Syringa oblata Lindl. was growing naturally on the campus of Northeast Agricultural University (N 45°44′33.64″, E 126°43′22.07″) in Harbin, Heilongjiang Province of China, and were authenticated by Professor Xiuju Wu from College of Life Sciences, Northeast Agricultural University. The raw materials were dried, pulverized, sifted through an 80-mesh sieve, and stored at 4°C before use. Voucher specimens were deposited at the Department of Animal Pharmacy, Northeast Agricultural University. Rutin, luteolin, oleanolic acid, and kaempferol standards (purity≥98%) were purchased from the Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). L-proline, L-lysine, 1,2-propanediol, glycerol, glycol, 1,4-butanediol, methanol, and other chemicals were obtained from the Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). D101 macroporous resin was procured from the Tianjin BSF Resin Technology Co. Ltd. (Tianjin, China).

A green and efficient deep eutectic solvent (DES) combined with ultrasound-assisted extraction (DES-UAE) was developed to extract active components of SOL. The detailed extraction method is described in Supplementary Material 1.

The main compounds of SOL extracts were analyzed using a Vanquish UPLC system coupled with a Q Exactive^TM HF-X Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer (UHPLC-HRMS; Thermo Fisher Scientific). The mass spectrometer was operated in negative or positive ion mode. LC separation was done on an ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) using a gradient of solvent A (10 mM ammonium formate, acetonitrile:water = 95:5, and 0.1% formic acid) and solvent B (10 mM ammonium formate, acetonitrile:water = 50:50, and 0.1% formic acid) in positive ion mode, and solvent A (10 mM ammonium acetate, acetonitrile:water = 95:5, and pH = 8) and solvent B (10 mM ammonium acetate, acetonitrile:water = 50:50, and pH = 8) in negative ion mode. The flow rate was 0.3 mL/min, the injection volume was 5 μL, and the column temperature was 25°C. In MS acquisition, the instrument was set to acquire the m/z range of over 70–1,050 with an MS resolution of 60,000. Raw data were collected by mass spectrometry, peak extraction, and retention time using the compound discoverer software (Thermo Fisher Scientific).

The contents of rutin, luteolin, oleanolic acid, and kaempferol of SOL extracts were determined using the high-performance liquid chromatography (HPLC) analysis. The samples were filtered through a 0.45 mm membrane filter and loaded for HPLC analysis. The determination was performed on a Thermo C18 column (4.6 mm × 250 mm, 5 μm) at a temperature of 30°C. The information on the mobile phase, UV wavelengths, and injection volumes are shown in Supplementary Table S2.

Fifty-five Kunming female mice (6–8 weeks, 32–36 g) were purchased from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. The research was conducted in accordance with the Institutional Animal Care and Use Committee of Northeast Agricultural University (No. NEAUEC20).

All animals were first acclimated in temperature (22 ± 3°C) controlled room with 12 h light cycle. All mice were randomly divided into 11 groups (with five mice each): (1) control group; (2) 0.5% CMC-Na group; (3) model group; (4) high-dose SOL-treated (H-SOL) group; (5) low-dose SOL-treated (L-SOL) group; (6) rutin-treated group; (7) luteolin-treated group; (8) oleanolic acid-treated group; (9) kaempferol-treated group; (10) monomer mixture-treated group; (11) DEX-treated group. To induce endometritis, 150 μL S. aureus ATCC29213 (1 × 10⁶ CFU/mL) was injected into the uteri of mice via the vagina with a blunt needle for 24 h (22). In the SOL-treated group, the mice received 3.13 g/kg and 1.56 g/kg optimized SOL extracts by uterine perfusion. Rutin (24.63 mg/kg), luteolin (21.56 mg/kg), oleanolic acid (1.52 mg/kg), kaempferol (129.91 μg/kg), and monomer mixture (47.84 mg/kg) were given by uterine perfusion and these drugs were dissolved in 0.5% CMC-Na, then the 0.5% CMC-Na was given separately to exclude solvent interference. The dose of monomeric administration was converted according to the content results in SOL extracts. In addition, 5 mg/kg dexamethasone (DEX) was administrated as a positive control (23, 24). Twenty-four hours later, the mice were euthanized and the uterus tissues were collected and stored at −80°C for follow-up experiments.

