Effects of Shaofu Zhuyu decoction on intestinal flora and fibrosis in a mouse model of endometriosis

Bing-Bing Li: Writing – review & editing, Methodology, Funding acquisition. Qing-Qing Xun: Methodology. Chao Wei: Methodology, Investigation, Formal analysis. Bin Yu: Visualization, Software, Methodology. Xue Pan: Writing – original draft, Visualization. Qian Shen: Writing – review & editing, Writing – original draft.

All animal experiments were approved by the Animal Experiment Ethics Subcommittee of Jining Medical University (No. JNMC-2023-DW-150).

This study was supported by grants from the 10.13039/501100001809 National Natural Science Foundation of China (No. 82104920 ), the 10.13039/501100007129 Natural Science Foundation of Shandong Province (No. ZR2021QH129 ), the Shandong Provincial Key Laboratory of Traditional Chinese Medicine (No. [2022]4 ), and the Shandong Medical and Health Science and Technology Development Plan Project (No. 202002040939 ).

The total ion flow diagram of SFZYD was obtained via UPLC-MS/MS analysis, and the data were processed using UNIFI software (Waters, Shanghai). A total of 157 SFZYD components were identified (Supplementary File), some of which included galloylpaeoniflorin, dehydrofumarin, quercetin, and kaempferol ( Fig. 2 A-B). Fig. 2 The total ion flow diagram for SFZYD. (A) Positive ion mode; (B) Negative ion mode. SFZYD: Shaofu Zhuyu decoction; TIC, total ion chromatogram. Fig. 2 The total ion flow diagram for SFZYD. (A) Positive ion mode; (B) Negative ion mode. SFZYD: Shaofu Zhuyu decoction; TIC, total ion chromatogram. The body weights of the mice in the control, model, and SFZYD groups were measured on days 0, 7, 14, and 21 of the drug administration timeline; the results showed that the body weights of all three groups of mice decreased gradually over time ( Fig. 3 A). Gross observation revealed that the ectopic lesions were predominantly localized within the left abdominal wall of the mice. The cysts were either singular in number or grouped into multiple bead-like structures, with an irregular shape and a transparent or red–brown color. The surface and surrounding neovascularization of the cysts were visible, and adhesion with the surrounding tissues was occasionally evident. The release of cystic fluid was observed in cases in which rupture occurred during dissection, and the lesions exhibited evidence of angiogenesis on and around the surface. Following dissection, the ectopic cysts were revealed to be irregular in shape, polycystic in form, and transparent in appearance, with the longest diameter being approximately 15 mm ( Fig. 3 B). The weight of the ectopic tissue in the SFZYD group was significantly lower than that in the model group ( P  < 0.05) ( Fig. 3 C). The HE staining revealed that the uterine structure of the control group was clear and composed of the endometrial layer, myometrium, and outer membrane. The ectopic lesions of the mice in the model and SFZYD groups differed from the appearance of the normal uterus; for example, the tissue structure of the ectopic lesions was not transparent and could be distinguished based on the morphology of the histiocytes ( Fig. 3 D). Fig. 3 Effect of SFZYD on body weight and lesion morphology in mice with endometriosis. (A) Body weight curve; (B) Representative images of ectopic lesions; (C) Lesion weights in the model and SFZYD groups; (D) Representative images of HE staining in each group. The scale bars represent 100 μm. Data are presented as the mean ± SD (n = 6). ∗ P  < 0.05 compared to the model group. SFZYD: Shaofu Zhuyu decoction; HE: hematoxylin & eosin. Fig. 3 Effect of SFZYD on body weight and lesion morphology in mice with endometriosis. (A) Body weight curve; (B) Representative images of ectopic lesions; (C) Lesion weights in the model and SFZYD groups; (D) Representative images of HE staining in each group. The scale bars represent 100 μm. Data are presented as the mean ± SD (n = 6). ∗ P  < 0.05 compared to the model group. SFZYD: Shaofu Zhuyu decoction; HE: hematoxylin & eosin. To obtain more robust evidence, 16S rRNA sequencing was conducted to assess the biological characteristics of the intestinal microbiota from the stool samples of mice in the control, model, and SFZYD groups (n = 6/group). A total of 10,629 operational taxonomic unit (out) sequences were collected; of them, only 2648 sequences were annotated as intestinal microbiota, including 1305 OTUs in the control group, 1822 OTUs in the model group, and 1268 OTUs in the SFZYD group ( Fig. 4 A). As shown in Fig. 4 B, the curve gradually flattened out, indicating that the volume of sequencing data was sufficient, as verified by the rank abundance curves ( Fig. 4 B-C). Fig. 4 SFZYD affects the abundance of intestinal microbes in mice with endometriosis. (A) Venn diagram of the number of OTUs in each group; (B) Rarefaction curve in each group; (C) Rank abundance curve in each group; (D) Shannon indices showing the α-diversity in each group; (E) PCoA plots on the OTU level in each group. Data are presented as the mean ± SD (n = 6/group). ∗∗∗ P  < 0.001 and ∗∗ P  < 0.01 compared to the model group. SFZYD: Shaofu Zhuyu decoction; Sobs, observed species; OTU: operational taxonomic unit; PCoA: principal coordinate analysis. Fig. 4 SFZYD affects the abundance of intestinal microbes in mice with endometriosis. (A) Venn diagram of the number of OTUs in each group; (B) Rarefaction curve in each group; (C) Rank abundance curve in each group; (D) Shannon indices showing the α-diversity in each group; (E) PCoA plots on the OTU level in each group. Data are presented as the mean ± SD (n = 6/group). ∗∗∗ P  < 0.001 and ∗∗ P  < 