2.1. Patient selection and sample processing
A total of 90 UC patients were selected for the study according to the opinions of the third European Crohn’s and Colitis Organization (ECCO) consensus on the diag-nosis and management of UC. The patients were treated at the First Affiliated Hospital of Kunming Medical University from January 2018 to December 2019. Disease severity was classified by the Mayo Score as mild, moderate, or severe. The lesions were all of left semicolon type and only mesalazine was used for treatment within 3 months before hospitalization (all patients who needed other drugs, such as glucocorticoids and immunosuppressants, to control the disease underwent colonoscopy and biopsy as soon as possible before medication, so as to reduce the interference of those drugs on the results of this study). At the same time, 60 healthy volunteers for colon cancer screening were chosen as controls. The required age range was between 18 and 60 years. Table 1 shows the information and characteristics of enrolled participants. All colonoscopy examinations were performed by specialists in the Department of Gastroenterology, and the same number of tissue samples were collected from the same fixed site (sigmoid colon). All enrollees signed the informed consent form.
The hsf2−/− C57BL/6 mice that had been successfully established were reared in the SPF laboratory animal house. The genotypes of the mice were identified by PCR. Homozygous mice were used in this study. A total of 40 male mice (7–8 weeks) were divided into wild-type (WT, 20) and knockout (KO, 20) groups. Then each group was randomly divided into 2 subgroups, with 10 mice in each (WT + H2O, WT + DSS, KO + H2O, KO + DSS). The WT + H2O and KO + H2O groups were given distilled water for 7 days, and the WT + DSS and KO + DSS groups were given 3% dextran sulfate sodium(DSS) for 7 days. The disease activity index(DAI) of all mice was measured every day. On the 8th day, all mice were killed, and colon specimens were taken to measure their length. Each mouse colon was cut into 5 segments: one was immediately put into 2% glutaraldehyde for electron microscope observation, 2 were stained with hematoxylin eosin(HE) and detected by immunohistochemistry, and 2 were stored at -80 ℃ for RT-PCR and Western blotting.
2.3. Cell culture
Human colon adenocarcinoma(Caco-2) cells were used in this study. The Caco-2 cells were obtained from the cell culture library of the Kunming Institute of Zoology, Chinese Academy of Sciences. After the cells were resuscitated in a water bath at 37 ℃ constant temperature, they were cultured in 1640 medium containing 10% fetal bovine serum and then incubated under 5% CO2 at 37 ℃.
2.4. Regulating HSF2 expression by transfecting lentiviral
HSF2 overexpression and knockdown and negative control lentiviruses were purchased from Shanghai GeneChem Co., Ltd. Viruses were transfected into Caco-2 cells according to the lentivirus product instructions. The MOI of Caco-2 cells was set at 20. In addition, a certain amount of HittransG P was added to the transfection group to make a final concentration of 1 ug/mL, so as to improve transfection efficiency. Ac-cording to the experimental method of Wang et al., an appropriate amount of LPS was added to the cell culture medium to stimulate cell inflammation.
2.5. Transmission electron microscope (TEM) cell culture
TEM (JEM-1400 Flash, Nippon Electronics Corporation) was provided by Kunming Medical University. The collected colonic mucosal tissues were immediately placed in 2.5% glutaraldehyde and stored at 4 ℃ in a refrigerator. PBS was first used for repeated washing 3 times, then osmium acid(1%) was used for fixation for 2 hours, and then washing again 3 times for 10 min each time. Dehydration was then carried out by eth-anol and acetone(paying attention to the temperature). Finally, after handling the embedding solution, staining by uranyl acetate and lead citrate, and drying, the speci-mens were observed under TEM.
The intestinal mucosal tissue specimens were embedded with paraffin, fixed with 4% paraformaldehyde, and sliced continuously with a slicer. They were then defatted and hydrated in a solution of xylene, then treated with a citrate buffer(0.01M) under 800 W microwave, and incubated for 10 minutes in 3% hydrogen peroxide at room temperature. After that, 50 uL of primary antibody (PARL or PINK1 or Parkin, diluted 1:200) was added to the slices, followed by incubation at 4 ℃ overnight. Another 50 uL DAB was added for color development, and the dyeing time and degree were controlled under microscope observation. Double steam water was used to wash twice, one minute each time, and hematoxylin was used for re-staining(1 minute), followed by rinsing with 1% ammonia after removal. The slides were successively immersed in 95% and 100% eth-anol, and were dehydrated twice in total. After blow-drying, they were sealed with neutral resin and the results were observed under the microscope.
The ROS levels in tissues were observed by laser confocal microscopy. First, appropriate OCT encapsulating agent was poured into the premarked specimen box. Then, the mucosal colon tissues were quickly placed in the specimen box and stored at -80 ℃. The second part was staining. After the frozen section was taken out, ROS dye was added, and the sample was incubated in darkness for 30 minutes at 37 ℃. Second, nuclear staining was carried out. Finally, the tablets were sealed with anti-fluorescence sealing tablets. The slices were observed under a laser confocal microscope and images were collected (blue light: DAPI ultraviolet excitation wavelength 330–380 nm, emission wavelength 420 nm; red light: CY3 excitation wavelength 510–560 nm, emission wave-length 590 nm).
2.8. Reverse transcription polymerase chain reaction (RT-PCR)
This part of the experiment was conducted according to the Platinum® SYBR® Green qPCR kit (TaKaRa, Japan) instructions. Each part of the experiment was repeated at least 3 times. The primer sequence in this study was designed and synthesized by TaKaRa Bio Inc. as follows:
PARL (Forward): 5’-CCTATAAGAACACTCGTGAAGCC-3’
PARL (Reverse): 5’-CCAGTCAGCTTTTATGCCATC-3’
PINK (Forward): 5’-GGTGTCAGGCTGGGGCAA-3’
PINK (Reverse): 5’-TGGCTTCATACACAGCGGC-3’
Parkin (Forward): 5’-TCTTCGGCATCTTGTCTG-3’
Parkin (Reverse): 5’- CTGGGAGTCGTAGTTCTAACG − 3’
GAPDH (Forward): 5’-CAAGTTCAACGGCACAGTCA-3’
GAPDH (Reverse): 5’-CACCCCATTTGATGTTAGCG-3’
2.9. Western blotting
Mouse tissue samples or cells were homogenized in lysis buffer containing 1% protease inhibitors. Protein assay kit was used to determine the protein concentration. The antibodies used in this study were anti-PARL (1:1000, Santa Cruz), anti-PINK1 (1:1000, Santa Cruz), anti-Parkin (1:1000, Santa Cruz), anti-β-actin (1:5000, Abcam), and anti-GAPDH (1:5000, Abcam). The results were analyzed by ImageJ.
2.10. Statistical analysis
SPSS 25.0 software was used for statistical analysis of all data. Measurement data were expressed as mean ± standard deviation. One-way ANOVA was used for measurement of data between groups, and LSD was used for pairwise comparison between groups. In the figures, * P < 0.05, ** P < 0.01and **** P < 0.0001.