RSC96, the Schwann cell line of the rat sciatic nerve, was purchased from Procell (Wuhan, China). Briefly, cells were maintained in DMEM (Gibco Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies, CA, USA) and 1% penicillin-streptomycin (Gibco Life Technologies, CA, USA) at 37 ˚C in a humidified atmosphere of 5% CO_2. CDDP was acquired from APExBIO (Houston, USA) and dissolved in normal saline.

Cell viability was evaluated at different time points (48 hours, 72 hours) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dip-henyltetrazolium bromide (MTT) assay. RSC96 cells were seeded in 96-well plates at a density of 3×10³ cells/well and cultured for 24 hours. Then, the cells were cotreated with different concentrations of CDDP in the medium. At different time points, 10 μL of MTT was added to each well and incubated at 37°C for an additional 4 hours. Then, the supernatant was carefully removed, and dimethyl sulfoxide was added to each well to dissolve the crystals by gentle agitation for 10 minutes. For each well, the absorbance at 490 nm was estimated on a microplate reader (Bio-Tek, VT, USA).

Cells were seeded in 6-well plates at a density of approximately 20 000 cells/well for 24 hours, and then treated with different concentrations of CDDP for 2 days. Thereafter, the medium was removed and replaced with complete medium (without CDDP) for another 2 days. Then, the cells were fixed and stained using a Senescence β-Galactosidase Staining Kit (Solarbio Life Science, Beijing, China).

For qPCR, total RNA was extracted using the RNA Kit (Omega Bio-Tek, USA) according to the manufacturer’s instructions. Reverse transcription was performed using a HiScript II 1^st strand cDNA synthesis kit (Vazyme, China). qPCR was performed on a step-one plus real-time PCR system (Bio-Rad, CA, USA) using ChamQ universal SYBR qPCR master mix (Vazyme, China). All primers were synthesized by Ribobio Co., Ltd. (Guang Zhou, China). Primer sequences are listed in Table 1 . β-actin was used as an internal control. The relative levels of RNAs were calculated using the 2-ΔΔCt method.

Bilateral MPG/CN (entire MPG with a 2-mm length CN attached) complexes from each rat were isolated and excised intact. Each MPG/CN complex was placed in a 6-well plate and then covered with 50 μL of Matrigel (Corning, NY, USA) as previously described (14). After incubation at 37 °C for 5 minutes, 2.0 mL of complete culture medium and 2.0 ml supernatant from RSC96 treated with or without CDDP were added according to the grouping. RSC96 cells were treated with CDDP for 2 days, and the medium was removed and replaced with complete culture medium (without CDDP) for another 2 days. The supernatant was then used to culture the MPG/CN complex.

All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Fifth Affiliated Hospital of Sun Yat-sen University.Twenty 12-week-old male Sprague−Dawley rats were used for this study. The rats were divided into 2 groups: The CDDP group rats (n = 10) were intraperitoneally administered 2 mg/kg body weight of CDDP on Days 1, 2, 8, 9, 15, 16, 22, and 23 ( Figure 1A ). And the others (n=10) injected with normal saline as the control group. The dose was based on some scholars’ recommendation about the dose translation from animals to humans (15, 16). The erectile function was evaluated after a one-week washout.

Intracavernous pressure (ICP) was measured by electrical stimulation, as previously reported (17, 18). All rats were anesthetized by injection with pentobarbital (40 mg/kg). Then, the carotid artery was exposed and cannulated with a PE-50 tube which was connected to a pressure transducer to continuously monitor arterial pressure. A 25-gauge needle containing 100 IU/mL heparin solution was inserted into the right penile crura and the other end of the tube was also connected to a pressure transducer to monitor intracavernous pressure (ICP). The cavernous nerve was electrically stimulated at 1 V, 20 Hz, with a pulse width of 5 ms for 1 minute and a 5-minute interval before subsequent stimulation. The ratios of maximal ICP (MaxICP) to mean arterial pressure (MAP) were calculated to evaluate erectile function in vivo.

After the ICP test was completed, clinical needles and vacuum tubes were used to collect blood through the inferior vena cava.The serum was separated immediately and stored at 80°C. The levels of testosterone were assessed using ELISA kits from Solarbio (Beijing, China).

Results were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and expressed as the mean ± standard deviation (SD). Statistical analyses were performed using the two-tailed Student’s t test. Differences among groups were considered significant at a P value less than 0.05.

As shown in Figures 1B, C , the mean body weight and testosterone levels did not differ significantly between the two groups, but erectile function had obvious difference ( Figures 1D, E ). Erectile function was assessed by MaxICP and MaxICP/MAP. The results showed that both MaxICP and MaxICP/MAP revealed a significant decrease in the CDDP group compared to control rats.

The morphological changes and smooth muscle (SM)-to-collagen ratios of the different groups were detected with H&E and Masson’s trichrome staining, as shown in Figures 2A – C . CDDP significantly caused structural disorders and decreased smooth muscle mass in the corpus cavernosum. Moreover, Immunofluorescence staining with an anti-α-SMA antibody showed a significant reduction in smooth muscle content in CDDP-treated rats compared with normal control rats ( Figure 2D ). Cavernosal endothelial dysfunction is recognized as a hallmark of the disease pathology. Tomoya Kataoka found that oxaliplatin causes erectile dysfunction in rats due to endothelial dysfunction (11). Consistent with the findings, we found the endothelial cell content and eNOS was severely decreased in the CDDP group ( Figure 2E ).

CDDP binds to DNA to cause a biological effect by forming CDDP-DNA adducts and induceing DNA damage response. Phosphorylated H2AX (γ-H2AX) is known to be a marker to investigate the effects on DNA damage (20). As shown in Figure 3A , CDDP treatment significantly increased γ-H2AX levels compared with the control group in the corpus cavernosum. The apoptotic pathway may be triggered in cells after CDDP treatment. We measured the apoptosis level in corpus cavernosum by TUNEL ( Figures 3B, C ). In the CDDP group, there were a dramatically larger percentage of apoptosis cells than the control group. The aforementioned results indicates that CDDP can induces DNA damage and apoptosis in corpus cavernosum.

ED is considered a complication of CDDP-induced peripheral neuropathy (12). Aina Calls found that CDDP-induced peripheral neuropathy was associated with neuronal senescence-like response (21). In senescent cells, the levels of cell cycle inhibitors, including p16, p21, and p53 were augmented. Following CDDP treatment, the expression levels of the p16, p21, and p53 were increased in CN ( Figure 4 ).

To determine RSC96 cells response to CDDP, an MTT assay was performed. Notably, the proliferation viability of cells decreased with increasing concentration ( Figure 5A ). The SA-β-gal staining assay confirmed our hypothesis that senescence occurred in RSC96 cells treated with CDDP ( Figure 5B ). Immunofluorescence illustrated that CDDP upregulated the expression of senescence-related genes: p16, p21 and p53 ( Figure 5C ). SASP is another typical feature of senescent cells. CDDP promoted the expression of SASP-related genes, IL-6, TGFβ1, ICAM1, TGFβ2, CCL2, IL-1α and CXCL1 ( Figure 5D ).

To investigate the effect of senescent Schwann cells on axonal growth of the CN, we used an in vitro model of MPG/CN culture (14). As shown in Figure 6A , the supernatant of RSC9 cells was used to treat the MPG/CN system. We dissected the MPG with 2 mm of CN attached and cultured the tissue in vitro. After 120 hours, new neurite outgrowths from the end of the CN were measured. CDDP significantly restrained the neurite outgrowth of CN ( Figures 6B–D ).