Uterine tissues of each mouse were carefully taken and kept in a 50-mL centrifuge tube which was fixed in 4% paraformaldehyde. After that, uterine tissues were dehydrated and embedded in paraffin wax. Then, uterine sections were cut into 4 μm slices and paraffin-embedded samples were de-waxed with xylene and rehydrated with alcohol. Finally, the sections were stained with hematoxylin and eosin (H&E) and examined under an optical microscope. Histology scores were determined in a blinded fashion. A combined score of tissue damage, hyperemia, edema, and inflammatory cell infiltration was determined. The major histopathological indicators were evaluated by tissue damage, hyperemia, edema, and inflammatory cell infiltration (graded 0–3, from normal to severe, including normal, mild, moderate, and severe) (25, 26).

The mice were sacrificed after drug treatment for 24 h. The uterine tissues were harvested and homogenized in sterile saline solution (1:10 w/v) on ice. Then, the tissue homogenate was serially diluted in sterile PBS. The homogenates and their serial log dilutions were quickly plated onto TSB agar plates and incubated for 12 h at 37°C. Finally, the S. aureus numbers of CFUs remaining in each uterus were evaluated, and bacterial colony counts were presented as mean log10 CFU/ uterus (± SD).

Total RNA from uterine tissue samples was extracted using an RNAprep Pure Tissue Kit (DP431, Tiangen Biotech, Beijing). The concentration and purity of the total RNA were determined by spectrophotometry (NanoDrop 2000, Thermo, USA) at 260/280 nm. Then, reverse transcription of total RNA into cDNA was performed using a reverse transcription kit (Tiangen Biotech (Beijing) Co., Ltd.). The real-time PCR program was: 1 cycle at 95°C for 10 s, 40 cycles at 95°C for 5 s, followed by 55°C for 15 s and 72°C for 30 s. The primers for qRT-PCR are listed in Table 1. The expression levels of TLR2, TLR4, NF-κB, IκBα, TNF-α, IL-6, and IL-1β were quantified relative to the expression of GAPDH as the endogenous control by the 2^−ΔΔCT method.

The uterine tissues homogenate was centrifuged at 12,000 rpm for 15 min at 4°C, and then the supernatant was collected. Biochemical estimations of TLR2, TLR4, NF-κB, IκBα, TNF-α, IL-6, and IL-1β were performed using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.), according to the manufacturer's instructions. Briefly, a solid-phase antibody is made by coating the microtiter plate with purified mouse target antibody, adding samples to the coated microtiter wells in sequence, and then combining it with HRP-labeled detection antibody to form an antibody–antigen enzymatic complex, which is thoroughly washed and then colorized with the substrate TMB, which is converted to blue by HRP enzyme and transformed by the action of an acid to the yellow color. Absorbance was observed using an automatic enzyme standard instrument at a test sample of 450 nm. All absorbance results were normalized via standard curves.

All data were shown as mean ± standard deviation (SD). Data analyses were carried out using a student's t-test, and a value of p < 0.05 was considered statistically significant. The statistical analyses were performed using the SPSS 11.0 software (IBM, USA), and pictures were done using the GraphPad Prism 8 software.

A total of 254 SOL targets were screened, and then the C–T network was constructed. As shown in Figure 2A, kaempferol (degree = 140), luteolin (degree = 135), and oleanolic acid (degree = 65) were connected to the most targets, indicating that these three compounds are important active components in SOL. In addition, 90 endometritis-related targets were obtained, and 28 common targets of SOL and endometritis were screened (Figure 2B). The C–T–D network results showed that the active components in SOL with more targets were luteolin (degree = 18), kaempferol (degree = 15), oleanolic acid (degree = 11), and rutin (degree = 10), indicating that these four active components are the main components that exert an anti-endometritis effect (Figure 2C). Therefore, in the next in vivo experiments, we will further evaluate the pharmacological effects of these four active components on endometritis. The detailed target information is shown in Supplementary Table S3.

