Materials and methods
Cell culture and experimental design
Mouse fibroblast L929 cells were obtained from Boster Co, Ltd (Wuhan, China). Cells were cultured in 1640 (Jenom Biotech Co, Ltd Hangzhou, China) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1% antibiotics (100 IU penicillin and streptomycin) in a humidified incubator at 37°Cand 5% CO2. Cells in the logarithmic growth phase were used in this experiment.Cells were divided into six groups: Control group,cells without intervention; CMSgroup, cells were subjected to cyclicmechanical strain of 5333 μ; shNfe212 group (Nrf2 silencing), cells were transfected with LV-shNfe212; shNfe212CMSgroup, cells transfected with LV-shNfe212 were subjected to cyclic mechanical strain of 5333 μ; LV-Nfe212 group (Nrf2 over-expressing), cells were transfected with LV-Nfe212; LV-Nfe212+CMSgroup, cells transfected with LV-Nfe212 were subjected to cyclicmechanical strain of 5333 μ.
Cell transfection
Lentiviruses carrying shRNA targeting mouse Nrf2 lentiviral vectors(shNfe212) and lentiviruses carrying Nrf2expressing lentiviral vectors (LV-Nfe212) were from GeneChem (Shanghai, China). Three short hairpin RNA (shRNA) duplexes were designedto target different sites of mouse Nrf2 gene (GenBanknumber NM_010902). The sequences of shRNA targeted toNrf2 were shown as follows: Nfe2l2-RNAi (30845-1), 5′-CTT ACT CTC CCA GTG AAT A-3′; Nfe2l2-RNAi (30846-1), 5′-TCG CAT TGA TCC GAG ATA T-3′;Nfe2l2-RNAi (30847-1), 5′-AGG CAC AAT GGA ATT CAA T-3′. L929 cells were seeded into 6-well plates and infectedby lentivirus at MOI of 10 PFU/cell with the presence of Polybrene. The cells were cultured in medium containing puromycin of 4 μg/mL for the selection of stable clones. The clones stably up or down regulated Nrf2 expression were identified and verified by quantitative real-time polymerase chain reaction(q-PCR) and Western Blot. Aftercells transfected by lentivirus, western Blot showed that the protein expressionof Nrf2 effectively increased in LV-Nfe212 group and decreased in shNfe212 group (Figure S1A-B). q-PCRshowedthe same trend in the mRNAexpression as western Blot (Figure S1C). Cells are successfully constructed confirmed by q-PCR and western Blot.
Mechanical strain
Cells were seeded onto 15 cm2 (5 cm×3 cm) cell culture plates at a density of 5×104 cells per cm2 and cultured in regular medium. After reaching 80% confluence, cells were synchronized with serum-free medium for 12 hours and prepared for mechanical strain.Afterwards, the quiescent cells were maintained in regular medium. Strain applicationaccording to previously described methods[1], the cells were exposed to strain of 0, 5333 μ for 4 h at the frequency of 1.0 Hz using the four‑point bending device (Miracle Technology Co., Ltd., Chengdu, China). The experiment was independently repeated 3 times.
Cell apoptosis and cell cycle analysis
Cell apoptosis and cell cycle was determined by a cell apoptosis kit (Beyotime,China) and a cell cycle analysis kit (Beyotime,China)respectively. After digested from culture plates, cells were washed with phosphate-buffered saline(PBS) twice and centrifuged for 5 min at 2000 rpm. then, cells were treated according to the manufacturer’s instructions. Then, Samples were immediately analyzed using a FACS Calibur flow cytometer.The experiment was independently repeated 3 times.
Measurement of mitochondrial membrane potential
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanineiodide (JC-1, Beyotime,China) was used to detect mitochondrial membranepotential (m). In healthy cells with high m, JC1 spontaneously forms complexes known as JC1 aggregates with intense red fluorescence. By contrast, in apoptotic cells with low m, JC1 remains in the monomeric form, which shows only green fluorescence. After the cells had been subjected tomechanical stress stimulation, they were stained with JC-1 solution(10 mg/ml) for 20 min at 37°C inthe dark and washed twice with buffer (precooled at 4°C), followed by observed under the inverted fluorescence microscope andanalyzed with the Image J software.m was shown as the ratio of red to greenfluorescence intensity.
Determination of reactive oxygen species (ROS)
2',7'‑dichlorodihydrofluorescein diacetate(DCF-DA, Beyotime,China) was used to evaluate mitochondrial ROS levels. After the cells had been subjected tomechanical stress stimulation, DCF‑DA were added to cells for 25 min at 37°C in the dark, then they were rinsed with serum-freemedium three times. The ROS-associated fluorescence was observed under the inverted fluorescence microscope and Image J software was employed to analyze the images. The integrated intensity which represented the fluorescence intensity of each cell was used to reflect ROS production in different groups.
