Male Sprague-Dawley rats and C57BL/6J mice came from the Animal Center of the Southern Medical University. Animal studies were approved by the Southern Medical University Committee on the Use and Care of Animals and were performed following Committee guidelines and regulations.

Adult male Sprague-Dawley rats (250–300 g) were used to construct subacute incomplete SCI models at the thoracic level, as previously described (40). Briefly, rats were acclimatized in the housing facility for at least 3 days prior to experimentation. Animals were randomly grouped and intraperitoneally anesthetized with 1% pentobarbital sodium (25 mg/kg). In the sham group, the spinal cord at the thoracic level was exposed after T9–T11 laminectomy, and then sutured. In the SCI group, a spinal cord compression injury was generated using an aneurysm clip (Sugita, Japan) with a clamping force of 60 g at T10 for 1 min. After surgery, rats were intramuscularly injected with penicillin (50 000 units/kg/day) for 3 days. Manual urination and nursing were performed twice daily to prevent complications. The Basso-Beattie-Bresnahan (BBB) locomotor rating scale was implemented at 24 h after surgery (41), and rats with scores < 3 were considered successful SCI candidates for further analyses. All rats were euthanized on the seventh day after BBB scoring for following experiments.

For in vivo treatments and after BBB scoring confirmed SCI, SCI rats were intraperitoneally injected with the NO scavenger, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO, 20 mg/kg/day; C221, Sigma, Shanghai, China) (42). Two strategies were used: 1) Injections commenced immediately following scoring and lasted for 7 days - CPTIO-7d group and 2) Injections commenced at day 3 after scoring and lasted for 4 days – CPTIO-4d group. For the NOS inhibitor, SCI rats were intraperitoneally injected with NG-Nitroarginine methyl ester (L-NAME) hydrochloride (100 mg/kg/day; A7088, APExBIO, Shanghai, China) for 7 consecutive days. Control SCI rats were injected with saline. All rats were euthanized on the seventh day. For in vitro treatments, a working concentration of 150 μM CPTIO or 1 mM L-NAME in culture medium was used.

LCs were isolated as previously described (43). Briefly, adult C57BL/6J mice were euthanized by cervical dislocation and testes aseptically removed. The tunica albuginea was carefully removed and seminiferous tubules and interstitial cells were dispersed by treating decapsulated testes with collagenase IV (1 mg/mL; C5138, Sigma) in DMEM/F12 plus 5% fetal bovine serum (FBS; 164210, Procell, Wuhan, China) in a shaking water bath at 37°C for 5 min. After incubation, cold DMEM/F12 was added to terminate collagenase IV activity. Seminiferous tubules were separated from interstitial cells via gravity sedimentation. Crude interstitial cells were collected by centrifugation at 300×g for 5 min and resuspended in 2 mL DMEM/F12 plus 5% FBS after washing three times in phosphate buffered saline (PBS, pH 7.4). To obtain purified LCs, resuspended cells were loaded onto a four-layer discontinuous Percoll gradient (17-0891-02, cytiva, Sweden) consisting of 5%, 30%, 58%, and 70% Percoll dissolved in Hanks’ balanced salt solution (HBSS) and centrifuged at 800×g at 4°C for 30 min. Highly purified LCs were distributed in the 30%–58% Percoll fraction. LCs were then carefully collected, washed in PBS, and centrifuged at 300×g for 5 min. LC purity was > 95% as determined by 3β-hydroxysteroid dehydrogenase 2 (LCs marker) immunofluorescence. Purified LCs were cultured in DMEM/F12 plus 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ atmosphere.

Peritoneal macrophages were isolated as described previously (26). Briefly, adult C57BL/6J mice were intraperitoneally injected with 4% sodium thioglycolate (3 mL). After 3 days, mice were euthanized by cervical dislocation and soaked in 75% ethanol for 5 min. Using sterile procedures, cells were obtained by peritoneal lavage using calcium- and magnesium-free HBSS. Cell suspensions were centrifuged at 300×g for 5 min at 4°C and supernatants discarded. Then, 5 mL DMEM plus 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin were added to resuspend cell pellets, and cell counting and culturing performed. After 6 h, culture supernatants were discarded, cells washed twice in PBS to remove non-adherent cells, and complete medium added to continue culturing. To stimulate NO production, 100 ng/mL lipopolysaccharide (LPS; L4391, Sigma) and 50 ng/mL interferon-γ (IFN-γ; AF-315-05, PeproTech) were added to induce PMs to M1 phenotypes. For PM and LC co-culture, transwell plates were used; PMs were cultured in lower plates while LCs were cultured in inserts.