This study is based on the STRING database to analyze interactions between targets of SOL for the treatment of endometritis. The 28 interaction targets of SOL against endometritis were imported into the Cytoscape software for analysis. As shown in Figure 3, the size and color of the node were positively correlated with the Degree value; the larger the node, the larger the Degree value corresponding to the color change from green to red, indicating that this target was more important in this network. In the interaction network, tumor necrosis factor (TNF, Degree = 13), Interleukin-6 (IL-6, Degree = 12), Interleukin-8 (IL-8, Degree = 12), and Interleukin-10 (IL-10, Degree = 10) were at the heart of the direct target of action in SOL treatment of endometritis and may play an important role in SOL against endometritis.

Using the DAVID database for the core targets related to active components and disease, the threshold value of p < 0.05 was set to filter the top biological progress, and then GraphPad Prism 8.0 was used for plotting. Gene Ontology (GO) is the de facto standard for gene function description and was widely used in functional annotation and enrichment analysis (27). It includes three branches: biological process (BP), molecular function (MF), and cellular component (CC). As shown in Figure 4A, the items of BP annotations included inflammatory response, immune response, positive regulation of NF-kappa B transcription factor activity, and so on. For MF, the targets were enriched in cytokine activity, transcription factor activity, steroid binding, etc. CC analysis showed more targets were enriched in extracellular space, an integral component of plasma membrane and cytosol. It is reported that the innate immune system was activated when bacterial infestation and endometrial cells secreted large amounts of cytokines and chemokines in response to the inflammatory response (28, 29). The above BP or MF can infer the pathogenic endometritis.

We further conducted a KEGG pathway analysis on the 28 common targets shared by SOL active components targets and endometritis-associated targets. The KEGG functional enrichment totally identified 43 signaling pathways. The top 20 KEGG pathways are shown in Figure 4B; they mainly consisted of four signaling-related pathways associated with inflammation, including TNF signaling pathway, NF-κB signaling pathway, Toll-like receptor signaling pathway, and T cell receptor signaling pathway. Toll-like receptor (TLR) signaling is a central component of the primary innate immune response to pathogenic challenges (30). TLR2 and TLR4, members of the TLR family, are highly expressed in the endometrial cells of the uterus (31, 32). In this study, S. aureus was used to induce endometritis in mice, and it has been shown that S. aureus could be recognized by TLRs; following TLRs recognition, the NF-κB signaling pathway was activated (33, 34). In view of this, TLRs/NF-κB signaling pathway was selected as the validation pathway (Figure 4C).

Of the four potential active components against endometritis, three of them (luteolin, kaempferol, and rutin) are flavonoids. Considering the effect of the quantitative–effective relationship, the extraction yields of total flavonoid contents (TFC), luteolin, and rutin were further optimized. The optimal solvent was composed of L-Proline/1,4-butanediol with 40% water content (Supplementary Figure S1). The single-factor test results indicated that the extraction yield of TFC, luteolin, and rutin were higher than the results of extraction with other parameters at the solid/liquid ratio of 1:20 g/mL, ultrasound power of 500 W, extraction temperature of 50°C, and extraction time of 30 min (Figure 5A).

To optimize DES-UAE parameters, a 17-run BBD with three conditions (solid/liquid ratio, extraction temperature, and extraction time) and three levels (Supplementary Table S4) were carried out, and the extraction yield of TFC, luteolin, and rutin were taken as the responses (Figure 5B). By solving the regression equation, the optimal value of the chosen variable can be obtained. Design-Expert 12 software calculated that the optimal formulation of TFC, luteolin, and rutin under following condition: solid/liquid ratio was 1:21.50 g/mL, extraction for 33.30 min at 44.40°C, with corresponding Y1 = 151.51 mg/g, Y2 = 6.98 mg/g and Y3 = 7.86 mg/g. To confirm this result, the experiment was conducted under the following conditions three times: solid/liquid ratio was 1:22 g/mL, and extraction for 33 min at 44°C, in which the total flavonoid extraction yield, rutin extraction yield, and luteolin extraction yield were 151.08 ± 1.22 mg/g, 6.97 ± 0.05 mg/g and 7.86 ± 0.08 mg/g, respectively. The predicted values matched the actual results, indicating that the optimization parameters were available. In addition, the surface morphology of SOL with different extraction solvents was observed by SEM (Figure 5C). Different solvents have different degrees of damage to plant cells, and the microstructural damage degree is as follows: DES extracts>methanolic extracts>water extracts>raw samples. These results suggested that plant cells were easily destroyed in DESs conditions, largely due to the fact that DESs could promote the dissolution of plant cell wall fibers (35). Therefore, DES can achieve more efficient extraction of target components than conventional solvents, and the DES-UAE provided a green method for extracting the main components of SOL against endometritis.

