1.Cell identification and culture
HUCMSCs were donated by Shandong Qilu Cell Therapy Technology Co., Ltd and suspended in specialised medium (Dulbecco’s modified Eagle’s medium [DMEM, Gibco, USA] with supplements for HUCMSCs [Qilu Cell Therapy Technology, China]) and cultivated at 37°C in a 5% CO2 incubator. The fourth passage HUCMSCs were identified by flow cytometry. Flow cytometry was used to identify the HUCMSCs in the fourth passage. For surface marker verification, the following antibodies were used: CD29, CD31, CD34, CD45, CD90, and CD105 (BD Biosciences, USA).
The multilineage differentiation potential of HUCMSCs was examined using third passage cells, as previously described. For 14 days, cells were grown in adipogenic medium (DMEM containing 10% FBS, 2 mM dexamethasone, 2 mg/L insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin) and for 28 days in osteogenic medium (DMEM containing 10% FBS, 2 mM dexamethasone, 1 M sodium glycerol phosphate, 10 mM vitamin C). The capacity to develop into adipocytes and osteoblasts was then assessed using Oil red O and Alizarin red S staining.
Our team's typical approach involved isolating corpus cavernosum smooth muscle cells (CCSMCs) from rat penis and purifying them using a differential adhesion method over the course of two weeks. The CCSMCs were then grown in DMEM with 10% FBS added. CCSMCs were detected by immunofluorescence using antibodies against α-smooth muscle actin (α-SMA; 1:200; Affinity Biosciences, AF1032, USA) and desmin (1:200; Affinity Biosciences, AF5334, USA).
The co-culture system was constructed by a polycarbonate membrane of Costar Transwell Inserts (Corning, No.3412, USA). The upper and lower chambers were seeded with CCSMCs and HUCMSCs, respectively.
2. Experiments with animals
Thirty 7-week-old male Sprague-Dawley (SD) rats and thirty 7-week-old male Zucker Diabetic Fatty (ZDF) rats were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., and they were all fed adaptively for 7 days after fasting overnight. For type 1 diabetes mellitus (T1DM), streptozotocin (STZ; 60 mg/kg; Sigma-Aldrich) was diluted in citrate phosphate buffer (50mM; pH = 4.5) and injected intraperitoneally into SD rats. The rest 6 SD rats were administrated with the same buffer and placed in the control group. The blood glucose levels of all SD rats were measured three and seven days later, and rats with fasting blood glucose > 16.7 mmol/L at both tests were classified as T1DM. Purina5008 was given to ZDF rats for 4 weeks to induce type 2 diabetic mellitus (T2DM). The remaining seven rats were designated as the control group and continued to be fed with standard chow. The blood glucose levels of ZDF rats were tested three- and seven-days following induction feeding, and animals with fasting blood glucose levels > 16.7 mmol/L at both assessments were defined as T2DM.
Ten weeks later, all of DM rats survived. Following that, an apomorphine (APO) test was performed to identify the rats with DM ED, and those with a negative erection were classified as DMED rats, as in the prior study. Through the APO test, 18 rats each were diagnosed with T1DM and T2DM ED. SD rats were further separated into four groups (DMED group, n = 5; DMED + corpus cavernosum injection (CI) group, n = 7; DMED + tail vein injection (VI) group, n = 6; control group, n = 6). Meanwhile, ZDF rats were seperated into four groups (DMED group, n = 5; DMED + corpus cavernosum injection (CI) group, n = 7; DMED + tail vein injection (VI) group, n = 6; control group, n = 7). Two of the groups (the DMED + CI group and DMED + VI group) received HUCMSCs injections at different sites, whereas the DMED group and the control group received saline treatment. Moreover, HUCMSCs were labelled with 5-ethynyl-2’-deoxyuridine (EdU; Beyotime, China).
3. Erectile function evaluation
The intracavernous pressure (ICP) was monitored four weeks following injection, as previously described. Ketamine (100 mg/kg) and midazolam (5 mg/kg) were used to anaesthetize rats. To measure ICP, a 25-gauge needle was placed into the left penile crus. With micro scissors, a V-shaped incision was created in the carotid artery, a PE-50 tube was inserted, and it was ligated and fastened. The PE tube was pre-filled with heparinized saline (200 IU/mL) and attached to a signal collection device (AD Instruments, Australia), allowing for continuous monitoring of mean arterial pressure (MAP). After that, bipolar electrodes were used to electrically activate the cavernous nerve (15.0 Hz; 5.0 V; for 1 minutes). The ratio of maximum ICP (max ICP) to MAP (max ICP/MAP) was used to determine erectile function. The rats were slaughtered after their erectile function was assessed, and the penises were dissected from the original tissue. For histologic research, one third of the penis was preserved in paraffin, while the rest were kept at 80°C for other experiments.
4. Immunohistochemistry, hematoxylin-eosin (H&E) staining, and Masson's trichrome staining
Immunohistochemistry was performed as previously reported. After antigen retrieval and dewaxed, sections of corpus cavernosum were incubated with antibodies against endothelial nitric oxide synthase (eNOS; 1:100; Affinity Biosciences, AF0096, USA) or neuronal nitric oxide synthase (nNOS; 1:100; Affinity Biosciences, AF6249, USA) antibodies, followed by biotinylated secondary antibodies.