Following euthanasia, the testes and epididymides were removed and weighed, fixed in modified Davidson fixative, and processed in paraffin according to standard methods. At least three sections from each testis and epididymis (3 μm, taken 100 μm apart) were stained with hematoxylin and eosin (H&E) for regular histological examination. Photoshop CS6 Extended (13.0.1; Adobe) was used to analysis the thickness of seminiferous epithelium, diameter of seminiferous lumen and diameter of seminiferous tubule, and area of intestitium.

One cauda epididymis was removed from each rat and minced in 1 mL of M2 medium (M7167, sigma) containing 3% bovine serum albumin at 37°C for 20 min to allow the sperm to be released into the medium. The total number of sperm in the final suspension was counted with a hemocytometer.

Immunofluorescence (IF) was performed according to standard procedure, using the primary antibodies summarized in Supplementary Table 1 and Alexa-Fluor-488-labeled or Alexa-Fluor-594-labeled secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). 4, 6-diamidino-2-phenylindole (DAPI) was used to visualize nuclei. Immunofluorescent images were obtained and staining intensity was analyzed using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan). The fluorescence intensity was quantified using the Image J software (1.53t, National Institutes of Health, USA).

For BODIPY staining, 20 µm frozen sections or primary LC cells were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS, and subsequently incubated in 1 µg/ml BODIPY (D3922, Invitrogen, Carlsbad, CA, USA)-PBS solution at 37 °C for 15 min in dark, alone or followed by immunofluorescent staining with indicated antibodies.

For LysoTracker staining, 50 nM LysoTracker reagents (lysosome fluorescent probe, C1046, Beyotime Biotechnology) were added to LCs culture medium and incubated for 30 min at 37°C. After washing by PBS, fresh culture medium was added and subsequently observed under a confocal microscope.

Cell apoptosis was evaluated by the terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay for in situ visualization of DNA fragmentation using a commercial kit (DeadEndTM Fluorometric TUNEL System, G3250, Promega, Madison, WI, USA). At least three sections from each testis (3 μm, taken 100 μm apart) were stained. Signals were captured by a confocal microscope. TUNEL-positive germ cells were quantified in each tissue section by counting the number of TUNEL-positive cells in each seminiferous tubule.

Following euthanasia, rat testes were immediately removed, triturated, and lysed on ice with RIPA lysis buffer (P0013B, Beyotime Biotechnology) supplemented with protease and phosphatase inhibitor cocktail (P1046, Beyotime Biotechnology). After centrifugation at 12 000 ×g at 4 °C, the supernatant was collected and the protein concentration was quantified by a BCA kit (C503021, Sangon Biotech, Shanghai, China), and finally boiled in sodium dodecyl sulfate (SDS) loading buffer. Thirty microgram of protein was then subjected to 6–12% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (10600001, GE Healthcare Life Sciences). The membranes were then blocked in 5% nonfat dry milk for 1 h at room temperature, washed, and incubated with primary antibodies summarized in Supplementary Table 1 at 4 °C overnight. The membranes were further washed, incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch) for 1 h at room temperature, washed again, and finally visualized using an enhanced chemiluminescence kit (NEL105001EA, PerkinElmer, Waltham, MA, USA). Quantification was performed by measuring the gray value of bands using the Image J software (1.53t, National Institutes of Health, USA).

Total RNA was purified using the Trizol Reagent (Invitrogen), and processed to cDNA using the Hifair™ II 1^st Strand cDNA Synthesis Kit, followed by amplification and quantification using the Hieff^® qPCR SYBR Green Master Mix (all from YEASEN Biotech, Shanghai, China) with a StepOne Plus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Gapdh was used as the endogenous control transcript. Three technical replicates were applied for each transcript. The 2-^ΔΔCt method was used to calculate the fold changes in gene expression. The primer sequences are summarized in Supplementary Table 2.