To identify the compounds of SOL extracts, LC/MS test was performed. As shown in Table 2, a total of eight compounds, including kaempferol, rutin, luteolin, hyperoside, oleanolic acid, quercetin, isoquercetin, and caffeic acid, were identified. Among them, kaempferol, rutin, luteolin, hyperoside, quercetin, and isoquercitin belong to flavonoids; oleanolic acid is a pentacyclic triterpenoid compound; and caffeic acid is classified as a hydroxycinnamic acid. Kaempferol, rutin, luteolin, and oleanolic acid are the main predicted components of SOL that exert an anti-endometritis effect, and the target components can be extracted with the DES-UAE approach. The total ion current chromatograms (TIC) of SOL extracts are shown in Supplementary Figure S2.

The contents of rutin, luteolin, oleanolic acid, and kaempferol of SOL extracts after the treatment with D-101 macroporous were determined by HPLC (Supplementary Figure S3). The content of rutin, luteolin, oleanolic acid, and kaempferol were 10.0 ± 0.16 mg/g, 7.98 ± 0.03 mg/g, 485.70 ± 0.12 μg/g, and 41.57 ± 0.07 μg/g, respectively.

In H&E staining, the uterus in 0.5% CMC-Na group had a similar structure to the uterus in the control group (Figures 6A,B). The S. aureus group showed severe pathological changes, including endometrial congestion and edema, inflammatory cell infiltration, and decreased number of glands (Figure 6C). The pathological changes caused by S. aureus could be attenuated after treatment with SOL extracts, rutin, luteolin, oleanolic acid, kaempferol, and monomer mixture to varying degrees (Figures 6D–J). The degree of inflammatory damage was also slightly reduced in the DEX group (Figure 6K). In addition, tissue in the S. aureus group had the highest histological score compared to the control group (p < 0.01), and other groups' score was lower than the S. aureus group (Figure 6L).

In the reproductive tract, the endometrium's initial defense against invasive bacteria depends on the innate immune system (28). Once the innate immune system is activated, endometrial cells secrete large amounts of cytokines and chemokines that can recruit neutrophils and macrophages to clear pathogens (29). As shown in Figure 7, the SOL-treated groups, the monomer groups, the monomer mixture group, and the DEX group could reduce colony counts in uteri vs. the model group.

To further elucidate the molecular mechanism of SOL against endometritis, we evaluated the regulation of TLRs/NF-κB signaling pathway by SOL. Compared with the control group, the mRNA and protein levels of TLR2, TLR4, IκBα, NF-κB, TNF-α, IL-6, and IL-1β of the model group were significantly upregulated (Figures 8, 9). After drug treatment, TLR4 mRNA expression, IL-6, and IL-1β protein expression did not show significant differences in the L-SOL group compared with the model group, and other treatment groups downregulated the TLR4 mRNA expression and IL-6 and IL-1β protein expression compared with the model group. In addition, all drug treatment groups could downregulate the mRNA expression of TLR2, IκBα, NF-κB, TNF-α, IL-6, IL-1β, and protein expression of TLR2, TLR4, IκBα, NF-κB, TNF-α as compared to the model group (Figure 9). These results demonstrated that SOL and predicted active compounds in SOL could alleviate the inflammatory response generated in S. aureus-induced endometritis via TLRs/ NF-κB signaling pathway.