Drugs and reagents
TFA was extracted from flowers of Abelmoschus manihot by the Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing, China. Abelmoschus Manihot flowers was purchased from Anhui xiehecheng co.,ltd (Batch No 20092701). The source and production process of the Abelmoschus Manihot flowers are in accordance with Chinese Pharmacopoeia standards (2015 version). Dr Fengyu Zhu identified the Abelmoschus Manihot flowers in Department of Pharmacy, Jiangsu Provincial Hospital of Traditional Chinese Medicine. Abelmoschus Manihot flowers was immersed in 75% ethanol for 60 min. refluxed the mixture for 60 mins at 90℃,then filtered with analytical filter paper. Finally, rotary evaporation was used to evaporate the extracts under vacuum at 60℃5. DSS (molecular weight of 36-50 kDa) was provided by German MP Biopharmaceutical Company. Prednisone acetate tablets (PAT) was purchased from Cisen Pharmaceutical Co. Ltd. (Jining, Shandong, China). Mucin (from porcine stomach) was purchased from Sigma (USA).
Animals
Six-week-old male C57BL/6J mice were purchased from Beijing Si Pei Fu Laboratory Animal Technology Co. Ltd. The mice were raised in the laboratory of Basic Pharmacology, Affiliated Hospital of Nanjing University of Chinese Medicine (Nanjing, Jiangsu, China). Sterilized standard rodent chow food and sterilized water were not restricted during the experiment, the temperature was controlled at 23 ± 1°C, the humidity was controlled at 50 ± 5%, and the light system was set at 12 hours/day. The care and use of the animals were followed the animal welfare guidelines, and all the experimental protocols were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine.
Induction of colitis and treatment
As shown in Fig. 1A, colitis was induced by 2.5% DSS in the drinking water adlibitum for 7 consecutive days (days 1-7). The mice were randomly allocated after modeling. The mice were supplemented daily with 200µL of phosphate buffered saline (vehicle), TFA (125mg/kg, 62.5mg/kg) or PAT(2.5mg/kg) by intragastric gavage for 7 consecutive days (days 8-14). Mice were sacrificed on 15 day, and the colon was obtained to measure the colon length. 1 cm of the distal colon tissues was collected for histologic examination. All samples were stored at -80℃ for further analysis.
Disease activity index (DAI)
The changes of DAI were measured using the following criteria: (1) weight loss (%), (2) stool consistency and (3) blood in feces as previously described (Table1)6.
Table 1. Disease activity index(DAI)
Weight loss (%)
|
Stool consistency
|
Occult blood
|
Score
|
None
|
Normal
|
Negative
|
0
|
1-5
|
-
|
-
|
1
|
5-10
|
Loose stools
|
Hemoccult+
|
2
|
10-20
|
-
|
-
|
3
|
>20
|
Diarrhoea
|
Gross bleeding
|
4
|
Hematoxylin and eosin(H&E) Staining
Distal colon specimens were fixed for 48 h in 4% formalin after mice were sacrificed. Then, the distal colon specimens were paraffin-embedded. Finally, the sections were segmented and stained with hematoxylin and eosin, and pathological changes were observed with a light microscope.
Immunohistochemical staining
First, paraffin sections were dewaxed in water; antigen repair was performed, and endogenous peroxidase was blocked. The sections were blocked in serum, which was followed by primary antibody application, secondary antibody application, DAB color development, nuclear staining, dehydration and sealing. Finally, the positive expression of mucin-2(MUC2); Kruppel-like factor 4(KLF4) and zonula occludens-1(ZO-1) in the colonic mucosal epithelial cells was observed under the microscope.
Assessment of cytokine level in serum
The level of tumor necrosis factor-α(TNF-α); interleukin-1β(IL-1β) and interleukin-6(IL-6) were measured using commercial ELISA kits (Jin Yi bai Biological Technology Co. Ltd., Nanjing, China) according to the manufacturer’s instructions.
Quantitative Real-time Polymerase Chain Reaction (qPCR)
Total RNA was extracted from colon tissues using TRIzol reagent, and the concentration of RNA was measured and then reverse transcribed according to the manufacturer's instructions using 5x PrimerScript. The primer sequences are shown in Table 2. GAPDH was used as the reference gene. The ΔΔCt method was used to compute the relative expression. The abundance of A. muciniphila in stool samples was quantified by quantitative PCR as described in Everard et al7.
Table 2. Primers used in the real-time PCR assays
Gene
|
Primer Sequences (5’-3’)
|
TNF-α
Forward
Reverse
IL-1β
Forward
Reverse
IL-6
Forward
Reverse
IL18
Forward
Reverse
IL-17a
Forward
Reverse
CCL2
Forward
Reverse
MUC2
Forward
Reverse
KLF4
Forward
Reverse
ZO-1
Forward
Reverse
GAPDH
Forward
Reverse
|
CACCACGCTCTTCTGTCTACTG
GGGCTACAGGCTTGTCACTC
CTCGTGCTGTCGGACCCAT
GCTTGTGCTCTGCTTGTGA
GAGGATACCACTCCCAACAGACC
AAGTGCATCATCGTTGTTCAT
GTGAACCCCAGACCAGACTG
CCTGGAACACGTTTCTGAAAGA
GTTAGGGTGCTTTAGGTCC
TAACAATGAGTTTCTGTACG
TGCCCTAAGGTCTTCAGCAC
AAGGCATCACAGTCCGAGTC
TGCCCACCTCCTCAAAGAC
TAGTTTCCGTTGGAACAGTGAA
CAGGATTCCATCCCCATCCG
GAGAGGGGACTTGTGACTGC
GGGGCCTACACTGATCAAGA
TGGAGATGAGGCTTCTGCT
AGAACATCATCCCTGCATCC CTGGGATGGAAATTGTGAGG
|
16S rDNA Gene High-throughput Sequencing
The V3-V4 variable region of the bacterial 16S rRNA gene was amplified by F338 (5'-ACTCCTACGGGAGGCAGCA-3') and R806 (5'-GGACTACHVGGG TWTCTAAT-3'). On the Illumina MiSeq platform, the extracted PCR products were analyzed by isomolecular 250-bp double-terminal sequencing. The original pyrophosphate sequence was uploaded to the NCBI Data Center database SRA (Sequence Read Archive). High-quality sequence merge overlaps generated fastq files. QIIME (version 1.9.1) software was used to multichannel decode and quality control filter the fastq file output.
Bacterial strains and growth curve
A. muciniphila strain ATCC was cultured in brain heart infusion (BHI) medium in tubes at 37◦C in anaerobic chamber. A. muciniphila were collected in log phase and diluted with sterile phosphate-buffered saline (PBS) to 3 × 108 colony-forming units/mice for gavage. To acquire the growth curve of A. muciniphila, different concentrations (1, 10, and 100 µg/mL) of TFA was added into BHI medium, and then add A. muciniphila suspension to make the final concentration of bacteria 106cfu /ml. The growth profile was evaluated by intermittently measuring absorbance at 600 nm every 5 hours8. Mucin was added to the final concentration of 4 g/L. TFA was dissolved in PBS. Each experiment was repeated three times.
Statistical analysis
Graphing was performed using GraphPad Prism 8 (GraphPad Software, Inc.). One-way analysis of variance was applied to compare differences between multiple groups. When only two groups were compared, Student’s t-test was conducted. A value of P < 0.05 indicated that the difference was statistically significant. All plots are shown as the mean ± standard error of the mean (S.E.M). P<0.05 was considered as statistically significant.