0.01 compared to the model group. SFZYD: Shaofu Zhuyu decoction; Sobs, observed species; OTU: operational taxonomic unit; PCoA: principal coordinate analysis. The Shannon and Simpson indices represent the diversity and richness of the intestinal microbiota, respectively. The statistical analysis of the alpha diversity revealed that compared to that of the control group, the model group exhibited a decreasing trend in alpha diversity, whereas compared to that of the model group, the SFZYD group exhibited an increasing trend, indicating that SFZYD treatment increased the abundance of the microbiota in mice with endometriosis ( Fig. 4 D). The beta diversity of the intestinal microbiota was evaluated based on the unweighted UniFrac-based principal coordinate analysis clustering results, which revealed separation between each of the groups and indicated that there were differences in the clustering results; these findings suggested a disorder of the intestinal microbiota in mice with endometriosis and that these changes could be affected by SFZYD treatment ( Fig. 4 E). Thus, modulation of the intestinal flora is one of the mechanisms through which SFZYD alleviates endometriosis. Differences in the taxonomic composition of the intestinal microbiota between the three groups were analyzed at the phylum, family, and genus levels. Bacteroidota, Firmicutes, and Verrucomicrobiota constituted a major proportion of all phyla in the three groups. In the control and model groups, the abundances of Bacteroidota, Firmicutes, and Verrucomicrobia were 34.67 % and 56.20 %, 56.29 % and 41.85 %, and 7.44 % and 0.10 %, respectively. The Firmicutes/Bacteroidetes ratio of the control group was 1.62, whereas that of the model group was 0.74. The abundance of Bacteroidota in the SFZYD group was 82.70 % higher than that in the model group, whereas the abundance of Firmicutes in the SFZYD group was 16.29 %, which was lower than that in the model group. The abundance of Verrucomicrobiota in the SFZYD group was 0.03 %, which was also lower than that in the model group. This resulted in a lower Firmicutes/Bacteroidetes ratio in the SFZYD group (0.20) than in the model group (0.74) ( Fig. 5 A). At the family level, the top six microorganisms were Muribaculaceae, Lactobacillaceae, Lachnospiraceae, Bacteroidaceae, Akkermansiaceae, and Rikenellaceae ( Fig. 5 B). At the genus level, the top six microorganisms were norank_f__Muribaculaceae, Lactobacillus, Bacteroides, Akkermansia, Lachnospiraceae_NK4A136_group, and Alistipes ( Fig. 5 C). Fig. 5 SFZYD alters the composition of intestinal microbes in mice with endometriosis. (A) Bacterial abundance at the phylum level; (B) Bacterial abundance at the family level; (C) Bacterial abundance at the genus level; (D) Test for significance differences in bacterial composition between groups at the phylum level; (E) Test for significant differences in bacterial composition between groups at the family level; (F) Test for significant differences in bacterial composition at the genus level. Data are presented as the mean ± SD (n = 6/group). ∗∗ P  < 0.05, ∗∗∗ P  < 0.01. SFZYD: Shaofu Zhuyu decoction. Fig. 5 SFZYD alters the composition of intestinal microbes in mice with endometriosis. (A) Bacterial abundance at the phylum level; (B) Bacterial abundance at the family level; (C) Bacterial abundance at the genus level; (D) Test for significance differences in bacterial composition between groups at the phylum level; (E) Test for significant differences in bacterial composition between groups at the family level; (F) Test for significant differences in bacterial composition at the genus level. Data are presented as the mean ± SD (n = 6/group). ∗∗ P  < 0.05, ∗∗∗ P  < 0.01. SFZYD: Shaofu Zhuyu decoction. Intergroup differences in intestinal microorganisms were further analyzed at the phylum, family, and genus levels. At the phylum level, there were significant differences in Bacteroidota ( P < 0.01) between the three groups, and significant intergroup differences were observed at the family level for Muribaculaceae ( P < 0.01) and Rikenellaceae ( P < 0.01). Significant intergroup differences were observed at the genus level for norank_f_Muribaculaceae ( P < 0.01) and unclassified_f__Ruminococcaceae ( P  < 0.01) ( Fig. 5 D–F). Subsequent pairwise comparisons between the control and model groups and between the model and SFZYD groups revealed significant differences in Rikenellaceae ( P < 0.01), Oscillospiraceae ( P < 0.05), Tannerellaceae ( P < 0.05) at the family level and in Alistipes ( P < 0.05), norank_f__Oscillospiraceae ( P < 0.05), and Rikenella ( P < 0.05) at the genus level between the control and model groups, indicating that these microorganisms were closely related to the occurrence of endometriosis ( Fig. 6 A–B). There were also significant differences in Rikenellaceae ( P < 0.01), Ruminococcaceae ( P < 0.01), and Muribaculaceae ( P < 0.05) at the family level, and in norank_f__Ruminococcaceae ( P < 0.01), norank_f__Muribaculaceae ( P < 0.05), and Desulfovibrio ( P  < 0.05) at the genus level between the control and model groups, suggesting that these microorganisms may play an important role in the mechanism through which SFZYT alleviates endometriosis ( Fig. 6 C–D). Furthermore, the linear discriminant analysis scores from the linear discriminant analysis effect size calculations revealed high expression levels of the microorganisms Desulfovibrio, Paludicola, Lachnospiraceae_NK4A136_group, Desulfovibrio, and Rikenellaceae in the intestines of mice with endometriosis, and treatment with SFZYD not only improved the symptoms of endometriosis but also enriched the levels of