Modulating effects of Astragalus polysaccharide on immune disorders via gut microbiota and the TLR4/NF-κB pathway in rats with syndrome of dampness stagnancy due to spleen deficiency 黄芪多糖通过肠道菌群和TLR4/NF-κB途径对脾虚水湿不化大鼠免疫功能紊乱的调节作用 1 corresp first-author Wenxiao ZHAO, zhaowx@sdutcm.edu.cn Wenxiao ZHAO, zhaowx@sdutcm.edu.cn zhaowx@sdutcm.edu.cn https://orcid.org/0000-0001-7165-5870 1 1 2 2 3 1 1 2 2 corresp Haijun ZHAO, haijunzhao@sdutcm.edu.cn Haijun ZHAO, haijunzhao@sdutcm.edu.cn haijunzhao@sdutcm.edu.cn https://orcid.org/0000-0003-0858-7486 山东中医药大学护理学院， 中国济南市， 250355 山东中医药大学护理学院， 中国济南市， 250355 School of Nursing, Shandong University of Traditional Chinese Medicine, Jinan 250355, China School of Nursing, Shandong University of Traditional Chinese Medicine, Jinan 250355, China 山东中医药大学中医学院， 山东省中医经典名方协同创新中心， 中国济南市， 250355 山东中医药大学中医学院， 山东省中医经典名方协同创新中心， 中国济南市， 250355 College of Traditional Chinese Medicine, Shandong Co-Innovation Center of Classic Traditional Chinese Medicine Formula, Shandong University of Traditional Chinese Medicine, Jinan 250355, China College of Traditional Chinese Medicine, Shandong Co-Innovation Center of Classic Traditional Chinese Medicine Formula, Shandong University of Traditional Chinese Medicine, Jinan 250355, China 山东大学齐鲁医学院药学院， 中国济南市， 250012 山东大学齐鲁医学院药学院， 中国济南市， 250012 School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China The syndrome of dampness stagnancy due to spleen deficiency (DSSD) is relatively common globally. Although the pathogenesis of DSSD remains unclear, evidence has suggested that the gut microbiota might play a significant role. Radix Astragali , used as both medicine and food, exerts the effects of tonifying spleen and qi. Astragalus polysaccharide (APS) comprises a macromolecule substance extracted from the dried root of Radix Astragali , which has many pharmacological functions. However, whether APS mitigates the immune disorders underlying the DSSD syndrome via regulating gut microbiota and the relevant mechanism remains unknown. Here, we used DSSD rats induced by high-fat and low-protein (HFLP) diet plus exhaustive swimming, and found that APS of moderate molecular weight increased the body weight gain and immune organ indexes, decreased the levels of interleukin-1β (IL-1β), IL-6, and endotoxin, and suppressed the Toll-like receptor 4/nuclear factor-‍κB (TLR4/NF-‍κB) pathway. Moreover, a total of 27 critical genera were significantly enriched according to the linear discriminant analysis effect size (LEfSe). APS increased the diversity of the gut microbiota and changed its composition, such as reducing the relative abundance of Pseudoflavonifractor and Paraprevotella , and increasing that of Parasutterella , Parabacteroides , Clostridium XIVb , Oscillibacter , Butyricicoccus , and Dorea . APS also elevated the contents of short-chain fatty acids (SCFAs). Furthermore, the correlation analysis indicated that 12 critical bacteria were related to the body weight gain and immune organ indexes. In general, our study demonstrated that APS ameliorated the immune disorders in DSSD rats via modulating their gut microbiota, especially for some bacteria involving immune and inflammatory response and SCFA production, as well as the TLR4/NF-κB pathway. This study provides an insight into the function of APS as a unique potential prebiotic through exerting systemic activities in treating DSSD. 脾虚水湿不化证亦称为脾虚湿困证，可单独或伴随疾病存在，是一种较为常见的中医证型，尽管其发病机制尚不明确，但有证据表明肠道菌群起着重要作用。黄芪健脾益气，既可药用，又可食用。黄芪多糖（APS）是从黄芪中提取的一种大分子物质，具有多种药理作用。然而，APS能否通过调节肠道菌群改善脾虚水湿不化证机体免疫功能紊乱及相关机制尚未可知。本研究中，我们使用高脂低蛋白饮食结合力竭游泳方法诱导脾虚水湿不化模型大鼠。结果发现，中等分子量的APS增加了模型大鼠的体重和免疫器官指数，降低了IL-1β、IL-6和内毒素水平，并抑制了TLR4/NF-κB通路。此外，LEfSe分析结果显示，共有27个关键菌属被显著富集。APS增加了肠道菌群的多样性，改变了其组成。例如，减少了 Pseudoflavonifractor 和 Paraprevotella 的相对丰度，并增加了 Parasutterella 、 Parabacteroides 、 Clostridium XIVb 、 Oscillibacter 、 Butyricicoccus 和 Dorea 的相对丰度。同时，APS还提高了肠道短链脂肪酸含量。相关分析也表明，12种关键菌属相对丰度与体重增加和免疫器官指数有关。综上所述，APS通过调节脾虚水湿不化大鼠的肠道菌群[特别是涉及免疫和炎症应答、短链脂肪酸（SCFA）产生的菌群]，以及TLR4/NF-κB通路，改善了大鼠免疫功能紊乱，为我们深入了解APS治疗脾虚水湿不化证的机制及其作为益生元的潜力提供了实验依据。 