20 µm frozen testicular sections were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS. Sections were then incubated in 60% (vol/vol) isopropanol for 5 min and air-dried. Sections were immersed in Oil Red O solution (O0625; Sigma-Aldrich; Oil Red O saturated solution in 3:2 isopropanol/water) for 15 min. Sections were then rinsed in 60% isopropanol for 5-10 s to remove background staining. After rinsing with tap water, sections were counterstained with hematoxylin and placed in 1:1 glycerol/PBS for further analysis. The images were observed and acquired though a light microscope (Carl Zeiss, Germany). Image Pro Plus 6.0 software was used to calculate the ratio of oil red O staining lipid droplet area to total area.

Testosterone level was measured by an enzyme-linked immunosorbent assay (ELISA) kit (ELK1332, ELK Biotechnology, Wuhan, China; detection range: 62.5-4000 pg/mL). For testis, values were normalized to tissue weights and for Leydig cells were to protein concentration. Rat serum luteinizing hormone (LH) level was measured by an ELISA kit (ELK2367, ELK Biotechnology, detection range: 0.47-30 mIU/mL).

The concentration of free cholesterol in LCs was measured using an Amplex^® Red Cholesterol Assay Kit (A12216, Invitrogen, Detection Range: 200 nM–20 μM). In brief, the supernatant of LC lysates was added to a working solution of 300 μM Amplex^® Red reagent containing 2 U/mL horseradish peroxidase, 2 U/mL cholesterol oxidase, and 0.2 U/mL cholesterol esterase and incubated for 30 min at 37°C in dark. Fluorescence was measured in a microplate reader using excitation in the range of 530–560 nm and emission detection at 590 nm. Free cholesterol levels were calculated based on the standard curve and normalized to protein concentration.

NO level was measured by a NO assay kit (BC1475, Solarbio, Beijing, China, Detection Range: 0.00078-0.25 μmol/mL). The cellular NO in LCs was monitored using the fluorescent NO probe DAF-FM DA (S0019, Beyotime Biotechnology). LCs were cultured in medium with 50 μM DAF-FM DA for 30 min at 37°C. The fluorescence was visualized by a confocal microscope or measured by a FACScan flow cytometer (BD Immunocytometry Systems).

At least three testis samples from SCI and sham groups were taken for Tandem mass tag (TMT)-based proteomic analysis. The primary experimental procedures performed by Applied Protein Technology (Shanghai, China) include protein preparation, trypsin digestion, TMT labeling, HPLC fractionation and LC-MS/MS analysis. The MS raw data for each sample were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis.

The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST+ client software (ncbi-blast-2.2.28+-win32.exe) and InterProScan to find homologue sequences, then gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO. The GO annotation results were plotted by R scripts.

Following annotation steps, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/) to retrieve their KEGG orthology identifications and were subsequently mapped to pathways in KEGG.

Enrichment analysis were applied based on the Fisher’ exact test, considering the whole quantified proteins as background dataset. Benjamini-Hochberg correction for multiple testing was further applied to adjust derived p-values. And only functional categories and pathways with p-values under a threshold of 0.05 were considered as significant.

All studies were performed in triplicate. Data were presented as the mean ± standard deviation, and differences between groups analyzed using t-tests for two groups, or one-way analysis of variance for more than two groups, followed by the Dunnett test for between-group differences in SPSS Ver. 13.0 (SPSS, Chicago, IL, USA). A P<0.05 value was considered statistically significant.