Muribaculaceae, norank_f__Muribaculaceae, Bacteroidales, and Bacteroidota ( Fig. 6 E). Fig. 6 Tests for significant differences between two groups and functional predictions. (A) Tests for significant differences between the Control and SFZYD groups at the family level; (B) Tests for significant differences between the Control and SFZYD groups at the genus level; (C) Tests for significant differences between the Model and SFZYD groups at the family level; (D) Tests for significant differences between the Model and SFZYD groups at the genus level; (E) LeFSe multi-stage discriminant analysis of species differences between the Model and SFZYD groups for the prediction of metabolic pathways using PICRUSt software; (F) KEGG functional abundance statistics; (G) GO functional abundance statistics. Data are presented as the mean ± SD (n = 6/group). ∗ P  < 0.05, ∗∗ P  < 0.01. SFZYD: Shaofu Zhuyu decoction; LDA: linear discriminant analysis: LeFSe, linear discriminant analysis effect size; PICRUSt: Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology. Fig. 6 Tests for significant differences between two groups and functional predictions. (A) Tests for significant differences between the Control and SFZYD groups at the family level; (B) Tests for significant differences between the Control and SFZYD groups at the genus level; (C) Tests for significant differences between the Model and SFZYD groups at the family level; (D) Tests for significant differences between the Model and SFZYD groups at the genus level; (E) LeFSe multi-stage discriminant analysis of species differences between the Model and SFZYD groups for the prediction of metabolic pathways using PICRUSt software; (F) KEGG functional abundance statistics; (G) GO functional abundance statistics. Data are presented as the mean ± SD (n = 6/group). ∗ P  < 0.05, ∗∗ P  < 0.01. SFZYD: Shaofu Zhuyu decoction; LDA: linear discriminant analysis: LeFSe, linear discriminant analysis effect size; PICRUSt: Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional abundance analyses were performed for the model and SFZYD groups using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 software. The analysis of the top 15 KEGG modules in terms of total abundance demonstrated that the differences in intestinal microbiota before and after SFZYD treatment were mainly related to carbohydrate, amino acid, and lipid metabolism pathways, among others ( Fig. 6 F). The GO analysis showed that the biological functions of the intestinal microbiota in the model and SFZYD groups were similar and were predominantly related to amino acid transport and metabolism, translation, ribosomal structure, biogenesis, cell cycle control, cell division, and chromosome partitioning ( Fig. 6 G). To further explore the mechanisms through which SFZYD alleviates endometriosis, its effects on energy metabolism were evaluated. Previous clinical studies have shown that patients with endometriosis commonly exhibit a lower body mass index and dysfunctional lipid metabolism, and the progression of the disease is closely related to the development of fibrosis. Masson's trichrome staining revealed that SFZYD treatment significantly reduced fibrosis in the ectopic lesions of mice ( P < 0.001) ( Fig. 7 A). The results of the IHC labeling in ectopic lesions showed that the protein expression levels of TGF-β1 ( P < 0.001) ( Fig. 7 B), collagen type I alpha 1 chain (COL1A1) ( P < 0.05) ( Fig. 7 C), and α-SMA ( P < 0.01) ( Fig. 7 D), all of which are related to fibrosis, were significantly decreased following SFZYD treatment. In addition, the ELISA results revealed that, compared to those in the model group, the serum levels of adiponectin were significantly higher ( P  0.05) ( Fig. 7 F) in the SFZYD group; both of those proteins are related to lipid metabolism. SFZYD treatment also significantly reduced the expression levels of TGF-β1 ( P < 0.001) ( Fig. 7 G) and CTGF ( P  < 0.05) ( Fig. 7 H), consistent with the IHC labeling results. Fig. 7 SFZYD alleviates fibrosis in mice with endometriosis. (A) Representative images of Masson's trichrome staining and quantification of the proportion of collagen fiber area in each group; (B – D) representative images of IHC labelling and quantification of the expression of TGF-β1, COL1A1, and α-SMA, respectively, in each group; (E – H) Quantification of the protein expression levels of adiponectin, leptin, TGF-β1, and CTGF, respectively, in each group via ELISA. The scale bars represent 100 μm. Data are presented as the mean ± SD (n = 6/group). ∗ P  < 0.05, ∗∗ P  < 0.01, ∗∗∗ P  < 0.001. SFZYD: Shaofu Zhuyu decoction; IHC: immunohistochemistry; TGF-β1: transforming growth factor beta 1; COL1A1: collagen type I alpha 1; α-SMA: alpha-smooth muscle actin; ELISA: enzyme-linked immunosorbent assay; CTGF: connective tissue growth factor. Fig. 7 SFZYD alleviates fibrosis in mice with endometriosis. (A) Representative images of Masson's trichrome staining and quantification of the proportion of collagen fiber area in each group; (B – D) representative images of IHC labelling and quantification of the expression of TGF-β1, COL1A1, and α-SMA, respectively, in each group; (E – H) Quantification of the protein expression levels of adiponectin, leptin, TGF-β1, and CTGF, respectively, in each group via ELISA. The scale bars represent 100 μm. Data are presented as the mean ± SD (n = 6/group). ∗ P  < 0.05, ∗∗ P  < 0.01, ∗∗∗ P  < 0.001. SFZYD: Shaofu Zhuyu decoction; IHC: immunohistochemistry; TGF-β1: transforming growth factor beta 1; COL1A1: collagen type I alpha 1; α-SMA: alpha-smooth muscle actin; ELISA: enzyme-linked immunosorbent assay; CTGF: connective tissue growth factor.

