A total of forty-eight 10-week-old Sprague-Dawley male rats weighing 260–280 g were purchased from the Animal Center of Shandong University. The care and treatment were approved by the Animal Care and Use Committee of Shandong University (Jinan, China). Rats were maintained in specific pathogen-free environment around 23 ± 1 °C with a 12 hours light-dark cycle and supplied food and water ad libitum. All animals were adapted to the new environment for 1 week before the experiment. To establish diabetes, thirty-six rats were fasted for 12 hours, then received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ, Sigma-Aldrich Chemical Co, St. Louis, MO). Twelve rats were administered vehicle only and were used as a Sham Group. Random blood glucose levels were monitored 72 hours later after STZ or vehicle injection. Only those STZ-treated rats with random blood glucose concentrations consistently greater than16.7mmol/L were accepted as being diabetic. These rats were divided randomly into following 3 groups:1.Probucol (Sigma-Aldrich, St. Louis, MO, 500 mg/kg/day) was administered daily after intracavernous injection of MSCs (P+M group, n = 12); 2. Intracavernous injection of MSCs (1×106) in diabetic rats (M group, n = 12); 3. Diabetic group (DM group, n = 12). Initial and final blood glucose levels and body weight of all rats were recorded.
2. Isolation and cultivation of MSCs
Isolation and cultivation of bone marrow derived stem cells were performed according to previous descriptions . Typically, male Sprague-Dawley rats (80–120 g) were killed unconsciously; bone marrow tissues were washed with PBS until the bloodiness was eluted. After digestion with collagenase I and centrifugation, supernatant was discarded, and the residual cells were suspended in DMEM then cultured in humidified atmosphere and 5% CO2 at 37˚C for 48h. Cells were passaged when they reached about 90% confluence and passage 3 cells were used in vivo and vitro experiments. To verify the cellular identity of cells, fluorescence-activated cell sorting was employed via the usage of CD90, CD29, CD34 and CD45 markers.
3. The label, intracavernous injection and observation of MSCs
The label of MSCs using Cm-Dil is performed under protocol. After anesthesia, the prepuce was rolled up to expose penis, the needle was inserted by 3–4mm under the microscope. MSCs (1.0 × 106 cells in fresh PBS 100 μl) or with vehicle (fresh PBS 100 μl only) was injected into the middle of the left corpus cavernosum. An elastic band was placed at the base of the penis immediately before MSCs injection and was removed 3 minutes after the injection. Post-transplantation in 3days, 1week, 2weeks, penis was harvested to make frozen section and underwent observation in fluorescent microscope.
Protein extraction and Western blotting were performed as previously described . tissues and cells were lysed with RIPA buffer containing protease inhibitor cocktail, and the protein concentrations of tissue lysates and cell lysates were determined by BCA assay. Samples containing 20 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk and incubated at 4°C overnight with primary antibodies against LC3 (1:3,000, Abcam), p62 (1:1,000, Cell Signaling Technology), Nrf2 (1:1,000, Cell Signaling Technology), HO–1 (1:1,000, Abcam),Beclin1 (1:1,000 Cell Signalling Technology), Bcl–2 (1:1,000,Cell Signaling Technology), Bax (1:1,000, Abcam), Caspase3 (1:1,000, Cell Signaling Technology),Cleaved-Caspase3 (1:2000;Cell Signaling Technology). After hybridization of secondary antibodies, the resulting was visualized with an LAS3000 Image Analyzer (Fujifilm, Tokyo, Japan) and data were analyzed using Multigauge software (Fujifilm).
Penile tissue was fixed in fresh 4% paraformaldehyde, and then were immersed in 30% sucrose in PBS overnight at 4°C. The fixed tissues were cry embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cut into 5 μm sections before mounting on slides. After permeabilization and blocking, the slides were incubated with primary antibodies, including rabbit anti-α smooth muscle actin (α-SMA, 1:1000; Abcam) and rabbit anti-von Willebrand factor (vWF, 1:2000; Abcam) at 4°C overnight. At room temperature, the sections were rinsed and incubated with Alexa Fluor–594 conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were stained by 4′,6-diamidino–2-phenylindole (DAPI, Invitrogen) for 5 min. Slides were visualized under a fluorescence microscope (Leica, Heidelberg, Germany).
6. Masson’s Trichrome Stain
Masson’s trichrome stain was used to evaluate the smooth muscle cell and collagen fibril expression in cavernous tissue. Three micrometer sections of formalin-fixed, paraffin embedded tissues were deparaffinized in xylene (3 washes for 3 minutes each) and hydrated in graded ethanol to distilled water. The slides were then stained with Masson’s trichrome stain kit (Dako Sciences, Glostrup, Denmark), followed by dehydration in graded ethanol to xylene.
7. Determination of cell viability using CCK8
The CCK–8 assay was used to measure MSCs cell viability. MSCs (1, 000/well) were seeded in 96-w plates overnight. Cells were incubated with different concentrations (100, 150, 200 and 250 μM) of H2O2 for 12 h; normal culture media were used for the control group. Then, cells were first co-cultured with Probucol (50, 100 and 150 μM) for 12 h and then exposed to H2O2 (250 μM) for 12 h. Normal culture media were used for the control group. At the prespecified time points, 10μL of CCK–8 solution (DOJINDO, Kumamoto, Japan) was added to the cells. After incubation for another 4 h, the optical density (OD) values were determined at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). Each group was tested in triplicate in three replicates.
8. Erectile Function evaluation
Measurements of maximal intracavernous pressure (max ICP) and the ratio of max ICP/mean systemic arterial pressure (MAP) were used to assess erectile function. After treatment, rats were anesthetized with 5% sodium pentobarbital. PE–50 (Intramedic; Becton Dickinson & Co., Sparks, MD) tubes were inserted into the left carotid artery of each rat to detect continuous measurement of MAP. A 26-gauge needle filled with heparin (250 U/ml) was inserted into the cavernous body of the penis to detect ICP. After finding and dissociating cavernous nerve (CN), an electrical stimulus with a frequency of 15 Hz and a pulse width of 5 ms was used to stimulate 60s. ICP and MAP were measured continuously by a BL–420V pressure transducer system (AD instrument).
9. Autophagic flux measurements
To detect autophagic flux, RFP-GFP-LC3 reporter plasmid (1 μl/ml, Addgene, Cambridge, MA, USA) was transfected into MSCs using lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. Then, the transfected cells were grouped into 3 groups: Sham Group, H2O2 Group (200 μM), Probucol Group (150 μM). The cell images were obtained using Olympus FV1000 laser scanning confocal microscopy (Olympus, Tokyo, Japan).
10. ROS assay
Cellular ROS levels were measured using cell permeable probe 5-(and–6)-chloromethyl–2’,7’-dichlorodihydrofluorescein diacetate (CM- H2DCFDA, Thermo Fisher, Waltham, MA, USA). Cells were loaded with 10 μM H2DCFDA in DMEM (phenol red-free) for 1 h. After washing cells twice with DMEM, fluorescence was measured with an Envision 2104 Multilabel reader (Perkin Elmer, Waltham, MA, USA).
11. Statistical analysis
All experiments were repeated at least three times, and all data were presented as means ± S. E. Statistical significance was analyzed by the SPSS version 22.0 software (SPSS, IL, USA). Differences between two groups were assessed using the Student t-test, and between multiple groups using one-way ANOVA. Values of P < 0.05 were considered significant.