黄芪多糖 肠道菌群 TLR4/NF-κB通路 脾虚水湿不化 免疫功能紊乱 短链脂肪酸 Astragalus polysaccharide Gut microbiota Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) pathway Dampness stagnancy due to spleen deficiency Immune disorder Short-chain fatty acid 1 Introduction 1 According to the theory of traditional Chinese medicine (TCM), the spleen, regarded as the “official of granaries,” dominates the transportation and transformation of refined substances in the diet and is the foundation and source of blood biochemistry ( Yang and Jia, 2013 Yang and Jia, 2013 ). Improper diet and prolonged exhaustion will lead to the syndrome of dampness stagnancy due to spleen deficiency (DSSD), which is characterized by a series of symptoms, such as dizziness, fatigue, indigestion, weight loss, loss of appetite, thick and slimy tongue, and edema ( Zhao et al., 2017 Zhao et al., 2017 ; Liu et al., 2022 Liu et al., 2022 ). DSSD is a common syndrome globally, which often exists alone or accompanying other diseases, with a certain value for disease prognosis ( Liu et al., 2022 Liu et al., 2022 ). At present, the pathogenesis of DSSD syndrome remains unclear, while Chinese materia medica with the function of fortifying the spleen and eliminating dampness provide crucial drugs for treatment and prevention. Radix Astragali (Huangqi), the dried root of Astragalus membranaceus (Fisch.) Bge. or A. membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao, is traditionally considered as Chinese materia medica for its spleen-fortifying and qi-tonifying effects against fatigue, loss of appetite, anemia, infection, and edema, and has been approved as a food resource. Astragalus polysaccharide (APS) is a substance containing macromolecules extracted from the dried root o f Radix Astragali , which are responsible for its therapeutic efficacy ( Fu et al., 2014 Fu et al., 2014 ). Modern scientific research has demonstrated that APS possess various pharmacological activities, including immunopotentiation, anti-inflammatory, antioxidant, and antitumor ( Zhou et al., 2017 Zhou et al., 2017 ; Du et al., 2022 Du et al., 2022 ). APS is not only widely used in the clinic but also extensively applied as an immunopotentiator in farmed animal industries. Recently, APS has become new raw materials of a dietary supplement for reinforcing body immunity ( Song BC et al., 2022 Song BC et al., 2022 ). In our previous studies, we established that APS is the main component of Radix Astragali for its spleen-fortifying effect ( Zhao et al., 2017 Zhao et al., 2017 ); moreover, APS enhances the immune function of rats with DSSD syndrome induced by the method of high-fat and low-protein (HFLP) diet plus exhaustive swimming ( Zhao et al., 2019 Zhao et al., 2019 ). Numerous studies have suggested that the gut microbiota and their metabolites play an important role in immune disorders and low-grade inflammation triggered by high-fat diet (HFD) intake or exhaustion ( Dahl et al., 2020 Dahl et al., 2020 ; Dupuit et al., 2021 Dupuit et al., 2021 ). Research has also confirmed that APS could alleviate HFD-induced metabolic disorders via the gut microbiota and exert an immunomodulatory effect via the Toll-like receptor 4 (TLR4) signaling pathway ( Zhou et al., 2017 Zhou et al., 2017 ; Hong et al., 2020 Hong et al., 2020 ). However, whether APS mitigates DSSD via regulating the gut microbiota to remedy the immune disorder and the relevant mechanism have not been elucidated. To further clarify whether the gut microbiota and TLR4 pathway are the therapeutic targets of APS, thus mitigating immune disorders in DSSD syndrome, we investigated the effects of APS on gut microbiota community and their metabolites and the TLR4/nuclear factor-‍κB (NF‍-‍κB) pathway, and identified the changes in critical bacterial genera closely associated with APS treatment. 