Neutrophil extracellular traps promote erectile dysfunction in rats with diabetes mellitus by enhancing NLRP3-mediated pyroptosis

Erectile dysfunction (ED) is the most prevalent consequences in men with diabetes mellitus (DM). Recent studies demonstrates that neutrophil extracellular traps (NETs) play important roles in DM and its complications. Nevertheless, whether NETs are involved in ED remains unknown. This work intended to explore the role and mechanisms of NETs in ED in the context of DM. Here, we observed that NET generation and pyroptosis were promoted in DM rats with ED compared with controls. Mechanistically, NETs facilitated NLRP3 inflammasome activation and subsequently triggered pyroptosis under high glucose stress, ultimately leading to ED. Intriguingly, DNase I (a NET degrading agent) alleviated ED and corpus cavernosum injury in DM rats. Overall, NETs might induce ED in DM by promoting NLRP3-mediated pyroptosis in the corpus cavernosum.


NLRP3-mediated pyroptosis was promoted in the corpus cavernosum tissues of diabetic ED rats
NLRP3 expression in the corpus cavernosum tissues of rats was detected firstly to determine the involvement of NLRP3 in diabetic ED development.Immunohistochemistry showed an elevation of NLRP3 expression in the corpus cavernosum tissues of diabetic ED rats relative to that in controls (Fig. 3A).Accordingly, we hypothesized that NLRP3 dysregulation might affect pyroptosis in diabetic ED and evaluated pyroptosis markers (GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, ASC, IL-1β, and IL-18).Western blotting revealed that NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC protein expression levels were significantly augmented in the corpus cavernosum tissues of diabetic ED rats relative to those in controls (p < 0.01; Fig. 3B).www.nature.com/scientificreports/Moreover, ELISA showed markedly higher plasma IL-1β and IL-18 levels in diabetic ED rats than those in controls (p < 0.01; Fig. 3C).

NET degradation alleviated diabetic ED symptoms and inhibited pyroptosis in corpus cavernosum tissues
To further determine the promotive function of NETs in NLRP3-mediated pyroptosis in diabetic ED, diabetic ED rats were intraperitoneally injected with the NET degrading agent, DNase I. Here, DNase I treatment markedly reduced NET generation in diabetic ED rats (Fig. 4A, B).Through blood routine examination, the white blood cells (WBC) level in diabetic ED rats was significantly higher than that in controls, while the red blood cells (RBC), hemoglobin (Hb), and platelets (PLT) levels were lower, which was reversed following DNase I treatment (p < 0.01; Fig. 4C).Notably, ED (p < 0.01; Fig. 4D, E) and histopathological injury (Fig. 4F, G) of diabetic ED rats were effectively mitigated following DNase I treatment.Furthermore, DNase I treatment reduced NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, ASC, IL-1β, and IL-18 expression in the corpus cavernosum tissues of diabetic ED rats (Fig. 5A-C).

NETs facilitated NLRP3-mediated pyroptosis of high glucose (HG)-treated rat corpus cavernosum smooth muscle cells (CCSMCs)
We further performed in vitro experiments to confirm our hypothesis by treating rat CCSMCs with HG and co-culturing them with or without rat neutrophils (N).Here, the cell viability of HG-treated CCSMCs was markedly reduced relative to that of controls, and the decreased viability was aggravated when co-cultured with neutrophils (p < 0.01; Fig. 6A).Moreover, HG treatment significantly promoted CCSMC apoptosis, which was further enhanced when CCSMCs were co-cultured with neutrophils (p < 0.01; Fig. 6B).
In terms of NET generation, H3Cit and MPO expression were elevated in HG-induced CCSMCs relative to those in controls, which were further upregulated when CCSMCs were exposed to neutrophils (Fig. 6C).Notably, we observed that HG-induced CCSMCs displayed pyroptotic-like morphological characteristics and membrane ballooning relative to controls, especially when co-cultured with neutrophils (Fig. 6D).Furthermore, western blotting revealed notable increases NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC protein expression in HG-treated CCSMCs, especially when co-cultured with neutrophils (p < 0.05; Fig. 6E).ELISA showed IL-1β and IL-18 levels were markedly higher in HG-treated CCSMCs than those in controls, which were further elevated with neutrophils co-cultured (p < 0.05; Fig. 6F).

