2.1. Materials

Cornuside (Figure 1(a); HPLC ≥98% purity) was obtained from Chengdu Ruifensi Biotechnology Co. Ltd. (Chengdu, China). The hematoxylin and eosin (H&E) assay kit was purchased from Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China). The testosterone ELISA kit was obtained from Anhui Joyee Biotechnics Co. Ltd. (Hefei, China). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) ELISA kits were obtained from Shanghai Meilian Biotechnology Co. Ltd. (Shanghai, China). The TUNEL assay kit was obtained from Beyotime Biotechnology Co. Ltd. (Shanghai, China).

2.2. Animal Experimental Procedures

In this study, 14-week-old male KK-Ay and C57BL/6J mice were purchased from Beijing Huafukang Bioscience Co. Ltd. (Beijing, China) (license no. SCXK Beijing 2014-0004). They were housed in a standard animal care facility (25°C ± 1°C; 55% ± 5% relative humidity; and 12-h light/dark cycle). The flow diagram of the animal experiment design is demonstrated in Figure 1(b). The animals were given a 2-week acclimatization period prior to experimental use. The Animal Ethics Committee of the Nanjing University of Chinese Medicine approved the present study (approval number: ACU-13 (20161011)).

According to a random grouping number table, KK-Ay mice were randomized into two treatment groups (n = 6/group): the model (DM) group and the cornuside treatment (Cor) group, both fed daily a high-fat diet. The Cor group received cornuside [100 mg/(kg day)] by oral administration, and the DM group received the same volume of normal saline daily equal to that of the Cor group. Six C57BL/6J mice were used as the normal group, which were fed with a normal diet and given the same volume of normal saline daily equal to that of the other groups for an 8-week period. During this period, a high-fat diet (60.5% standard rat feed, 10% sugar, 0.2% cholesterol, 24% lard, and 5% egg yolk powder) obtained from Beijing Huafukang Bioscience Co. Ltd. (Beijing, China) was fed to KK-Ay mice, whereas C57BL/6J mice were fed standard chow. The fasting plasma glucose level in all animals was measured after 4 and 8 weeks using blood collected from the tail vein of these animals following a 12-h fasting period. In addition, the bodyweight, 24-h food consumption, and 24-h water intake were measured. Prior to the sacrifice, the fecal sample of each mouse was collected and stored at −80°C for the analysis of gut microbiota. Also, testis and epididymis were collected and tested.

2.3. Histopathological Evaluation

After fixation using 10% formalin, the testicular tissue sections were paraffin-embedded, cut into 5 μm sections, and stained using H&E. A microscope was then used to evaluate islet histology (200×). Images were blindly taken from randomly selected areas, and the representative images of these parts were displayed. Testicular tissue injury was analyzed and semiquantitatively scored by pathologists on the basis of the hierarchical arrangement and structure of spermatogenic cells, interstitial vasodilation, interstitial cell hyperplasia, hemorrhage, and inflammatory cell invasion. Data were presented as mean ± SEM. A score of 0 represented no lesions, and a score of 6 represented the most severe lesions.

2.4. Measurement of Testosterone, LH, and FSH Levels

The testosterone, LH, and FSH levels in testes were assessed following the manufacturer’s protocols. In brief, the samples were added for 2 h at 28°C to 96-well plates coated with primary antibodies. Then, the wells were washed three times, and a 100 μL volume of the chromogenic substrate was added for 20 min at 28°C. The absorbance at 450 nm was then evaluated within 10 min of the addition of 50 μL of stop solution to each well.

2.5. Measurement of the Apoptosis of Testicular Cells in KK-Ay Mice

The TUNEL assay was used for testing apoptotic testicular cells. After fixation using 10% paraformaldehyde, the testicular tissue sections were paraffin-embedded and cut into 5 μm sections. In brief, the testicular tissue sections were dewaxed, rehydrated in xylene and ethanol, and then incubated with the working solution of proteinase K at 37°C for 20 min. Further, 50 μL of TUNEL reaction mixture was added after washing the samples with PBS at 37°C for 1 h. The samples were again washed with PBS to stop the reaction. Apoptosis was found to occur in TUNEL-positive cells. ImageJ software was used for quantitative analysis.

2.6. Measurement of Sperm Count and Sperm Motility

The epididymis was cut, and the sperm suspension was dissolved in saline. The sperm suspension was placed in the Neubauer counting chamber, and the sperms were counted by using a light microscope. Sperm motility was detected by eosin-nigrosin one-step staining technique and distinguished by staining (Bjorndahl et al. 2003).