2 Materials and methods 1 2.1 Animals 2 A total of 45 specific pathogen-free Wistar male rats (four weeks old, (120±10) g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Three animals were housed in each individually ventilated cage at 24 ℃ and 55% relative humidity. All rats were maintained under standard lighting conditions with free access to regular feed and water. 2.2 Drugs 2 The APS formula was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China), with more than 90% (mass ratio) polysaccharide content. The relative molecular weight and homogeneity of APS were determined using the size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) method and calculated by Astra 5.3.4 software (Waytt Technology, CA, USA). The homogeneities were evaluated by the polydispersity index. 2.3 Animal model and intervention strategy 2 After 3 d of acclimatization, the rats were randomly divided into five groups: normal group (NG), model group (MG), APS high-dose (APSH) group, APS medium-dose (APSM) group, and APS low-dose (APSL) group, with nine rats per group. Rats in the normal group were fed with AIN-76A purified rodent diet. The other four groups of rats were used to establish the DSSD rat model, which were fed with HFLP diet and given exhaustive swimming once a day for six weeks based on our published method ( Zhao et al., 2017 Zhao et al., 2017 ). Then, the rats in the APSL, APSM, and APSH groups were administered 300, 600, and 1200 mg/kg APS, respectively, via oral gavage at 1 mL/100 g once a day for two weeks according to our previous study ( Zhao et al., 2019 Zhao et al., 2019 ). The rats in the normal and model groups were given an equal volume of normal saline. The changes in the body weight of all rats were observed during the experiment. At the end of the eighth week, the rats were fasted for 12 h and anesthetized with isoflurane inhalation. The blood, colon tissue, feces, and cecal contents were collected for future analyses. 2.4 Body weight gain and immune organ index analysis 2 Body weight gain was defined as the difference between the body weight of rats at the end of the sixth week and the eighth week. The spleen and thymus were cut off and weighed after anesthesia. 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The plasma endotoxin was measured according to the instructions of the limulus amoebocyte lysate (LAL) Chromogenic Endotoxin Quantitation kit (Bioendo, Xiamen, China). 2.6 Western blot 2 Proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer with protease inhibitors from colon tissue, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA). The membranes were incubated with the primary antibodies overnight and the secondary antibodies for 1 h, followed by the detection of chemiluminescence. Antibody against TLR4 was purchased from Abcam (MA, USA), and antibodies against NF‍-‍κB p65, inhibitor of NF‍-‍κB‍-‍α (IκBα), phospho-IκBα (p-IκBα), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (MA, USA). The intensity of the bands was quantified by ImageJ software ( https://imagej.net/downloads https://imagej.net/downloads ). 2.7 Gut microbiota detection and analysis 2 Total genomic DNA was extracted from the feces using a QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s directions. The microbial 16S ribosomal DNA (rDNA) V3‍–‍V4 region was amplified with indexes and adaptors linked to the universal primers 341F and 806R. The high-throughput sequencing protocol was performed on the Illumina MiSeq PE250 platform at the Realbio Genomics Institute (Shanghai, China). The chimeric sequences were filtered, and sequences with more than 97% similarity were regarded as the same operational taxonomic units (OTUs) by USEARCH software ( https://www.drive5.com/usearch https://www.drive5.com/usearch ). Alpha and beta diversity analyses were carried out by the Quantitative Insights into Microbial Ecology (QIIME) software ( http://qiime.org http://qiime.org ). The rank sum test for inter-group significant difference analysis was performed to identify species with differences. The linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed to assess the differential species abundance among groups. The Spearman’s correlation coefficients were analyzed by R software ( https://www.r-project.org https://www.r-project.org ). 