We generated a rat SCI model (Figure 1A). Basso-Beattie-Bresnahan (BBB) scoring showed that hindlimb movements in injured rats were very subtle or had disappeared, indicating successful model generation (Figure 1B). In SCI rats, we observed slightly reduced body weights, while testis weights remained steady, therefore slight increases in testis/body weight ratios were recorded (Supplementary Figures 1A–C). Further histomorphological examinations showed that when compared with sham rats, seminiferous tubules in SCI rats had thinner germ cell layers with a larger tubular lumen, but a comparable tubule diameter (Figures 1C–F). Interstitial cells in SCI rats were severely degenerated (Figure 1G). These observations suggested that spermatogenic defects had arisen in SCI rats. Furthermore, defects were confirmed by the reduced expression of the germ cell marker DDX4 (Figure 1H), while increased germ cell loss was identified by TUNEL staining (Figures 1I, J). Concordantly, epididymides in SCI rats had significantly lower sperm numbers (Figures 1K, L). We excluded injury to epididymides as causing this sperm reduction (Figure 1K and Supplementary Figure 1D). Additionally, we detected a sharp decrease in testicular testosterone levels in SCI rats (Figure 1M), which explained spermatogenic disorders. The serum luteinizing hormone (LH) level in the SCI rats was notably raised, and the mRNA level of Lhcgr, a receptor for LH, was also elevated (Supplementary Figures 1E, F), indicating that the pituitary gonadotropin-LH pathway is functional and the raise of LH is probably a feed-back compensation. The testosterone decline in SCI rats likely due to impairment in testosterone synthesis within testis. Overall, our rat SCI model exhibited spermatogenic defects and testosterone insufficiency, which largely recapitulated conditions in SCI human males.

To unveil the global molecular changes underlying the SCI-induced testicular injury, we performed proteomics analyses by isobaric tandem mass tag (TMT) labeling with the sham and SCI testes. As shown in Figure 2A and Supplementary Figure 2B, volcano plot and heat map indicated 129 differentially expressed proteins (DEPs) in the SCI rats, of which 51 were upregulated and 78 downregulated. Gene Ontology (GO) enrichment analysis showed that the down-regulated DEPs were mostly enriched in steroid and cholesterol metabolism and hormone biosynthesis, as well as sperm flagella (Figures 2B, C). Besides, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the down-regulated DEPs were also significantly enriched in steroid and hormone biosynthesis pathways (Figures 2D, E). These data globally suggested that after SCI, steroid metabolism and hormone biosynthesis in testis were disrupted, leading to testosterone deficiency and spermatogenic disorders.

Autophagy participates in testosterone synthesis. We next tested if SCI impaired autophagy in testes. Immunoblotting showed that SCI testes displayed marked decreases in LC3B isoform protein levels and in LC3B-II to LC3B-I ratios (Figures 3A, B), whereas SQSTM1/p62 accumulation indicated blocked autophagy (Figures 3A, C). To further analyze these phenomena, we performed LC3B immunofluorescence on testis cross-sections. In sham testes, LC3B positive signals were primarily localized around tubule base membranes and lumen, and also the interstitium. In SCI testes, signals around the base membrane were moderately decreased, while those around the lumen and interstitium had almost vanished (Figures 3D, E). Changes in the lumen may have been due to reduced spermatids owing to spermatogenic arrest, as autophagy is required for sperm release. We next focused on lipid deposition and autophagic activity in interstitial cells. Oil red O staining showed that lipid droplets (LDs) were barely detected in sham testes but were distinctly deposited in interstitial cells from SCI rats (Figures 3F, G). 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-bora-3a and 4a-diaza-s-indacene (BODIPY - fluorescent dye which labels natural lipids) staining in combination with LC3B immunofluorescence verified increased BODIPY signals, while reduced LC3B signals were observed in SCI testes (Figures 3H, I). Combined, these observations suggested that SCI rats had low testosterone levels and impaired spermatogenesis similar to SCI male patients, and their testes showed depressed autophagic activity and lipid accumulation in interstitial cells.