Discussion
NETs contribute to the pathogenesis of diabetes and its complications 11,22,23 .Nevertheless, whether NETs are involved in diabetic ED is still unknown.The current research fills this void in understanding.Herein, we constructed a DM rat model induced by STZ, and rats without any erection after APO testing were thought to suffer from ED.As a result, NET generation was found to be elevated in diabetic ED rats, which was associated with ED.Mechanistically, it was demonstrated that the pathogenic role of NETs in diabetic ED might be partly owing to the promotion of NLRP3-mediated pyroptosis.Our findings emphasize a new mechanism of ED in the context of DM, which exacerbates corpus cavernosum injury.
NETs are frequently characterized as a crucial part of the anti-bacteria process; nevertheless, due to the non-specific nature of NET components, an overabundance of NETs can often lead to inflammation and tissue lesions 24 .Chronic inflammation is a vital part of ED pathogenesis and a possible middle phase of endothelial dysfunction, particularly in metabolic disorders, including diabetes 25 .At the beginning of this study, the NET formation markers (H3Cit, NE, cf-DNA, and MPO) were observed to be upregulated in diabetic ED rats compared with those in controls.In line with our findings, previous research revealed increased NET generation in     www.nature.com/scientificreports/DN 26,27 .Thus, we hypothesized a significant role of NETs in diabetic ED pathogenesis and subsequently explored related mechanisms.
Increasing studies reveal NETs can interact with NLRP3 inflammasome to aggravate inflammation in various diseases 10,11,28 .Herein, we found that NLRP3 expression was higher in diabetic ED rats than that in controls, which conformed to the previous investigations into diabetic ED 13 .Notably, neutrophils, the source of NETs, further enhanced NLRP3 expression in rat CCSMCs with decreased cell viability and enhanced apoptosis, under the condition of HG.This suggested that NETs might promote diabetic ED progression by activating NLRP3 signaling.The NLRP3 inflammasome is known to recognize internal danger signals and then activate caspase-1 and IL-1β, ultimately triggering inflammatory responses 29 .Additionally, NLRP3 activation conduces to caspase-1 maturation, facilitating IL-1β and IL-18 secretion, thereby triggering pyroptosis 30 .Accumulating evidence indicates that NLRP3 activation contributes to the development and progression of ED 20,31 .Inhibition of NLRP3mediated pyroptosis of nerve is associated with ameliorated ED 20 .On the other hand, prior data demonstrated that the promotion of pyroptosis in corpus cavernosum could conduce to ED in rats 15,32 .Based on these facts, we explored the effects of NETs on pyroptosis in diabetic ED.
Unlike apoptosis, pyroptosis is an inflammatory caspase-dependent form of programmed cell death, marked by cell membrane pore formation, membrane rupture, cell swelling, and cell content release 33 .Central to pyroptosis is the activation of the inflammasome, especially the NLRP3 inflammasome, which is among the most extensively researched 34 .Upon detecting danger or pathogen-associated molecular patterns, NLRP3 recruits pro-caspase-1 via ASC to assemble the NLRP3 inflammasome 16 .This assembly prompts the transformation of pro-caspase-1 into its active state, subsequently processing pro-IL-1β and pro-IL-18 into their mature forms 17 .GSDMD acts as the primary executor of pyroptosis.GSDMD is cleaved by caspase-1 into GSDMD-N, which creates pores in the cellular membrane 35 .This process ultimately induces cell death and facilitates the release of pro-inflammatory factors like IL-1β and IL-18, igniting a potent inflammatory reaction 35 .Herein, GSDMD, GSDMD-N, pro-caspase-1, c-caspase-1, ASC, IL-1β, and IL-18 levels were elevated in the corpus cavernosum tissues of diabetic ED rats relative to controls, indicating the promotion of pyroptosis in diabetic ED.These data were in line with the previous findings that pyroptosis was promoted in diabetic ED 13 .Significantly, neutrophils aggravated the pyroptosis of rat CCSMCs, under the condition of HG.However, NLRP3 knockdown abolished the effects of NETs on decreasing the viability and enhancing the apoptosis of HG-treated rat CCSMCs.Furthermore, NLRP3 downregulation eliminated the promoting function of NETs on the pyroptosis of HG-treated rat CCSMCs.A prior investigation demonstrated that NLRP3 reduction could inhibit the pyroptosis in corpus cavernosum tissues, thus improving the erectile function of a rat model of diabetic ED 13 .Collectively, our data imply that NETs might act as pathogenic factors in diabetic ED by promoting NLRP3-mediated pyroptosis in the corpus cavernosum.
Finally, to further ascertain the pathogenic role of NETs in diabetic ED, we conducted in vivo experiment by treating diabetic ED rats with DNase I, a degrading agent of NETs.DNase I treatment rescued blood routine abnormities and alleviated histopathological injury of diabetic ED rats.Previous research suggested that inhibition of NLRP3-mediated pyroptosis could alleviate ED in diabetic rats 13 .Nevertheless, there is little report of the involvement of NETs in the pyroptosis mechanism in ED.Here, we for the first time observed that NET degradation improved the erectile function of diabetic ED rats and inhibited NLRP3-mediated pyroptosis in the rat cavernosum tissues.Collectively, these findings indicate that NETs might induce ED in DM by promoting NLRP3-mediated pyroptosis.
This study has some limitations.Firstly, while the study verified the relationship between NETs and NLRP3 in diabetic ED in vivo and in vitro, the signaling pathway by which NETs induce NLRP3-mediated pyroptosis (such as the NF-κB pathway) is yet to be explored.Besides, other inflammasomes, such as AIM2 inflammasome 36,37 , pyrin inflammasome 38 are also involved in NETs-driven inflammation.Further experiments should be conducted to reveal whether other inflammasomes functions in the roles of NETs in diabetic ED.Secondly, we induced the type 1 DM rat models, and the relative mechanism of ED was investigated in the context of type 1 DM.It is still unclear whether such phenomenon is appropriate to elucidate the mechanism of ED in the context of type 2 DM, and therefore, further investigations are needed.Thirdly, the use of animal models to simulate diabetic ED might not fully capture the complexity and diversity of human diseases.Therefore, future studies should incorporate in-depth exploration of the specific molecular pathways involved in NETs-mediated pyroptosis and identification of downstream molecules.Besides, additional studies are required to assess the efficacy of NET-, NLRP3-, and pyroptosis-targeted strategies for the treatment of ED in DM patients, which is essential for the translation of these findings into clinically therapeutic approaches.

