Isolation of ADSCs and differentiation of ADSCs to SCs
The experiments were approved by the Animal care and Experiments committee of the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The SD male rats (65–75 g, 4 weeks) were obtained from the Kaixue Biotechnology (Shanghai, China), following sacrificed. ADSCs were isolated from SD rats as descripted in previous study [2], subsequently cultured in Dulbecco’s Modified Eagle’s Medium/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C with 5% of CO2. In addition, the ADSCs separated from rats differentiated into Schwann cells (SCs) as descripted in previous study [2]. The normal SCs acted as a control.
Cell treatment and transfection
ADSCs (at 80% confluence) were treated with ICAII in different concentrations (10−9–10−5 mol/L), The cells without ICAII treatment was as a control. In addition, let-7i mimic (let-7i), negative control mimic (miR-NC), let-7i inhibitor (in-let-7i), negative control inhibitor (in-miR-NC), pcDNA3.0 vector (pcDNA), or STAT3 overexpression plasmid (STAT3) were also transfected into ADSCs with or without ICAII (10−7 mol/L) treatment. The treated or transfected cells were used for following experiments.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay
Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For let-7i expression, total RNA was reversely transcribed into cDNA using One Step Prime Script miRNA cDNA Synthesis kit (Qiagen, Valencia, CA, USA). To quantify the level of STAT3, nerve growth factor (NGF), neurotrophin-3 (NT-3), S100β, and P75 neurotrophin receptor (P75), total RNA was reversely transcribed into cDNA using the prime-script reagent kit (Takara, Dalian, China). qRT-PCR was performed using SYBR green (Biosystems, Foster City, CA, USA). The primers were listed as follows: STAT3 forward, 5′-CACCCATAGTGAGCCCTTGGA-3′, and reverse, 5′-TGAGTGCAGTGACCAGGACAGA-3′; NGF forward, 5′-CCAAGGACGCAGCTTTCTATC-3′, and reverse, 5′-CTGTGTCAAGGGAATGCTGAAG-3′; NT-3 forward, 5′-TGTGGGTAGCCGACAAGTC-3′, and reverse, 5′-GAGTTCCAGTGTTTGTCATC-3′; S100β, forward 5′-TCACTGAGGGACGAAATCAACAC-3′, and reverse, 5′-GGTGCTATTGGTAGTCTGCCTTG-3′; P75 forward, 5′-GAGGGCACATACTCAGACGA-3′, and reverse, 5′-CTCTTCGCATTCAGCATCAG-3′; β-actin forward, 5′-ATGGATGACGATATCGCTGC-3′, and reverse, 5′- CTTCTGACCCATACCCACCA-3′; let-7i forward, 5′-GGGGTGAGGTAGTAGTTTGT-3′, and reverse, 5′-TGCGTGTCGTGGAGTC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. U6 and β-actin were used as internal controls for miRNAs or mRNAs, respectively. Relative expression was calculated using the 2−∆∆Ct method.
Western blot assay
Protein form treated or transfected cells were collected and subsequently measured using BCA Protein Assay Kit (Solarbio, Beijing, China). Then, each protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Roche Diagnostics, Indianapolis, IN, USA). After that, the membranes were blocked with 5% BSA for 1 h and then incubated with the primary antibody solution (STAT3, NGF, NT-3, S100β, P75, or β-actin; Abcam, Cambridge, MA, USA) at 4 °C overnight. The blots were probed by horseradish peroxidase-conjugated secondary antibody solution (Abcam) for 1 h. Finally, protein bands were visualized using PierceTM ECL western blotting substrate (Thermo Fisher Scientific). Captured the signals using films in dark room.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay
Cell proliferation was measured by MTT assay. Briefly, the treated or transfected cells were seeded into 96-well plates and cultured for 48 h. MTT reagent solution (Thermo Fisher Scientific) was added to each well and incubated at 37 °C in 5% CO2 for 4 h. The optical density (OD) was detected at a wavelength of 450 nm using an Elx800 reader (Bio-Tek Instruments Inc., Winooski, VT, USA).
Luciferase reporter assay
The 3′-UTR of STAT3 containing let-7i binding sites were amplified by PCR and inserted into pMIR-REPORT™ (Thermo Fisher Scientific) to construct STAT3 wild-type reporter vector (STAT3-WT). The mutant type (STAT3-MUT) was made with GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific). STAT3-WT or STAT3-MUT and let-7i mimic, inhibitor or their negative controls were transfected into ADSCs using Lipofectamine 3000 (Thermo Fisher Scientific). The luciferase activity was examined using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) according the protocols. The transfections were performed three in independent experiments.
Construction of the rat model with BCNI
Adult male SD rats (280–300 g, 9–10 weeks) were divided into six groups (n = 6): sham, BCNI, PBS, ADSCs, ICAII, and ADSCs + ICAII. The BCNI rat models were established as descripted in previous study [2]. Briefly, SD rats were anesthetized with pentobarbital sodium (Xinya Pharmaceutical Co., Ltd., Shanghai, China). In the sham group, the abdomen was then closed. In BCNI model group, the major pelvic ganglion and cavernous nerve were exposed on either side of the prostate, then direct perturbated the nerve 5 mm distal to the MPG using mosquito hemostatic forceps for 1 min. In PBS or ADSCs group: After the penis was exposed, an elastic band was applied to the base of the penis and maintained for 2 min, and a 1 × 106 rat ADSCs suspension in PBS (ADSCs group) or PBS alone (PBS group) was injected into both corpora cavernosa. In ICAII or ADSCs + ICAI group, ICAII (5 mg/kg/day) were treated with intragastric administration. The individual performing the BCNI blinded as to which rats would be receiving ICAII treatment.
Erectile function evaluation
Mean arterial pressure (MAP) and intracavernosal pressure (ICP) were applied to evaluate the EF of the rats at 4 weeks after the surgery and injection as descripted in previous study [2]. EF was analyzed with the ratio of ICP/MAP. The stimulation parameters were 16 Hz with duration of 5 ms at 5 V for 60 s with 5 min between subsequent stimulations.
Statistical analysis
Data were presented as the mean ± standard deviation. The significance of the in vitro data and in vivo data between experimental groups was determined by Student’s t test or one-way ANOVA. P < 0.05 was considered to be statistically significant.