PHN (>98% HPLC) was purchased from Chengdu Preferred Biotechnology Co., Ltd. (Chengdu, China). Glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals used in this study were of analytical reagent grade. Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical (St Louis, MO, United States).

Male C57BL/6 (n = 72) mice with similar body weight (21–23 g) were purchased from the Laboratory Animal Department of the Harbin Medical University (Harbin, China) and housed at 24 ± 1°C and received food and water ad libitum. The mice were housed in a clean environment to strict ensure animal welfare.

In this study, LPS (5 mg/kg) was intraperitoneally injected to induce acute orchitis in mice (11). Mice were randomly divided into six equal groups, that is, the control, LPS, PHN (25, 50, and 100 mg/kg) with LPS, and PHN (100 mg/kg) groups. Phloridzin was dissolved in distilled water and orally administered to animals at dosages of 25, 50 and 100 mg/kg body weight daily throughout the experimental period. The experimental timelines for the animal models are shown in Figure 1B. In the PHN (25, 50, and 100 mg/kg) + LPS groups, mice were intragastrically administered their respective PHN dose 21 d before LPS treatment daily. Mouse orchitis was induced by intraperitoneal LPS injection at 5 mg/kg (PHN weight/mouse body weight) for 24 h (from day 20 to 21). Twenty-four hours after injection of LPS, mice were euthanized and testes were collected to detect tissue structure, expression of factors associated with inflammation and testosterone synthesis, and integrity of the blood-testosterone barrier. In addition, blood is collected and then centrifuged to obtain serum, which is assayed for testosterone content. Moreover, mice in the PHN groups were continuously treated with their respective PHN doses during LPS treatment.

Glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) activity in the teste tissues from different groups was examined using their corresponding kits (Nanjing Jiancheng Bioengineering Institute) in accordance with the manufacturer protocols. The enzymatic activity was measured by a microplate reader (Bio-Rad, United States of America) according to the respective absorbance.

ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to measure testosterone levels in the serum samples. Approximately 500 μL of blood samples were obtained from each mouse, and serum samples were collected by centrifugation at 1000 g for 12 min at 4°C. Serum testosterone levels were measured using kits according to the manufacturer protocols by a microplate reader (Bio-Rad, United States of America).

Total RNA was isolated from teste samples from different treatment groups using TRIzol reagent (Invitrogen), according to the manufacturer instructions. The RNA concentration was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific), and complementary DNA (cDNA) was synthesized using a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, United States). Real-time fluorescence quantitative PCR was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) and SYBR Green Plus reagent kit (TransGen Biotech, AQ141, Beijing, China). Primer sequences used in this study are listed in Table 1 with β-Actin was used as the reference gene. Quantitative RT-PCR was conducted thrice and normalized to the expression of the reference gene (i.e., β-actin). The relative gene expression levels were calculated using the 2^-ΔΔCT method.

Teste tissues obtained from different treatment groups and were fixed in 4% (v/v) paraformaldehyde for 24 h, embedded in paraffin, and cut into 5 μm sagittal sections. The sections were de-paraffinized with xylene and ethanol, washed with phosphate-buffered saline (PBS), and permeabilized with 0.1 M citrate and 0.1% Triton X-100 permeabilization solution. The deparaffinized sections were stained with hematoxylin and eosin (H&E), and images were captured using an OLYMPUS BX53 microscope to examine pathological structural changes in the testes.

Immunofluorescence staining was performed on paraffin-embedded sections of testicular tissue. Tissue slices were deparaffinized, rehydrated, and washed with 1% PBS-Tween. Sections were deparaffinized in xylene for 24 h and rehydrated. After washing with 1% PBS-Tween, the sections were treated with 3% hydrogen peroxide, permeabilized with 0.3% Triton X-100, and blocked with 3% BSA. Next, the sections were incubated for 1 h at 37°C with primary antibodies directed against Zona occludens 1 (ZO-1) or occludin (1:200; Beijing Biosynthesis Biotechnology Co., LTD, Beijing, China). After washing with PBS three times, the slides were incubated with species-specific fluorescent secondary antibodies (1:200; Beijing Biosynthesis Biotechnology Co., LTD, Beijing, China) for 1 h at 23°C and stained with Hoechst 33342. Finally, the cover slips were mounted, and images were captured using a light microscope (Olympus, Tokyo, Japan).