Conclusions
This study revealed that NETs could induce ED in DM, which might be partly due to their promotive role in NLRP3-mediated pyroptosis.In general, our findings suggest that intervention strategies targeting NETs are expected to be potential treatments for diabetic ED.This work furthers the understanding of diabetic ED pathogenesis and furnishes research directions for diabetic ED treatment.

Experimental animals
Thirty-five specific pathogen-free male Wistar rats (8 weeks old; 240 g), supplied by the Institute of Comparative Medicine, Yangzhou University (Yangzhou, China), were used for research.All rats were bred under the following conditions: a 12-h light/dark cycle; 19-25 °C; 35-55% humidity, and provided with ad libitum food and water.The animal experiments were permitted by the Experimental Animal Ethics Committee of Yangzhou University (No. 202304016), which conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and methods were reported in accordance with ARRIVE guidelines.

Animal modeling and treatments
After 7-day adaptive feeding, 12 rats were randomly selected to induce a type 1 DM model.According to the previous research 39,40 , the 12 rats were intraperitoneally injected with STZ (60 mg/kg; #S0130, Sigma, USA), dissolved in cold citrate buffer (pH 4.5; 0.1 M).Moreover, six normal rats were used as controls (control group), which were treated with the equal volume of odium citrate buffer via intraperitoneal injection.One day later, the blood glucose of rats was monitored, and those presenting blood glucose exceeding 16.7 mM for three successive days were perceived to be type 1 diabetic 41 .After 8 weeks, diabetic rats were further screened for ED using APO (Sigma-Aldrich, St. Louis, MO, USA).Following spending 10 min in a dim, tranquil environment, the rats were subjected to a subcutaneous injection of APO (100 µg/kg) in the nape of their necks.Subsequent behavior was recorded over a 30-min span using a video camera (Nikon, Tokyo, Japan; D7500).An erection was characterized by noticeable penile engorgement or the exposure of the glans.Diabetic rats that did not exhibit any erection were thought to undergo ED (diabetic ED) based on the prior study 42 .Finally, 12 diabetic ED rats were successfully determined.
To further confirm the effects of NETs on ED in DM, the 12 diabetic ED rats were assigned to diabetic ED and diabetic ED + DNase I groups (n = 6/group).Rats in diabetic ED + DNase I group were intraperitoneally injected www.nature.com/scientificreports/with DNase I (a NET degrading agent; 1 mg/kg; #HY-108882, MedChemExpress, Monmouth Junction, NJ, USA) for 8 successive days to inhibit NET generation.At the same time, control and diabetic ED groups received an intraperitoneal injection with the equivalent amount of normal saline for 8 successive days.
The urine and blood samples were collected, and all rats were euthanatized after anesthetization by 100 mg/ kg pentobarbital via intravenous injection 43 following 8 days of treatment.Then, the kidney and cavernous tissues were collected for later assays.