NO levels were significantly elevated in SCI rat testes (Figure 4A). Therefore, we investigated if NO disrupted autophagy and subsequently impaired steroidogenesis and testosterone synthesis. We used mouse primary LCs (Supplementary Figure 2B), tested NO production in LCs, and examined to what extent an endogenous NO donor (L-arginine) produced NO in LCs. L-arginine failed to increase NO levels in LCs (DAF-FM DA staining, Supplementary Figure 2C) and barely affected LC3B-I/II protein levels (Figures 4B, C), even at 5 mM. Our qRT-PCR analyses showed that iNOS and nNOS genes were very weakly expressed, and eNOS was barely detectable in mouse LCs (Figure 4D). Thus, mouse LCs produced low NO levels. In contrast, an exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) (25) significantly increased NO levels in LCs (Figures 4E, F), decreased LC3B-II while increasing LC3B-I levels, and accumulated the autophagic substrate SQSTM1/p62 (Figures 4G–I). Consistently, LC3B-GFP^–mCherry⁺ (red autophagosomes) and LC3B-GFP⁺/mCherry⁺ (yellow autolysosomes) dual-immunofluorescence signals were significantly decreased after SNAP treatment (Figures 4J, K), indicating blocked autophagic processes in SNAP-treated LCs. Also, testosterone synthesis was largely disrupted (Figure 4L) as cholesterol (Figure 4M) and steroidogenesis enzyme levels were decreased after SNAP treatment (Figure 4N). Thus, the exogenous NO donor (not the endogenous NO donor) produced NO, inhibited autophagy, and impeded testosterone production in mouse LCs, consistent with in vivo observations in SCI rats.

As observed in SCI rat testis sections, SNAP also induced lipid accumulation in primary mouse LCs (Figures 5A, B). We next monitored NO effects on lipophagy. Double BODIPY/LC3B fluorescence further confirmed decreased LC3B signals while LDs had accumulated in SNAP-treated mouse LCs, and strikingly, BODIPY/LC3B co-localization was almost abolished (Figures 5C, D), indicating that SNAP had disrupted LD engulfment into autophagosomes, an early and essential lipophagy step. Autophagosomes are destined to fuse with lysosomes so as to degrade substrates for recycling. Consistently, double BODIPY/Lysotracker fluorescence confirmed reductions in co-localized LDs and lysosomes in SNAP-treated LCs (Figures 5E, F), further validating blocked lipophagy processes. The mRNA levels of several critical factors involved in lipid uptake or synthesis were decreased (Supplementary Figure 3A), indicating that the observed lipid accumulation was not due to increase in lipid synthesis. To corroborate our evidence, treatment with 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), a NO scavenger (26), significantly reversed SNAP-induced NO elevation, restored autophagy, especially lipophagy, and therefore promoted testosterone production (Figures 4E–L, 5A–F). Unexpectedly, CTIOP lowered cellular cholesterol levels (Figure 4M) and downregulated factors required for lipid uptake (Supplementary Figure 3B). These observations require further investigations. However, of note, CPTIO alone did not alter autophagic activity in LCs (Supplementary Figure 3C), indicating that CPTIO reversed SNAP-induced lipophagic impairment by specifically clearing NO. Cumulatively, these results demonstrated that NO suppressed lipophagy and testosterone production in mouse LCs, which were effectively rescued by CPTIO, a NO scavenger.

We next determined which autophagy-associated pathways were altered in SNAP-treated LCs. Immunoblotting showed that the mTOR pathway, which inhibits autophagy, was activated as indicated by upregulated p-mTOR (S2448) and the downstream p-S6 protein (S235/S236) (Figures 5G, H). ULK1 phosphorylation at S757, which is activated by mTOR to inhibit autophagy, was also upregulated. Both AMPK (T172) and downstream ULK1 (S555) activities, which promote autophagy, were also downregulated (Figures 5G, H). Accordingly, SQSTM1/p62 accumulated in cells. Critically, these signaling alterations were reversed by CPTIO (Figures 5G, H). When combined, NO inhibited LC autophagy, which upregulated anti-autophagic mTOR and downregulated pro-autophagic AMPK/ULK1.

We next asked if activating upstream autophagy signaling antagonized NO in mouse LCs. LYN-1604 treatment [ULK1 agonist (44)] activated SNAP-depressed AMPK/ULK1 (S555) signaling and autophagic activity (Figures 6A, B). Correspondingly, LYN-1604 efficiently restored LDs engulfment into autophagosomes and lysosome delivery (degradation) (Figures 6E–H; Supplementary Figures 4C, D), and importantly, cholesterol and testosterone levels were restored (Figures 6C, D). These results showed that NO inhibited lipophagy and testosterone synthesis in mouse LCs, and that LYN-1604 alleviated these phenotypes by specifically activating upstream autophagic signaling, as LYN-1604 did not alter cellular NO levels in LCs (Supplementary Figures 4A, B).