2.7. Extraction of Fecal Genomic DNA and Sequencing

Fecal DNA was extracted from cecum stool samples using E.Z.N.A. soil kit (Omega Bio-Tek, GA, USA) following the manufacturer’s instructions. The hypervariable V3-V4 region of the 16S-rDNA gene was amplified by PCR using the primers 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′. The cycling conditions were as follows: initial denaturation at 95°C for 3 min, 28 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 45 s, and final extension at 72°C for 5 min and 10°C until completion. Amplicons were recovered from 2% agarose gels and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) following the manufacturer’s instructions and quantified using a QuantiFluor-ST fluorometer (Promega, USA). The purified amplicons were pooled in equimolar ratio and paired-end sequenced (2 × 300 bp) using PE300 strategies on an Illumina MiSeq platform (Illumina, CA, USA). The raw reads were deposited into the NCBI Sequence Read Archive database (accession number: SRP168312).

2.8. Bioinformatics Analysis

Illumina paired-end reads were connected, filtered, and then processed using the software package of QuantiFluor-ST. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). The representative sequences for each OTU were screened, and the differential analysis of groups of dominant species was performed. Alpha and beta diversities were used to analyze the complexity of species diversity.

2.9. Statistical Analysis

Data were expressed as mean ± SD and compared using one-way ANOVA with Tukey’s post hoc test. SPSS 22.0 was used for all statistical analyses, and P < 0.05 indicated a statistically significant difference. SPSS was used to calculate Pearson’s correlation coefficient to verify the correlation between bacterial abundance and reproductive injury indexes such as FBG, testicular index, testosterone, and live sperm rate. GraphPad software was used to draw a heat map of Pearson’s correlation coefficient between the above indexes and bacterial abundance.

3.1. Cor Improved General Symptoms of KK-Ay Mice

The 24-h food consumption, 24-h water intake, FBG, and body weight were measured in all experimental animals after 0, 4, and 8 weeks of the study. The 24-h food consumption and 24-h water intake dramatically increased in the DM model mice compared with the control mice in the 8th week (Figures 1(c) and 1(d)). Moreover, the FBG level remarkably improved in the model group compared with the control group, which was markedly decreased by Cor treatment (Figure 1(e)). Cor administration ameliorated the symptoms of polydipsia and polyphagia and increased the body weight compared with those in the DM model mice (Figure 1(f)).

3.2. Cor Mitigated Testicular Lesions in KK-Ay Mice

The study next analyzed testicular tissue and testis/body weight ratio. H&E staining was used to assess morphological changes so as to examine how Cor treatment affected testicular histopathology in DM model mice. The testis of mice in the control group showed normal testicular tissue structure, including concentric layers of integral seminiferous tubules and organized germ cells. Spermatogenic cells in various stages, spermatogonia to spermatids, were observed, accompanied by some interstitial cells in the lumen of neatly arranged seminiferous tubules. The mice in the DM model group exhibited rupture of seminiferous tubules, degeneration of germ cells, and structural shrinkage and separation from each other. The pathological injury was remitted to different degrees after an 8-week treatment with Cor. Figure 2(a) shows the pathological score of testicular lesions. Additionally, the testis/body weight ratio significantly decreased in the DM model group compared with the control group (Figure 2(b)), while it significantly increased in the Cor group compared with the DM model mice. Testosterone is crucial in sperm production, formation, and maturation. It is mainly secreted by testicular interstitial Leydig cells and closely related to spermatogenesis (Wang et al. 2009). The testosterone level represents sperm motility to some extent. The testosterone level in testes among groups is shown in Figure 2(c). The testosterone level significantly decreased in the DM model group compared with the control group, which was significantly increased by Cor treatment. Consistent with the testosterone trend, the LH and FSH levels also significantly decreased in the DM model group compared with the control group, which was significantly increased by Cor treatment (Figures 2(d) and 2(e)).

3.3. Cor Ameliorated the Apoptosis of Testicular Cells in KK-Ay Mice

TUNEL assay was used to assess apoptosis so as to examine the anti-apoptotic effect of Cor treatment on testicular injury in KK-Ay mice. The DM model mice exhibited a large number of apoptotic cells in the testes compared with normal mice; the apoptosis was mitigated by Cor administration (Figures 3(a) and 3(b)). Therefore, Cor had anti-apoptotic effects on DM-induced testicular damage.

3.4. Cor Improved the Sperm Count and Sperm Motility

Sperm count and sperm motility were tested after the experiment; the sperm count and sperm motility were significantly reduced in the DM model group compared with the control group, which was significantly increased by Cor treatment (Figures 3(c) and 3(d)).