Biochemical index and blood routine examination
The rat fasting blood glucose was assessed at weeks 0, 4, 6, and 8 using a glucometer (A36B, Roche, Basel, Switzerland).The rats were moved to metabolic cages one day before euthanasia, 24-h urine and blood were collected.The fresh urine samples were subjected to 15-min centrifugation at 3000 rpm and kept at −20 °C.Then, the rats were injected intraperitoneally with pentobarbital (40 mg/kg) for anesthesia, and the arterial blood (about 5 mL) was drawn from the abdominal aorta.The serum was isolated after centrifugation at 3000 rpm for 5 min and stored at −20 °C.BUN, CREA, and UACR, WBC, RBC, Hb, and PLT were assessed via an Indiko automatic biochemical analyzer (Thermo Scientific, Waltham, MA, USA).

Neutrophil isolation
Following the supplier's directions, neutrophils were separated from peripheral blood of rats in normal group using a neutrophil isolation kit (#CLS1091, Sangon Biotech, Shanghai, China).Briefly, rat blood was mixed with ethyl starch (1:1) and incubated at 37 °C for 10 min.Then, the supernatant was collected, and the settling red blood cells were retained.The supernatant was mixed with the cell washing solution, then 15-min centrifugation at 500g.Subsequently, the supernatant was removed and the settling cells were retained.The cell precipitate was added with the whole blood diluent to make the cell concentration 2 × 10 8 -1 × 10 9 cells /mL, followed by 20-min centrifugation at 400g.A large number of neutrophils were obtained using red blood cell lysate and after removal of red blood cells.The obtained neutrophils were washed and centrifuged at 500g for 15 min.After the precipitate was washed twice, the required neutrophils were acquired.
All plasmids used in this study were synthesized and supplied by GenePharma (Shanghai, China).A sh-NLRP3 was employed for NLRP3 downregulation in CCSMCs.Scrambled shRNA (sh-NC) was used as the control.In brief, CCSMCs were subjected to 72-h transfection after seeding onto six-well plates (2 × 10 5 cells/ well).Transfection experiments were carried out via HighGene transfection kits (#RM09014, ABclonal, Wuhan, China).To evaluate the transfection effectiveness, we applied quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting.The primers of sh-NLRP3 are listed in Table S1.
To ascertain the role of NETs in diabetic ED, CCSMCs were subjected to HG (25 mM) treatment or/and co-cultured with 1 × 10 4 neutrophils for 24 h.Furthermore, to figure out how NETs affect NLRP3-mediated pyroptosis in CCSMCs, sh-NC-or sh-NLRP3-transfected CCSMCs were exposed to HG and co-cultured with neutrophils.CCSMCs cultured in 5 nM glucose served as controls.

HE and Masson staining
The kidney or corpus cavernosum tissues from rats were paraffin-embedded, sliced at 5 µm, deparaffinized, and hydrated.Subsequently, the sections were stained with HE (#C0105S, Beyotime, Shanghai, China) or Masson (#G1340, Solarbio, Beijing, China) staining kit following the provided protocols.Finally, the section images were captured by a microscope (CKX53, Olympus, Tokyo, Japan).

Assessment of erectile function
Rats were housed in a low-light and quiet setting, conducive for observation.Then, the rats received an injection of APO (100 µg/kg) through the neck.Finally, the number of erections and the erection latency within 30 min were tracked and recorded.The erection latency refers to the time span from APO treatment to erection.

Detection of cf-DNA
As per the manufacturer's directions, the cf-DNA level in rat plasma was assessed using a Quant-iTPicoGreen dsDNA kit (#P11496, Thermo Scientific).The cf-DNA level was determined by a microplate reader (DR-3518G, Hiwell Diatek), and the excitation and emission wavelengths were 480 nm and 520 nm, respectively.

