with PBS. Finally, the sections were incubated in DAB substrate solution for 5 min. After been washed thoroughly in tap water, counterstained in hematoxylin, dehydrated in absolute alcohol, cleared in xylene, and subjected to microscopy. The expression of proteins was quantified using an image analysis and measuring system (Image-Pro Plus 6.0). The mean area and mean integrated optical density (mean IOD) of the expression of these proteins were calculated.
Immunofluorescence staining of LC3 in vivo and in vitro
Tissue paraffin sections of the small intestine or Caco-2 cells were incubated with primary antibody at 4 ℃ for 24 h. Then, goat anti rabbit red fluorescent second antibody Cy3 was applied for 60 min at 37℃. Samples were fixed with mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) to stain the nucleus. Images were captured with fluorescence microscope immediately. Quantification of LC3 puncta was performed using the ImageJ software[11].
DNA extraction and sequencing of mouse feces
Mice Feces were harvested with 2 ml perfectly clean eppendorf tube under sterile conditions 2 h before sacrifice and stored immediately in liquid nitrogen until processing for DNA isolation. The Genomic DNA extracted detection by 1% agarose gel electrophoresis after DNA isolation, PCR amplification were performed by ABI GeneAmp® 9700 model PCR instrumen. First, the purified DNA templates were amplified using the following primers of 16S rDNA V3-V4 regions: 5’- ACTCCTACGGGAGGCAGCAG- 3’ and 5’ - GGACTACHVGG GTWTCTAAT − 3’. AxyPrep DNA gel Recovery Kit (AXYGEN company) was used to recover PCR products by gel cutting.The PCR reaction were used TransStart Fastpfu DNA Polymerase, The reaction system consisted of PCR Mix 20 µL Reaction program: 95℃ predenaturation for3 min, 95℃ denaturation for 30s, annealing-temperature of 55℃ denaturation for 30s, 72℃ extension for 45s, a total of 27 cycles, 72℃ final extension for 10 min. 10℃ until halted by user. According to the preliminary results of electrophoresis, the PCR products were analyzed by quantifluor ™ -St blue fluorescence quantitative system (Promega company) to detection and quantification, sodium hydroxide denaturation were used to produce single stranded DNA fragments. Finally, miseq pe300 sequencer (Illumina company) were used for sequencin.
Statistical analysis
The experimental results were analyzed using SPSS 19.0 and GraphPad Prism 8.0. The results were expressed as mean ± standard deviation and analyzed by ANOVA test. P < 0.05 indicated that the difference was statistically significant.