3.5. Cor Improved the Dysbiosis of Gut Microbiota in KK-Ay Mice

16S rDNA sequencing analysis was performed to detect the changing characteristics of gut microbiota so as to investigate the influence of DM-induced intestinal flora dysbiosis on testicular damage. The OTU clustering of unique sequences was based on 97% sequence similarity. Alpha diversity of the gut microbiota was determined using the Chao index, Shannon index, and rarefaction curve. The alpha diversity dramatically reduced in the DM model group compared with the control group (P < 0.05). At the same time, Cor treatment could notably restore the alpha diversity of DM model mice. The dynamics of alpha diversity indices, including Chao index and Shannon index, indicated that the abundance and diversity of gut bacteria increased significantly in the Cor group compared with the DM model group (Figures 4(a) and 4(b)). Moreover, the number of OTUs observed was lower in the model group than in the other two groups. The rarefaction curve analysis showed that the sequencing depth covered rare new phenotypes and species as far as possible (Figure 4(c)). As shown in Figure 4(d), a weighted PCoA clearly demonstrated the compositional differences in gut microbiota with an obvious difference along the PC1 axis (reaching 49.4% of overall variation).

The results of the hierarchical cluster tree analysis showed significant clustering between the control and model groups; the Cor group also showed a better separation trend (Figure 5(a)). LEfSe was used to identify the bacterial phenotypes with specific changes from phylum to genus so as to study further the differences among control, model, and Cor groups. The cladogram showed the dominant bacteria in each group (Figure 5(b)). The LDA score (log 10 > 2) showed significant changes in 68 bacteria in the 3 groups (Figure 5(c)). The composition of gut bacterial species in each group showed apparent differences. Differential microbial lineages in the control group included 20 bacteria belonging to Verrucomicrobiales, Akkermansia, Verrucomicrobia, Verrucomicrobiaceae, Verrucomicrobiae, Actinobacteria, Bifidobacteriaceae, and so forth. Differential microbial lineages in the model group included 30 bacteria belonging to Enterobacteriales, Gammaproteobacteria, Enterobacteriaceae, Escherichia Shigella, Enterococcaceae, Enterococcus, and so forth. Differential microbial lineages in the Cor group included 18 bacteria belonging to Rikenellaceae, Alistipes, Ruminiclostridium_5, Lachnospiraceae_UCG_001, Butyricicoccus, Odoribacter, Marvinbryantia, and so forth.

3.6. Cor Reverses the Changes in the Distribution of the Microbes in KK-Ay Mice

It is worth noting that among the 68 strains of microorganisms, 7 strains are significantly different among the 3 groups, including Weissella confusa (Weissella), Clostridium sp. ND2 (Clostridium sensu stricto 1), uncultured bacterium (Roseburia), Anaerotruncus colihominis DSM 17241 (Anaerotruncus), [Clostridium] leptum (Anaerotruncus), unidentified (Ruminococcus 1), and uncultured bacterium (Bilophila) (Figures 6(a)–6(g)). Interestingly, the relative abundance of the marked 7 strains of microorganisms were significantly increased, which were significantly decreased by Cor treatment. These results suggest that Cor reverses the changes in the distribution of these microbes, which may be a potential biomarker for diagnosing the testicular injury caused by DM.

3.7. Correlation Analysis of Different Flora and Reproductive Injury Indexes

In order to explore the relationship between the testicular injury caused by DM and gut microbiota, we analyzed the correlation between gut microbiota, FBG, testicular index, testosterone, and live sperm rate. As shown in Figure 7(a), the closer the color is to red, the greater the positive correlation between the two parameters, and the closer the color is to blue, the greater the negative correlation between the two parameters in the heat map. The heat map suggested that Akkermansia, Bacteroidales S24-7 group, and Lachnospiraceae were negatively correlated with FBG. Akkermansia, Bacteroidales S24-7 group, Lactobacillus, Bacteroidaceae, and Prevotellaceae were positively correlated with testicular index, while Roseburia were negatively correlated with testicular index. Alloprevotella was positively correlated with testosterone, while Roseburia were negatively correlated with testosterone. Bacteroidales S24-7 group, Akkermansia, and Bacteroidaceae were positively correlated with live sperm rate, while Prevotellaceae were negatively correlated with live sperm rate.

As shown in Figure 7(b), the heat map of phylum level suggested that Bacteroidetes were positively correlated with FBG. Bacteroidetes were negatively correlated with testicular index. Actinobacteria were positively correlated with testosterone. Bacteroidetes were negatively correlated with live sperm rate, while Firmicutes were positively correlated with live sperm rate. These results suggest that the testicular injury caused by DM is closely related to gut microbiota.