Flow cytometry and observation of pyroptosis by microscopy
Flow cytometry was employed to assess CCSMC apoptosis.CCSMCs were suspended in 195 µL Annexin V-fluorescein isothiocyanate (FITC) binding buffer.Then, cells were stained with Annexin V-FITC (5μL) and propidium iodide (10 µL) (#C1062S, Beyotime) at 25 °C for 15 min and 10 min, respectively.A flow cytometer (CytoFLEX S; Beckman, Miami, FL, USA) was utilized for cell apoptosis assessment, and Cell Quest software (BD Biosciences, Franklin Lakes, NJ, USA) was for quantification.
To assess pyroptosis morphology, CCSMC cells were subjected to HG conditions for 24 h.The images were taken from four arbitrarily chosen areas using a microscope (DM3000, Leica, Germany).

Statistical analysis
Data were displayed as mean ± standard deviation.Statistical analyses were carried out using GraphPad 7.0 software (La Jolla, CA, USA).If the data showed a normal distribution, student's t-test was used to evaluate the differences between two groups, and one-way ANOVA with Tukey's test was applied for comparisons of multiple groups.If the data exhibited a non-normal distribution, the Mann-Whitney U test was used to analyze the differences between two groups, and the Kruskal-Wallis test followed by the Student-Newman-Keuls test was employed for comparisons of multiple groups.A p value below 0.05 was deemed significant difference.

Figure 1 .
Figure 1.A rat model of diabetic ED was successfully constructed.(A) Assessment of the fasting blood glucose level in rats (n = 6/group).(B) Assessment of erection number and latency in rats (n = 6/group).(C) Assessment of the serum testosterone level using ELISA (n = 6/group).(D) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm).(E) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm).Data were expressed as mean ± standard deviation.**p < 0.01.ED erectile dysfunction, HE hematoxylin-eosin, ELISA enzyme-linked immunosorbent assay.

Figure 4 .
Figure 4. NET degradation alleviated diabetic ED symptoms of rats.(A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group).(B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm).(C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/ group).(D) Assessment of erection number and latency in rats (n = 6/group).(E) Assessment of the serum testosterone level using ELISA (n = 6/group).(F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm).(G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm).Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days.Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline.Data were presented as mean ± standard deviation.**p < 0.01.NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin-eosin.

Figure 5 .
Figure 5. NET degradation inhibited the pyroptosis in the corpus cavernosum tissues of diabetic ED rats.(A) Assessment of NLRP3 expression in the corpus cavernosum tissues of rats using immunohistochemistry (n = 6/ group; scale bar = 50 μm).(B) Assessment of the protein expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC in the corpus cavernosum tissues of rats using western blotting (n = 6/group); the uncropped protein blots can be seen in Supplementary file 2. (C) Assessment of plasma IL-1β and IL-18 levels in rats using ELISA (n = 6/group).Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days.Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline.Data were expressed as mean ± standard deviation.**p < 0.01.NET neutrophil extracellular trap, NLRP3 NOD-like receptor family, pyrin domain-containing protein 3, GSDMD gasdermin D, GSDMD-N N-terminal proteolytic fragment of gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, IL interleukin, ELISA enzyme-linked immunosorbent assay.

Figure 7 .
Figure 7. NETs inhibited the proliferation of HG-treated rat CCSMCs by NLRP3 activation.(A,B) Evaluation of sh-NLRP3 transfection efficiency in CCSMCs using qRT-PCR and western blotting; the uncropped protein blots can be seen in Supplementary file 4. (C,D) Assessment of NLRP3 expression in HG-treated CCSMCs using qRT-PCR and western blotting; the uncropped protein blots can be seen in Supplementary file 4. (E) Assessment of H3Cit expression in HG-treated CCSMCs using western blotting; the uncropped protein blots can be seen in Supplementary file 4. (F,G) Assessment of the viability and apoptosis in HG-treated CCSMCs using CCK-8 assay and flow cytometry, respectively.(C-G) sh-NC-or sh-NLRP3-transfected CCSMCs were subjected to HG (25 mM) treatment or/and co-cultured with 1 × 10 4 neutrophils (N) for 24 h.CCSMCs cultured in 5 nM glucose served as controls.Data were presented as mean ± standard deviation.*p < 0.05 and **p < 0.01.NET neutrophil extracellular trap, NLRP3 NOD-like receptor family, pyrin domain-containing protein 3, HG high glucose, CCSMCs corpus cavernosum smooth muscle cells, qRT-PCR quantitative real-time polymerase chain reaction, H3Cit citrullinated histone H3, CCK-8 cell counting kit-8.