Twenty-four 8-week-old female BALB/c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Animal Qualification Certificate: SCXK(Lu)20220006). All mice were maintained under standard conditions (12 h/12 h light/dark cycle at 22–24 °C and 50 % relative humidity) and provided unrestricted access to food and water. After a one-week acclimatization period, the mice were randomly assigned to one of the following four groups at the beginning of the experiment: control group (n = 6), donor group (n = 6), model group (n = 6), and SFZYD treatment group (n = 6). Drug administration via intragastric gavage was conducted while the mice were conscious; however, discomfort was minimized by using straight gavage needles that were appropriate for the 15–17-g body weight of the animals (22-gauge, 1-inch length, and 1.25-mm ball diameter). All animals were euthanized by barbiturate overdose (intraperitoneal injection, 30 mg/kg pentobarbital sodium) for tissue collection. All animal experiments were approved by the Animal Experiment Ethics Subcommittee of Jining Medical University (No. JNMC-2023-DW-150). Mouse models of endometriosis have been previously established via intraperitoneal injection of uterine fragments [ 9 , 10 ]. In the present study, estradiol valerate (0.5 mg/kg) was administered by gavage to donor and recipient mice for five consecutive days to, and the donor mice were subsequently anesthetized. After opening the abdominal cavity, the uterus was quickly removed and transferred into pre-cooled normal saline to eliminate the blood and mucus. The uterus was divided into two parts, and each half was placed into a Petri dish containing 0.5 mL of normal saline. The uterus was cut lengthwise with ophthalmic scissors to expose the endometrial surface, then cut into small 1-mm 2 pieces. Each bisected uterus of a donor mouse was injected into the disinfected abdomens of two recipient mice (one mouse from the model group and one mouse from the SFZYD group). After establishment of the model, estradiol valerate (0.5 mg/kg) was continuously injected into the stomach of the mice for 3 days to promote the growth of ectopic lesions. The stability of the model was confirmed three weeks after surgery. SFZYD was uniformly prepared in pellet form by Jiangyin Tianjiang Pharmaceutical Co., Ltd. SFZYD includes 10 kinds of Chinese herbs ( Table 1 ). The pellet was dissolved in saline and stored at 4 °C until further use. The required dose of SFZYD was calculated according to the equivalent dose formula for humans and animals described in the third edition of Experimental Methodology in Pharmacology [ 11 ]. Mice in the treatment group were administered SFZYD at a dose of 8.645 g/kg via oral gavage once daily for 21 days, whereas the mice in the control and model groups received an equivalent volume of normal saline by oral gavage. Table 1 The traditional Chinese medicine composition of SFZYD. Table 1 Chinese name Latin name Weight (g) Dang gui Angelicae Sinensis Radix 9 Chi shao Radix Paeoniae Rubra 6 Yan hu suo Corydalis Rhizoma 3 Mo yao Myrrha 6 Chuan xiong Chuanxiong Rhizome 9 Wu ling zhi Trogopterus Dung 6 Pu huang Pollen Typhae 9 Gan jiang Zingiberis Rhizome 3 Xiao hui xiang Foeniculi Fructus 3 Rou gui Cinnanmomi Cortex 3 The traditional Chinese medicine composition of SFZYD. Liquid chromatography (LC)-MS/MS detection was conducted using an ultra-performance liquid chromatography (UPLC) I-Class series Waters SYNAPT G2Si Quantitative Time-of-Flight mass spectrometer and a UPLC I-Class chromatographic system. Samples were precisely weighed, and 20-mg/mL solutions were prepared by adding 80 % methanol to each one. The samples were subsequently vortexed for 5 min and centrifuged at 13,000 rpm for 10 min before LC-MS/MS detection. A BEH C18 chromatographic column was used (100 mm × 2.1 mm, 1.8 μm), and the separation conditions were as follows: column temperature: 30 °C; and flow rate: 0.3 mL/min. The mobile phase contained 0.1 % formic acid (A) and acetonitrile (B), and the gradient elution procedure was as follows: 0–5 min, 80 % A; 5–23 min, 80%–0% A; and 23–25 min, 0–80 % A. The sample volume was 10 μL, and the detection wavelength was 254 nm. The following MS detection parameters were used: ion source: electrospray ionization; sample distribution detected in positive/negative electrospray ionization mode in the mass-to-charge ratio range of 50–1500; scanning mode: mode-scanning excitation; capillary voltage set to positive mode 2.5 kV/negative mode −2.5 kV; cone voltage: 20 V; source temperature: 150 °C; desolventizing temperature: 400 °C; flow rate of cone gas: 50 L/h; and flow rate of desolventizing gas: 500 L/h. The LockSpray was calibrated in real-time using leucine-enkephalin. MassLynx version 4.2 software (Waters, Shanghai) was used to extract and normalize the data detected in the positive and negative modes of the LC-MS/MS analysis. The tissue samples were fixed and preserved in 4 % formaldehyde solution, after which they were dehydrated and paraffin-embedded. The tissue was