2.8 Analysis of short-chain fatty acids (SCFAs) 2 About 50 mg cecal contents were added with 50 μL 15% (volume ratio) phosphoric acid, 125 μg/mL of isohexanoic acid solution, and 400 μL ether, homogenized for 1 min, and grinded for 60 s in a high-throughput tissue grinder. The sample was centrifuged followed by the collection of supernatant liquid for identifying and quantifying short-chain fatty acids (SCFAs) with the gas chromatography-mass spectrometry (GC-MS) system. The metabolite-specific parameters (retention time) of the chromatogram were determined using high-purity chemical standards. The linear regression equation ( R > 0.99) was employed to estimate the concentration of each SCFA from the standard curves obtained using seven different concentrations. 2.9 Statistical analysis 2 The quantitative data were presented as the mean±standard error of the mean (SEM). All statistical analyses were performed using SPSS 20.0 software (Chicago, USA). If the data met the assumption of homoscedasticity, the significance of differences in the mean value for groups was determined by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) method for multigroup comparisons. P value of < 0.05 was considered statistically significant. 3 Results 1 3.1 Molecular weight of APS 2 Three peaks were detected in the differential refractive index (dRI) chromatogram (Fig. S1a), and the molecular weight distribution was shown in Fig. S1b. The molar mass moments and the polydispersity corresponding to each peak were shown in Table S1. The proportion of samples with molecular weight in the range of 0‍–‍500×10 3 g/mol was 96.2%, and the number-average molecular weight and the weight-average molecular weight of these samples were 35.8×10 3 and 48.9×10 3 g/mol, respectively. 3.2 Effects of APS on the body weight gain and immune organ indexes 2 As shown in Figs. 1 1 a and 1 1 b, the body weight gain and the spleen index in the model group were lower than those in the normal group ( P < 0.01), and the thymus index showed a downward trend compared with the normal group ( Fig. 1 Fig. 1 c). Compared with the model group, the body weight gain, spleen index, and thymus index in the APS groups with different doses were significantly increased. 3.3 Effects of APS on IL-1β, IL-6, and endotoxin contents, and protein expression levels in the TLR4/NF-κB pathway 2 The contents of IL-1β, IL-6, and endotoxin in the model group were higher than those in the normal group ( P < 0.01; Figs. 2 2 a– 2 2 c). The IL-1β and IL-6 contents were significantly decreased in each APS group compared with the model group ( P < 0.01; Figs. 2 2 a and 2 2 b). The endotoxin content in each APS group showed a reduction compared to the model group, although the difference was not significant ( Fig. 2 Fig. 2 c). As shown in Figs. 2 2 d and 2 2 e, the expression levels of TLR4, NF-κB p65, p-IκBα, and p-IκBα/IκBα in the model group were increased when compared with the normal group ( P < 0.01), and the expression level of IκBα was decreased when compared to the normal group ( P < 0.01), while APS in different doses significantly reduced the expression levels of NF-‍κB p65, p-IκBα and p-IκBα/IκBα compared with the model group ( P < 0.01). Compared with the model group, APSM and APSH decreased the expression level of TLR4 ( P < 0.01), while APSM increased the expression level of IκBα ( P < 0.01). 