Before the experiment began, each mouse was marked for subsequent experiments, and fresh fecal pellets were obtained after 20 d. Total DNA was extracted from the samples, primers were designed and synthesized in accordance with the conserved block, and the ends of the primers were connected with sequencing connectors to conduct PCR amplification. A sequencing library was established using product purification, quantification, and homogenization. The original data from high-throughput sequencing were analyzed and converted into sequence readings through base calling. The same operational taxonomic unit (OTU) was defined as a sequence greater than or equal to 97% for bacterial classification.

Statistical analysis of experimental data was performed using SPSS 17.0. The values are presented as mean ± standard deviation (SD), and multiple comparisons were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test. The differences between the two groups of data were assessed using an unpaired two-tailed Student t-test. Significant differences are represented by p < 0.05 and p < 0.01.

Excessive LPS in the body can cause systemic sepsis in animals, which can cause serious weight loss over a short period; therefore, we measured body weight variations of the different treatment groups every 4 h after LPS injection, and our results showed a significant decrease in the LPS treatment group compared to that in the control group; however, those treated with PHN (50 and 100 mg/kg) significantly decreased the LPS-induced weight loss (Figure 1C). Additionally, testicular weight loss is also an important marker of orchitis; therefore, we examined the relative changes in testicular and body weights. The results showed that LPS treatment significantly reduced testicular weight compared with the control group, while 100 mg/kg PHN treatment significantly restored this change (Figure 1D). Moreover, from histopathological observations, we found that the number of spermatogenic cells were decreased (Figures 1E-a), most spermatogenic cells were exfoliated (Figures 1E-b), and the mature sperm were rare (Figures 1E-c) in the LPS treatment group; however, PHN treatment partially reversed these changes in a dose-dependent manner.

Myeloperoxidase is abundant in neutrophils and is closely related to inflammation. Here, we examined the effect of PHN pretreatment on LPS-induced MPO activity in the testes using ELISA. As shown in Figure 2, LPS treatment increased a nearly three-fold MPO activity in the testes compared with the control group; however, compared with the LPS group, different concentrations of PHN significantly reduced MPO activity in a dose-dependent manner (Figure 2).

Changes in pro-inflammatory cytokines damaged the Leydig cells and BTB, thereby resulting in decreased testosterone synthesis and spermatogenesis. Here, we examined the effect of PHN pre-treatment on LPS-induced inflammatory cytokine production in the testes. Our results indicated that LPS treatment significantly increased the mRNA expressions of Tumor necrosis factor α (TNF-α), Interleukin-17A (IL-17A),Interleukin-1β (IL-1β), and Interleukin-2 (IL-2) compared with those in the control (Figures 3A–D); however, different PHN concentrations significantly reduced the mRNA expressions of TNF-α, IL-17A, and IL-1β compared with those in the LPS treatment group (Figures 3A–C). Additionally, 25 mg/kg and 50 mg/kg PHN did not affect the mRNA expression of IL-2, while 100 mg/kg PHN significantly reduced the mRNA expression of IL-2 compared with that in the LPS treatment group (Figure 3D).

To test whether PHN treatment has an antioxidant function in LPS-induced orchitis, we tested three oxidative stress markers. Our results showed that the expression levels of SOD (25.5 vs. 50.0, p = 0.002) and GSH (7.5 vs. 26.1, p = 0.006) in the LPS-induced acute orchitis group were significantly lower than those in the control group (Figures 4A,B). In contrast, the mice in the LPS group showed a significant increase in MDA (1.4 vs. 8.4, p = 0.015) concentration (Figure 4C). Compared with LPS group, after 50 and 100 mg/kg PHN treatment, the activity of SOD was significantly up-regulated (25.5 vs. 34.9, p = 0.049; 25.5 vs. 42.5, p = 0.017; Figure 4B). Moreover 100 mg/kg PHN treatment, GSH and MDA were significantly up-regulated (7.5 vs. 16.8, p = 0.042; Figure 4A) and down-regulated (8.4 vs. 5.3, p = 0.044; Figure 4C) compared with LPS group, respectively.