subsequently cut into 5-μm-thick transverse sections and subjected to HE and Masson's trichrome staining using standard techniques (Servicebio, G1003; Masson's staining solution, BASO, Zhuhai, BA4079). The staining was observed under a microscope (BX51, Olympus, Japan) for image capture or scanning. The HE staining was conducted to observe tissue structures and pathological changes before and after treatment. Masson's trichrome staining was conducted to visualize collagen fibers and cell nuclei in tissues, which appeared as blue and darker blue staining, respectively. ImageJ software was used to measure the area of collagen staining (blue) and the total tissue area, which were used to calculate the collagen volume fraction. The specimen slides were scored by a pathologist who was blinded to the study groups. After euthanizing the mice with pentobarbital sodium, the colon contents were collected in sterile tubes, cryoprotected, and stored in a −80 °C freezer for follow-up testing. The fecal samples from the mice were analyzed by Majorbio Biotechnology Co., Ltd. (Shanghai, China). The extracted genomic DNA was subjected to 1 % agarose gel electrophoresis, and the barcoded universal bacterial primers 338F and 806R (5′-barcode-ACTCCTACGGGAGGCAGCA-3’/5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 variable region of the 16S rRNA gene in triplicate. The resulting polymerase chain reaction products were further purified and quantified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA) and QuantiFluor-ST (Promega, Madison, USA). The purified amplicons were pooled in equimolar amounts and sequenced using a paired-end configuration on an Illumina MiSeq system (Illumina, San Diego, USA). Taxonomic analysis of OTU representative sequences at a 97 % similarity level was performed using the RDP classifier Bayesian algorithm, and the community species composition of each sample was statistically analyzed at each taxonomic level: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and Species. The community diversity was reflected by using the Rank-abundance curve and Alpha diversity analysis. PCoA analysis was used to study the similarity or difference of sample community composition. Based on the community bar chart, analyze which dominant species each sample contains at a certain taxonomic level and the relative abundance (proportion) of each dominant species in the samples. The LEfSe analysis was used to identify the differential genera of microbiota. PICRUSt2 was used for functional prediction of the sequencing results. All further data analyses were evaluated using online tools from Majorbio ( www.majorbio.com ). After deparaffinization, drops of endogenous peroxidase blockers were added to the tissue for a 10-min incubation at room temperature, after which the tissue was washed with double-distilled water. The slices were subsequently placed in ethylenediaminetetraacetic acid antigen retrieval solution, heated in a microwave at high temperature for 8 min, cooled to room temperature for 8 min, and re-heated in a microwave a second time at the same conditions. A 5 % bovine serum albumin blocking solution was added to the tissue for a 30-min incubation at 37 °C, after which the samples were incubated at 4 °C overnight in appropriately diluted primary antibody solutions. The primary antibodies included an anti-alpha smooth muscle actin (α-SMA) antibody (AF1032 Affinity), an anti-collagen I antibody (AF7001 Affinity), and an anti-transforming growth factor beta 1 (TGF-β1) antibody (AF1027 Affinity). The next day, the tissue was incubated at 37 °C for 30 min in a biotin-conjugated sheep anti-rabbit immunoglobulin G secondary antibody solution (SA1022 BOSTER), followed by 3,3′-diaminobenzidine staining. Finally, the samples were photographed under a microscope (BX51, Olympus, Japan) and scanned (APERIO VERSA 8, Leica). ImageJ software was used to convert the image format and grayscale units into optical density units to quantify the area, density, and integrated optical density according to the manufacturer's protocol. Blood collected from the venous plexus of the medial canthus of the mice was centrifuged at 3000 rpm for 15 min, and the supernatant was collected for subsequent quantification of protein levels according to the ELISA kits’ instructions. The following commercial kits were used in the present study: mouse adiponectin ELISA kit (MM-0547M2, MEIMIAN), mouse leptin ELISA kit (MM-0622M2, MEIMIAN), mouse TGF-β1 ELISA kit (MM-0135M2, MEIMIAN), and mouse connective tissue growth factor (CTGF) ELISA kit (MM-0077M2, MEIMIAN). The absorbance was measured at an excitation wavelength of 450 nm using a microplate reader (Synergy, BioTek, USA). All data are presented as the mean ± standard deviation, with t -test analysis for comparisons between two groups and one-way ANOVA for multi-group comparisons. Statistical analyses were performed using GraphPad Prism 9.5.0 software (GraphPad Software, San Diego, USA). P values less than 0.05 were considered statistically significant.