Measurement of MDA levels
Lipid peroxidation was determined by malondialdehyde (MDA) levels, using a malondialdehyde assay kit (Beyotime,China).The level of MDA was measured according to the manufacturer’s instruction.
Quantitative real-time polymerase chain reaction(q-PCR)
The total RNA was extracted by Trizol regent (Thermo Fisher Scientific, Waltham, MA). 1 μg of total RNA was added to the reaction volume of 20 μL and the cDNA was synthesized according to the manufacturer’s instruction (Takara, Tokyo, Japan). Using the ABI 7500 Real-Time PCR System (Applied Biosystems), PCR cycling conditions were set as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 34 s. Samples were run in triplicate and the expression levels of the target mRNAs were calculated and normalized to the mRNA level of GAPDH. The following primers (Sangon Biotech Co, Ltd, China) were used: GAPDH (used for normalization), forward 5-AGG AGC GAG ACC CCA CTA ACA-3′ and reverse 5′-AGG GGG GCT AAG CAG TTG GT-3′; Nrf2,forward5′-CTG GCT GAT ACT ACC GCT GTT C-3′and reverse 5′-AGG TGG GAT TTG AGT CTA AGG AG-3′. Cycle threshold (Ct) valueswere normalized with housekeeping gene (GAPDH) and the fold changewas calculated using 2-Ct method. The experiment was repeated 3 times.
Western blot
The proteins were extracted from cells using RIPA buffer containing protease inhibitors. Protein concentrations were detected by BCA protein assay kit (Beyotime,China). The protein samples were separated by 10% SDS-PAGE and were transferred to a PVDF membrane. The membrane was blocked at room temperature for 1 h in Tris-buffered saline supplemented with 5% non-fat milk powder and 0.05% Tween-20. For immunoblotting the membranes were incubated overnight at 4°C with antibodies directed against GAPDH (1:1000), Nrf2 (1:500), Bax (1:2000), Bcl-2 (1:1000), Cyt-C (1:5000), Caspase-3 (1:1000), Caspase-9 (1:2000), Cleaved Caspase-3 (1:1000)andCleaved Caspase-9 (1:2000)used as loading controls. Antibodies above were obtained from the Abcam (Cambridge, UK). The fluorescence-labeled secondary antibody (IRDye700 and IRDye800, goat antimouse/rabbit, 1:10000) was incubated at room temperature for 1h. Protein was detected by an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Each experiment was independently repeated 3 times.
Animals and experimental design
Virgin female C57BL/6 mice (n = 30, 8 weeks of age) were divided into 2 groups of 15 mice each: Control group, mice without intervention; VD group, mice underwentvaginal distension (VD) for 1 hour with an 8 mm dilator(0.3 ml saline).
Vaginal distention and urodynamic test
Vaginal distention and urodynamic test were conducted as described in our previous study[2], briefly, after anesthetized, a modified 6-F Foley catheter was inserted intothe vagina and fixed with a 5-0 silk suture and 0.3 ml distilled water was infused into the balloon for 1 h.The control group did not undergo VD and sham group just with a catheterinserted intothe vagina and fixed with a 5-0 silk suture without distilled water infused. On the day 6 after VD, an epidural catheter was implanted in the bladder. On the day 7 after VD, Leak point pressure(LPP)were detected.
TUNEL analysis
The urethra and anterior vaginal wall were removed and embedded in paraffin and cut into4-μm-thick slices and fixed to glass slides. TUNEL assay was employed to quantify the degree of apoptosis in the urethra and anterior vaginal wall. The sections were incubated interminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reactionmixture (Milipore, USA) for 60 min at 37°C, washed and subsequently counterstained with 4',6-diamidino-2-phenylindole(DAPI, 1μg/ml, Sigma) for 30 min. Results above were visualized under the inverted fluorescence microscope and quantified using the Image J software.
Statistical analysis
All data were analyzed with SPSS software and represented as mean standard deviation (SD). The differences among groups were analyzed by one-way ANOVA. Differences between two groups were compared with Dunnet t-test. Multiple comparisons were analyzed with Tukey test. Statistical differences were considered significant at P0.05.
References:
[1] Hong S, Li H, Wu D, et al. Oxidative damage to human parametrial ligament fibroblasts induced by mechanical stress[J]. Mol Med Rep. 2015, 12(4): 5342-5348.
[2] Min J, Li B, Liu C, et al. Therapeutic Effect and Mechanism of Electrical Stimulation in Female Stress Urinary Incontinence[J]. Urology. 2017, 104: 45-51.