Testosterone plays an important role in spermatogenesis and maintenance of sperm motility in mammals; however, Leydig cells damaged during orchitis result in lower testosterone levels. As shown in Figure 5, our results indicate that the mRNA expression of genes associated with testosterone synthesis (3beta-hydroxysteroid dehydrogenase, 3β-HSD and steroidogenic acute regulatory protein, stARF) in the LPS group was significantly lower than that in the control group. Consistent with these results, the testosterone levels in the blood were also significantly decreased in the LPS group compared with that in the control group (2.8 vs. 8.6, p = 0.002; Figure 5A). However, these changes were significantly reversed after PHN treatment in a dose-dependent manner (2.8 vs. 3.9, p = 0.023; 2.8 vs. 4.7, p = 0.011; 2.8 vs. 6.9, p = 0.008; Figure 5A).

The BTB is important in ensuring the microenvironment for independent spermatogenesis in the testes. Therefore, we examined the mRNA and protein expression of the BTB-associated tight junction proteins ZO-1 and occludin and found that the mRNA expression levels of ZO-1 and occludin in of the LPS-treated mice testes were significantly decreased (Figures 6A–B), and the protein content was also significantly decreased by immunofluorescence (Figures 6C–E). However, the mRNA and protein expression levels of ZO-1 and oocludin significantly recovered after PHN treatment in a dose-dependent manner (Figures 6C–E).

The changes in microbial species, a clone library of the 16S rRNA gene, were established and sequenced to study the potential mechanism of PHN against LPS-induced orchitis in mice. For beta diversity analysis, principal coordinates analysis (PCoA) was used to analyze the microbiota communities. The data showed significant and distinct clustering of microbiota composition between the control and PHN treatment groups (Figure 7A), indicating that significant differences in microbial composition between the control and PHN treatment groups. Each sample contained 10 different phyla, including Bacteroidetes, Firmicutes, Verrucomicrobia, Proteobacteria, Epsilonbacteraeota, Patescibacteria, Actinobacteria, Tenericutes, Cyanobacteria, and Deferribacteres in descending order (Figure 7A). The four phyla with the highest abundances were Bacteroidetes, Firmicutes, Verrucomicrobia, and Epsilonbacteraeota. After PHN treatment, Firmicutes and proteobacteria did not change significantly (Figures 7C,F), but the Bacteroidetes abundance was significantly higher than that in the control group (Figure 7B). However, the ratio of Firmicutes to Bacteroidetes in the PHN-treated group was significantly lower than those in the control group (Figure 8D). Additionally, the Epsilonbacteraeota and Verrucomicrobia abundances in the PHN group were significantly lower than those in the control group (Figures 7E,G). Moreover, further analysis by LDA found that Bacteroidetes and Verrucomicrobia also showed significantly increase and decrease at phylum level (Supplementary Figure S1).

At the family level, Muribaculaceae was the most abundant in all fecal samples (Figure 8A). The proportion of Muribaculaceae in the PHN group was 50.52%, which was significantly higher than that in the control group (37.72%; Figure 8B). The second most abundant family in the PHN groups was Lachnospiraceae (9.20%),which was significantly lower than that in the control group (19.38%; Figure 8C). The third most abundant family in the PHN group was Lactobacillaceae (13.36%), which was significantly higher than that in the control group (5.40%; Figure 8D). The relative abundance of Ruminococcaceae (Figure 8E) did not change, but the relative Prevotellaceae and Akkermansiaceae abundances significantly increased and decreased (Figures 8F–G). At the genus level, uncultured bacterium f Muribaculaceae and Lactobacillus were significantly increased, and Akkermansia were significantly decreased in the PHN group compared with those of the control group (Figures 9A–E). Additionally, LDA analysis showed that similar results at the family and genus levels (Supplementary Figure S1).