Endometriosis is a disease in which endometriotic tissue forms at sites other than the uterus, and compounds used in traditional Chinese medicine such as SFZYD have played an important role in its treatment. SFZYD comprises 157 chemical components, including, but not limited to, galloylpaeoniflorin, dehydrofumarine, quercetin, and kaempferol. In the present study, the HE staining and lesionweight comparisons revealed that SFZYD treatment reduced the development of endometriotic lesions while delaying disease progression. Although the precise mechanisms of action are unknown, previous studies have explored the effects of SFZYD on a molecular level in the treatment of endometriosis, most of which have focused on inflammation, cell proliferation, angiogenesis, fibrosis, and invasion ( Table 2 ), whereas the present study focused on changes in the gut microbiota and differences in energy metabolism. Table 2 The mechanism of SFZYD in improving endometriosis. Table 2 Reference Animal type Molding method Detection molecule Phenotype [ 12 ] Sprague-Dawley rats Autotransplantation HIF-1α, microvessel density Cell proliferation, cell apoptosis [ 13 ] BALB/c mice Heterotopic transplantation α-SMA, collange-Ⅰ, PTEN、mTOR, Akt, p-Akt, p-mTOR Fibrosis [ 14 ] SD rats Autotransplantation IL-6, IL-10, PGE2, TNF-α, MSK1, MSK2, DUSP1 Inflammation [ 15 ] SD rats Heterotopic transplantation TNF-α, IL-6, NK1, GPER2, MAPK, STAT1 Inflammation [ 16 ] SD rats Autotransplantation NOD1, RIP2, NF-κB, p65, IL-8, IL-18, TNF-α, COX-2 Inflammation [ 17 ] Homo sapiens – VEGF, MMP-9, IGF-1, NGF, SP, CGRP, BDNF, sFlt-1, PGE2, COX-2, TNF-α, IL-6 Inflammation, angiogenesis [ 18 ] Homo sapiens – RBP4, HMGB1, MCP-1, RANTES, PGE2、PGF2α、OT Endometrial receptivity, inflammation [ 19 ] Wistar rats Autotransplantation 5-HT, NO, PGF2α, Wnt, GSK-3β, TCF4, Frizzled, p-β-catenin, β-catenin Invasion [ 20 ] SD rats Autotransplantation TNF-α, IL-6, IL-8, ERK, VEGF, MMP-9, NF-κB, MEK, MAPK Inflammation [ 21 ] Homo sapiens primary cells – MMP-2, MMP-9 Invasion [ 22 ] Homo sapiens primary cells – Ang-1, Ang-2, Tie-2 Angiogenesis [ 23 ] SD rats Autotransplantation HIF-1α – [ 24 ] – – IL6, PTGS2, VEGFA, FOS, IL-1B, ESR1 Inflammation, cell proliferation The mechanism of SFZYD in improving endometriosis. The microbes in the gut comprise one of the most abundant microbiomes in the human body and are closely related to the absorption of nutrients and the establishment and maintenance of the immune system [ 25 ]. In addition to playing a role in the induction of various diseases of the gastrointestinal system, disturbances in the intestinal microflora can also affect the development or progression of many inflammatory and proliferative diseases via the modulation of immunity [ 26 ]. In recent years, scholars have proposed the existence of an "estrogen–gut microbiome axis," which closely links the composition or function of intestinal flora with various gynecological diseases [ 27 ]. For example, an experimental animal study demonstrated significant changes in the intestinal microbiomes of mice before and after intraperitoneal injection of endometrial tissue [ 28 ]. In another study, intestinal flora transplantation in mice with endometriosis promoted the development and progression of the disease; collectively, these findings suggested that the gut flora is a crucial regulator of endometriosis [ 29 ]. Huang et al. and Cao et al. reported that the alpha and beta diversity of the intestinal flora in groups with endometriosis were altered compared with those of the respective control groups in both human studies and animal experiments, which was basically consistent with the results of this study [ 30 , 31 ]. In the present study, compared with that of the control group, the model group exhibited a significantly increased abundance of Rikenellaceae ( P < 0.01 ) at the family level and of Alistipes ( P < 0.05 ) and Rikenella ( P < 0.05 ) at the genus level. It is interesting to note that Alistipes and Rikenella are both members of the Rikenellaceae family and belong to the Bacteroidota phylum. Previous studies have shown that Bacteroidota could play a role in adiposity modulation through the production of short-chain fatty acids (SCFAs), acetate, and propionate [ 32 ]. Similarly, Rikenellaceae may contribute to a reduction in visceral fat mass [ 33 ]. At the family level, SFZYD treatment significantly reduced the abundance of Lachnospiraceae ( P < 0.05 ), Rikenellaceae ( P < 0.01 ), and Ruminococcaceae ( P < 0.01 ), and at the phylum level, significant changes were observed for Lachnoclostridium ( P < 0.05 ), Lachnospiraceae_NK4A136_group ( P < 0.05 ), LachnospiraceaE_NK4A136_group ( P < 0.05 ), Lachnospiraceae_UCG-006 ( P < 0.05 ), norank_f__Lachnospiraceae ( P < 0.05 ), unclassified_f__Ruminococcaceae ( P < 0.05 ), and norank_f__Ruminococcaceae ( P < 0.01 ), which belong to Lachnospiraceae and Ruminococcaceae, respectively. Similar to Rikenellaceae , Lachnospiraceae has the ability to hydrolyze starches and other sugars to produce butyrate and other SCFAs [ 34 ]. In addition, Shan et al. showed that Blautia and Dorea , which are members of Lachnospiraceae , were also highly expressed in patients with endometriosis and were associated with increased estradiol levels in the blood [ 35 ]. Blautia is a major producer of SCFAs, especially acetic acid, and reductions in Blautia luti and Blautia wexlerae are associated with insulin resistance in obese individuals. In addition, Blautia isolates prevent pathogen colonization by producing bacteriocins and exhibit anti-inflammatory properties and maintenance of glucose homeostasis by up-regulating the production of