The tissues were fixed for 24 hours in 4% paraformaldehyde (Servicebio, China) before being imbedded in paraffin. Serial 4 µm paraffin-embedded slices were obtained and dewaxed in xylene I and xylene II for 10 minutes each, before being rehydrated in a variety of ethanol concentrations (100% for 5 min, 100% for 5 min, 95% 5 minutes, 90% 5 minutes, 80% 5 minutes, 70% 5 minutes). The slices were then washed three times in distilled water (5 minutes each). Finally, according to the manufacturer's instructions, sections were stained with H&E solution (Servicebio, China).
Masson’s trichrome staining was conducted according to the standard protocol. The ratio of smooth muscle to collagen was taken as an indicator of the degree of fibrosis in the corpus cavernosum.
5. Fluorescence staining
A superoxide anion fluorescent probe (Dihydroethidium, DHE; Yeasen Biotechnology) and the Reactive Oxygen Species Assay Kit (2,7-Dichlorodi-hydrofluorescein diacetate, DCFH-DA; Yeasen Biotechnology) were used to measure the quantity of reactive oxygen species (ROS) in the corpus cavernosum. DHE and DCFH-DA were used to incubate tissue and cell sections, respectively.
As previously stated, penis samples were preprocessed. GPX4 (1:200; ABclonal, A1933, China) and ACSL4 (1:200; ABclonal, A1933, China) were used to incubate sections (1:200; Affinity Biosciences, DF12141, USA). The slides were then treated with the appropriate secondary antibodies before being stained with 4', 6-diamidino-2-phenylindole (DAPI, Invitrogen) for nuclear staining.
Sections were assessed for EdU labelling using the BeyoClick™ EdU Assay Kit with Alexa Fluor 647 (Beyotime, China), for 30 minutes at room temperature. The above sections were inspected under a fluorescence microscope.
6. Measurement of oxidative activity
The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) reflected the oxidative and antioxidant activities, respectively. The Micro MDA Assay Kit (Solarbio, BC0025, China) and SOD assay kit (Nanjing Jiancheng Bioengineering Institute, A001-3-2, China) were used to detect these two indicators according to the manufacturer’s instructions.
7. Assessment of iron content
The content of Fe in corpus cavernosum was evaluated by Tissue Iron Assay Kit (Nanjing Jiancheng Bioengineering Institute, A039-2-1, China) according to the manufacturer’s instructions.
8. Examination of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels
Nitrate-nitrite Assay Kit (Beyotime, S0024, China) was used to detect the level of NO, and the cGMP levels were determined ELISA kit (R&D Systems, F15182, USA) according to the standard protocol. The results were normalised to the protein concentration.
9. Transmission electron microscope (TEM) analysis
Cells were fixed with Electron Microscope Fixative Solution (Servicebio, G1102, China) for 1 hour, then were re-fixed with 1% osmic acid in 0.1 M phosphate buffered saline (pH 7.4) at room temperature for 2 hours. Next, adding 50%, 70%, 80%, 90%, 95%, 100% and 100% ethanol to dehydrate in sequence, 15min each time. Finally, they were embedded in SPI-Pon 812 (SPI, 0529-77-4, USA). An ultramicrotome (UC, Leica) was used to cut 60nm ultrathin sections, which were stained with 2% uranyl acetate and lead citrate, subsequently. Sections were examined under a Hitachi electron microscope (Hitachi, HT7800, Japan).
10. Western blotting
Proteins were extracted by RIPA lysis buffer (Boster, AR0102, China) supplemented with 1% PMSF (Boster, AR1178, China), 1% protease inhibitor (Boster, AR1182, China) and 1% protease inhibitor cocktail (MCE, HY-K0010, China). SDS-PAGE was used to separate the proteins, which were then transferred to PVDF membranes. After blocking with Tris-buffered saline-Tween with 5% bovine serum albumin for 1 hour at room temperature, membranes were incubated with primary antibodies against SLC7A11 (1:500; ABclonal, A13685, China), GPX4 (1:500; ABclonal, A1933, China), ACSL4(1:1000; Affinity Biosciences, DF12141, USA), LPCAT3 (1:500; ABclonal, A17604, China), ALOX15 (1:500; ABclonal, A6864, China) and β-Actin (1:200000; ABclonal, AC026, China) at 4°C overnight. Then, the membranes were hybridized with goat anti-rabbit secondary antibody (1:10000; Boster, BA1055, China) and visualized by ChemiDoc Touch Imaging System (Bio-Rad, USA).
RSL3 is one of ferroptosis agonists. CCSMCs were exposed to 500 nmol/L RSL3 (MCE, HY-100218A, China) for 24 hours in order to induce ferroptosis. Ferroptosis inhibitor ferrostatin-1 (Fer-1; 1 µmol/L; MCE, HY-100579, China) was added to medium to inhibit ferroptosis in CCSMCs.
12. Statistical analysis
The photos were examined by Image-Pro Plus 6.0 software, and results were analysed with GraphPad Prism 8.3. Statistical analyses were conducted using one-way ANOVA followed by the Tukey’s test for post hoc comparisons. P < 0.05 was defined as statistically significant.