We next evaluated in vivo CPTIO efficacy in restoring testosterone levels and spermatogenesis. In mice or rats, acute immune responses begin at ~24 h after SCI, with a duration of approximately 3 days (45). Based on this information and using BBB scoring to confirm SCI, we used two CPTIO treatment strategies in SCI rats: 1) treatment began immediately following scoring and lasted for 7 days - CPTIO-7d group; 2) treatment began at day 3 and lasted for 4 days - CPTIO-4d group (Figure 7A). We were keen to identify which approach improved testicular function/recovery; i.e., eliminating NO as soon as possible (CPTIO-7d) or allowing NO production during acute immune stages (CPTIO-4d). Both treatments significantly decreased NO levels (Figure 7B). H&E staining showed that tubular histomorphology was restored in both groups, with epithelium thickness and interstitial cell areas significantly recovered (Figures 7C, E, F). The CPTIO-4d strategy appeared to show better effects when compared with CPTIO-7d. Both CPTIO treatments also enhanced lipophagic activity in interstitial cells, avoided aberrant lipid accumulation (Figures 7I–L), and promisingly, restored testosterone levels (Figure 7H). Surprisingly, neither CPTIO treatments restored sperm release from epididymides (Figure 7G). However, we noted that sperm reservoirs in epididymides were morphologically recovered (Figure 7D). Overall, these observations, in combination with in vitro data, indicated that NO scavenging by CPTIO attenuated SCI-impaired lipophagy and testicular functions.

We observed the increased testicular expression of two macrophage markers CD68 and iNOS (Figures 8A, B), further suggesting that NO was predominantly produced by macrophages and mediated inflammation-impaired steroidogenesis. We therefore explored if NO clearance, by directly inhibiting NOS, effectively recovered testosterone levels and spermatogenesis in vivo. To this end, SCI rats were injected with a NOS inhibitor (L-NAME) (24). Unexpectedly, in vivo analyses showed neither restored testosterone levels, nor testis morphology, nor sperm counts (Supplementary Figure 5). This was likely due to the general effects of L-NAME in blocking all NOS isoforms in testes. To overcome this issue, we used a co-culture system, with LCs (2 × 10⁵ cells/well) in upper and peritoneal macrophages (PMs, 5 × 10⁵ cells/well) in lower Transwell chambers, to mimic the immune-activated microenvironment in vivo (Figure 8C). Both cell types had no direct contact and communicated with each other only via signaling. At first, macrophages were induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), under which conditions LCs were co-cultured for 24 h (Supplementary Figure 6A). Immunoblotting showed that iNOS protein levels were significantly raised after LPS and INF-γ treatments (Supplementary Figure 6A), indicating successful M1 transition. We observed decreased LC3B-II/I ratios (Supplementary Figure 6B), indicating that M1 macrophage signals had suppressed LCs autophagy. However, L-NAME treatments failed to rescue LC3B-II/I ratios (Supplementary Figure 6B). We hypothesized that LPS and IFN-γ probably compromised L-NAME effects. Indeed, LPS and IFN-γ alone, even without M1 macrophages, also decreased LC3B-II/I ratios (Supplementary Figure 6B). Therefore, LPS and IFN-γ were supplemented for the first 24 h for M1 induction and then removed, with M1 macrophages and LCs co-cultured in fresh medium for another 24 h (Figure 8C). We observed that M1 macrophages alone produced high NO levels (Figure 8D), decreased autophagic activity and pathway outputs (Figures 8E, F), disturbed lipophagy, induced lipid accumulation (Figures 8I–L), and reduced testosterone secretion in LCs (Figures 8G, H), all of which were reversed by L-NAME (Figures 8D–L). Thus, NO production by M1 macrophages was a critical incentive for lipophagic defects and testosterone deficiency in LCs, and the application of a NOS inhibitor to clear NO appeared to prevent these phenotypes.