regulatory T cells and SCFAs. Dorea also has immunomodulatory effects, which may regulate the intestinal immune response and maintain the integrity and stability of the intestinal mucosal barrier by inducing Treg and inhibiting the differentiation and function of Th17 cells [ 36 , 37 ]. Ruminococcus has been identified as a potential intestinal biomarker for the diagnosis of endometriosis [ 38 ]. It is worth noting that Ruminococcaceae is one of the major bacterial producers of SCFAs. Lu et al. showed that SCFAs produced by Ruminococcaceae can reduce the expression of DNA methyl transferase 1, 3a, and 3b, as well as that of methyl-CpG binding domain protein 2; suppression of the binding of these enzymes can help correct the abnormal expression of adiponectin [ 39 ]. All of the top 15 pathways identified in the KEGG and GO analyses were found to be related to metabolic processes, indicating that endometriosis is closely associated with changes in metabolism. There is increasing evidence suggesting that endometriosis is inversely associated with body mass index; however, the underlying mechanism remains unclear [ 40 ]. Dutta et al. and Zolbin et al. reported changes in fat metabolism in mice with endometriosis, and the BMI in these animals was affected by the presence of adipocytes as well as by fat metabolism [ 41 , 42 ]. Endometriosis is an estrogen-dependent disease, and adipocytes can affect disease progression by regulating estrogen secretion [ 43 ]. Adiponectin and leptin are both endogenously active adipocyte-secreted peptides that regulate energy in the body, with the levels of adiponectin and leptin being inversely proportional and directly proportional to the size of fat stores, respectively. Adiponectin is mainly involved in the regulation of energy homeostasis, especially lipid and glucose metabolism, as well as insulin sensitization, leading to improved insulin resistance [ 44 ]. Adiponectin contains a highly conserved complement factor C1q-like globular domain that binds to and activates its respective receptors, AdipoR1 and AdipoR2 [ 45 ], the former of which is predominantly expressed at high levels in skeletal muscle and the latter of which is highly expressed mainly in the liver. After binding to AdipoR1 and AdipiR2, adiponectin further exerts its effects mainly through the activation of adenosine monophosphate-activated protein kinase (AMPK) [ 46 ]. Processes downstream of AMPK mainly include those related to the inhibition of protein, lipid, and glycogen synthesis and fibrosis, the promotion of autophagy, and mitochondrial biogenesis [ 47 ]. Among the related factors, TGF-β is a key molecule involved in AMPK-mediated fibrosis, inducing the expression of CTGF in fibroblasts, thereby promoting myofibroblast differentiation and collagen synthesis [ 48 ]. The results of two meta-analyses of previous studies that investigated the roles of adiponectin and leptin showed that adiponectin was expressed at low levels, whereas leptin was highly expressed in patients with endometriosis [ 49 , 50 ]. Takemura and Choi et al. showed that AdipoR1 and AdipoR2 are expressed in the endometrium, and Takemura et al. demonstrated that adiponectin phosphorylates AMPK via AdipoR to maintain energy homeostasis and that it exerts anti-inflammatory effects during endometriosis [ 51 , 52 ]. A related feature of endometriosis is the presence of fibrotic tissue in and around the lesions, which can result in classic endometriosis-associated symptoms such as pain and infertility. The role of TGF-β in endometriosis-related fibrosis has been demonstrated, and CTGF is a key marker of fibrosis in fibrotic diseases, such as endometriosis and intrauterine adhesions [ 53 ]. In the present study, the Masson staining revealed that SFZYD treatment significantly reduced fibrosis in the ectopic tissues ( P < 0.001 ), and the IHC labeling and ELISA results showed a significant overexpression of adiponectin ( P < 0.05 ) in mice with endometriosis, along with higher levels of leptin ( P < 0.05 ), TGF-β1 ( P < 0.001 ), CTGF ( P < 0.05 ), COL1A1 ( P < 0.05 ), and α-SMA ( P < 0.01 ); SFZYD treatment attenuated the changes in expression of all of these molecules. There are still some shortcomings in this study: (1) The positive drug group was not set up during the experiment, and the efficacy of SFZYD in the treatment of endometriosis could not be objectively compared; (2) Although some key microbiota were identified in SFZYD treatment of endometriosis, whether SFZYD treatment of endometriosis is microbiota-dependent needs to be further verified by antibiotic inhibition experiments; (3) The anti-fibrosis effect of SFZYD on endometriosis requires further rescue experiments to verify; (4) in the process of SFZYD treatment of endometriosis, intestinal flora changes associated with the existence of the mechanism of anti-fibrosis still needs to be further defined. In conclusion, the 16S rRNA analysis in the present study demonstrated that SFZYD treatment attenuated the changes in the intestinal microbiota caused by endometriosis, and the underlying mechanisms were mainly related to the regulation of carbohydrate, amino acid, and lipid metabolism. In addition, SFZYD treatment reduced the volume and fibrosis of ectopic lesions and improved metabolism, making it an effective treatment for endometriosis. Based on the intestinal flora and fiber into a breakthrough point, to explore the underlying mechanisms of SFZYD treatment of endometriosis, which will provide a scientific basis for research of traditional Chinese medicine treatment of endometriosis and new ideas, and provide guidance for SFZYD in clinical individualized application.