3.4 Effect of APS administration on the gut microbiota 2 3.4.1　Changes of the taxonomic abundance and diversity of gut microbiota by APS 3 The species taxonomic composition of gut microbiota at the phylum level was observed, and nine major phyla were shown in Fig. 3 Fig. 3 a. Firmicutes and Bacteroidetes were the most abundant phyla. An increase in relative abundance of Firmicutes and Proteobacteria, and a reduction in Bacteroidetes and Verrucomicrobia in the model group were detected compared with the normal group. APS remarkably enriched the relative abundance of Proteobacteria; moreover, APS slightly increased the relative abundance of Bacteroidetes and decreased that of Firmicutes compared with the model group. For alpha diversity, the Shannon and Simpson indexes were analyzed (Figs. 3 3 b and 3 3 c). Compared with the model group, the Shannon index of the APSM group was increased ( P‍<‍ 0.05). Compared with the normal group, the Shannon and Simpson indexes of APSH group ( P‍<‍ 0.05) and APSM group ( P‍<‍ 0.01) were both significantly increased. For beta diversity, the analysis of similarities (Anosim) using unweighted UniFrac and the non-metric multidimensional scaling (NMDS) analysis were calculated to assess the difference in community composition between different samples. The Anosim result showed that the difference in inter-group was greater than that in the intra-group ( R =0.442, P =0.001; Fig. 3 Fig. 3 d). The NMDS result indicated the degree of difference among each sample ( Fig. 3 Fig. 3 e). 3.4.2　Effect of APS on critical phylotype of the gut microbiota in different groups 3 3.4.2.1　Inter-group difference and LEfSe analysis of gut microbiota 4 We found 290 OTUs with significant differences among different groups by the rank-sum test, and the principal component analysis (PCA) score plot based on the differential OTUs was shown in Fig. 4 Fig. 4 a. The differentially abundant species in the cladogram created by LEfSe analysis were shown in Fig. 4 Fig. 4 b, while Fig. 4 Fig. 4 c illustrated the LDA scores of differential species and a total of 27 significantly different species at the genus level were identified. The genus Pseudoflavonifractor was enriched in the model group. Meanwhile, there was higher abundance of genera Oscillibacter , Lactonifactor , Butyricicoccus , and Staphylococcus in the APSH group, Parabacteroides , Clostridium XI , Roseburia , Clostridium XIVb , Flavonifractor , Pseudomonas , Gemella , Bilophila , and Dorea in the APSM group, and Helicobacter , Ruminococcus , Anaeroplasma , and Alistipes in the APSL group (lg(LDA score) > 3). Paraprevotella , Ruminococcus 2 , Psychrobacter , Lactobacillus , Eubacterium , Barnesiella , and Parasutterella were enriched in the normal group (lg(LDA score) > 3). 3.4.2.2　Comparison of relative abundance of the critical bacterial genera among groups 4 We performed a pairwise comparison analysis of variance on the enriched intestinal flora in the model and APS administration groups. The relative abundance of the critical bacterial genera among different groups was shown in Fig. 5 Fig. 5 . In the model group, the relative abundance of genera Pseudoflavonifractor , Flavonifractor , and Alistipes was increased ( P < 0.05), and that of Eubacterium , Barnesiella , Lactobacillus , Psychrobacter , Parasutterella , and Corynebacterium was decreased compared with the normal group ( P < 0.05). APS administration groups, especially APSH group and APSM group, decreased the relative abundance of Pseudoflavonifractor and increased that of Parasutterella and Gemella compared with the model group ( P < 0.05). The relative abundance of Anaeroplasma in the APSL group, relative abundance of Parabacteroides , Clostridium XIVb , Roseburia , Pseudomonas , and Dorea in the APSM group, and relative abundance of Oscillibacter , Butyricicoccus , and Staphylococcus in the APSH group increased compared with the model group ( P < 0.05). Additionally, the abundance of Barnesiella , Lactobacillus , and Corynebacterium showed different degrees of contrary variation tendency in the model rats after APS administration, although no significant difference was indicated. The relative abundance of Paraprevotella in the APSH and APSM groups was decreased compared to the normal group ( P < 0.05). 