Endometriosis predominantly occurs in women of childbearing age, with an incidence of approximately 6%–10 %. As a chronic neuroinflammatory and hormone-dependent gynecological disease, it mainly manifests as dysmenorrhea, chronic pelvic pain, and infertility [ 1 ]. At present, endometriosis is mainly treated via surgical resection of ectopic lesions or through the administration of non-steroidal anti-inflammatory or hormonally active drugs; however, these pharmacological agents often fail to effectively improve the symptoms women with the disease experience. In addition, more than 50 % of patients require further surgical intervention within 5 years, and these individuals are more susceptible to the development of ovarian cancer [ 2 ]. Therefore, there is an urgent need for more effective clinical treatments for patients with endometriosis. Shaofu Zhuyu decoction (SFZYD), which is used in traditional Chinese medicine, could be an effective treatment option. SFZYD was derived from the Correction of the Errors of Medical Works , which was written by Wang Qingren during the Qing Dynasty. SFZYD is composed of 10 herbs and can promote blood circulation, alleviate blood stasis, warm meridians, and relieve pain. These effects have led to SFZYD being widely used for the clinical management of various gynecological diseases, although the mechanisms are unclear. Intestinal microbes form a complex microbial community of bacteria, fungi, and viruses that not only play an important role in intestinal physiology and pathology, but they can also act as a kind of endocrine organ, influencing the function of parenteral organs through various biological pathways. Increasing evidence has shown that homeostatic imbalances in the intestinal microbiome can lead to the development of various diseases, and there is clear evidence of a link between intestinal microbes and the development of endometriosis [ 3 ], mainly through the regulation of estrogen levels, immunity, and inflammatory pathways [ 4 ]. For example, Chadchan et al. demonstrated that the intestinal microbiome is necessary for the development of endometriosis in mice, affecting its progression by regulating the peritoneal immune cell population, and the quinic acid produced by intestinal microbes can promote the growth of ectopic endometrial tissue [ 5 ]. Those researchers have also shown that gut-derived n-butyrate protects against endometriosis by activating the expression of Rap1 guanosine triphosphatase activating protein and by inhibiting the Rap1 oncogenic pathway [ 6 ]. Escherichia coli has been shown to be abundant in the intestinal tract of patients with endometriosis and can affect the development of the disease by stimulating β-glucuronidase expression and increasing the concentration of circulating estrogen [ 7 ]. Additionally, some probiotics, including Lactobacillus gasseri , have been used to alleviate symptoms in patients with endometriosis [ 8 ]. Given the link between the intestinal microbiome and endometriosis and the ability of SFZYD to alleviate symptoms of gynecological diseases, the aim of this study was to investigate the effects and potential mechanisms of SFZYD in a mouse model of endometriosis, which involved allograft transplantation, using 16S ribosomal ribonucleic acid (rRNA) sequencing and other techniques ( Fig. 1 ). Fig. 1 Graphic summary. The top panels depict the analysis of the key constituents of SFZYD using UPLC-MS/MS. The lower panels depict the generation of the mouse model of endometriosis and quantification of changes in expression in ectopic tissues using 16S rRNA, IHC, and ELISA, which revealed improvements in the intestinal flora and enhancement of anti-fibrotic signaling. SFZYD: Shaofu Zhuyu decoction; UPLC-MS/MS: ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry; rRNA: ribosomal ribonucleic acid; IHC: immunohistochemistry; ELISA: enzyme-linked immunosorbent assay; AdipoR: adiponectin receptor; AMPK: adenosine monophosphate-activated protein kinase; TGF-β: transforming growth factor beta; CTGF: connective tissue growth factor; COL1A1: collagen type I alpha 1; α-SMA: alpha-smooth muscle actin. Fig. 1 Graphic summary. The top panels depict the analysis of the key constituents of SFZYD using UPLC-MS/MS. The lower panels depict the generation of the mouse model of endometriosis and quantification of changes in expression in ectopic tissues using 16S rRNA, IHC, and ELISA, which revealed improvements in the intestinal flora and enhancement of anti-fibrotic signaling. SFZYD: Shaofu Zhuyu decoction; UPLC-MS/MS: ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry; rRNA: ribosomal ribonucleic acid; IHC: immunohistochemistry; ELISA: enzyme-linked immunosorbent assay; AdipoR: adiponectin receptor; AMPK: adenosine monophosphate-activated protein kinase; TGF-β: transforming growth factor beta; CTGF: connective tissue growth factor; COL1A1: collagen type I alpha 1; α-SMA: alpha-smooth muscle actin.

BBL designed and coordinated the study, performed the experiments, and acquired and analyzed the data; QQX and CW performed the experiments and acquired and analyzed the data; BY interpreted the data; and XP and QS wrote the manuscript. All authors have approved the final version of the article. The authors have no competing interests to declare that are relevant to the content of this article. All animal experiments were approved by the Animal Experiment Ethics Subcommittee of Jining Medical University (No. JNMC-2023-DW-150).

Data included in article/supp. material/referenced in article.