3.4.3　Correlation analyses among significant differential genera, body weight gain, and immune organ indexes 3 The correlation analysis found that 12 of 27 significant differential genera were associated with the body weight gain and the immune organ indexes. As shown in Fig. 6 Fig. 6 , Pseudoflavonifractor was negatively correlated with the body weight gain ( P< 0.01) and the spleen index ( P< 0.05). At the same time, Paraprevotella showed a negative correlation with the spleen index ( P< 0.01) and thymus index ( P< 0.05). In contrast, Parasutterella ( P‍<‍ 0.01) and Barnesiella ( P‍<‍ 0.05) were positively correlated with the body weight gain. Nine genera were positively associated with the spleen index and/or thymus index, including Dorea , Clostridium XIVb , Oscillibacter , Butyricicoccus , Gemella , Parabacteroides , Staphylococcus , Corynebacterium , and Parasutterella ( P< 0.05) . 3.5 Effect of APS on SCFA production 2 As shown in Table 1 Table 1 , the contents of seven SCFAs decreased to varying degrees, and the differences in the contents of propionic acid ( P < 0.05), butyric acid, valeric acid, isobutyric acid, and isovaleric acid ( P < 0.01) in the model group were statistically significant compared with the normal group. Compared with the model group, APS increased the levels of six SCFAs to some extent, except acetic acid. The content of caproic acid in each APS group ( P < 0.01), isobutyric acid in the APSL group ( P < 0.01), and isovaleric acid in the APSL and APSH groups ( P < 0.05) were higher than those in the model group. All data were expressed as mean±SEM, n =6. * P < 0.05, ** P < 0.01 vs. NG; # P < 0.05, ## P < 0.01 vs. MG. APS: Astragalus polysaccharide; APSH: APS high-dose group; APSL: APS low-dose group; APSM: APS medium-dose group; NG: normal group; MG: model group; SEM: standard error of the mean. Group Acetic acid (μg/g) Propionic acid (μg/g) Butyric acid (μg/g) Valeric acid (μg/g) Caproic acid (μg/g) Isobutyric acid (μg/g) Isovaleric acid (μg/g) NG 905.06±111.15 445.81±87.42 309.65±65.83 63.93±15.81 1.11±0.09 59.80±7.04 37.85±6.77 MG 756.63±74.89 254.98±6.52 * 123.43±12.56 ** 28.99±2.11 ** 0.95±0.09 34.57±4.62 ** 20.85±1.72 ** APSL 627.56±79.62 * 320.72±50.52 170.35±23.84 * 39.98±6.57 1.57±0.11 *## 56.37±4.32 ## 33.99±3.29 # APSM 730.51±40.54 339.48±65.79 177.82±35.73 * 39.08±7.77 * 1.62±0.17 **## 44.63±5.11 * 29.84±5.00 APSH 722.14±68.48 381.28±39.73 203.59±41.14 48.14±5.95 1.70±0.14 **## 47.97±3.95 33.39±2.57 # 4 Discussion 1 Earlier studies found that the pharmacological effects of TCM polysaccharides are closely related to their molecular weight (Biao et al., 2020; Li Y et al., 2020 Li Y et al., 2020 ). Most reported APS had a molecular weight of 8.7‍–‍4800.0 kDa, with the largest percentage of 20‍–40 kDa polysaccharides in the Inner Mongolia APS ( Jin et al., 2014 Jin et al., 2014 ; Sheng et al., 2021 Sheng et al., 2021 ). Among them, APS with moderate molecular weight provided the most potent effects of immunomodulation ( Jiang et al., 2016 Jiang et al., 2016 ; Li K et al., 2020 Li K et al., 2020 ). The average molecular weight of APS used in this study was 35.8×10 3 g/mol, which is similar to the reported result. We found that APS increased the body weight gain and the immune organ indexes of model rats, and reduced the levels of endotoxin, IL-1β, and IL-6, which suggested that APS alleviated the immune disorders of rats with DSSD syndrome and were consistent with the findings of our previous study ( Zhao et al., 2019 Zhao et al., 2019 ). The gut microbiota acts as a central signaling hub that integrates environmental factors (e.‍g., diet, exercise) with immune signals, and the microorganisms or their metabolites stimulate the TLRs on the gut epithelium and trigger downstream NF‍-‍κB to regulate the immune response ( Thaiss et al., 2016 Thaiss et al., 2016 ; Lu et al., 2022 Lu et al., 2022 ; Zhao et al., 2022 Zhao et al., 2022 ). Moreover, studies found that A. membranaceus polysaccharides can modulate the gut microbiota to alleviate diabetic symptoms in db/db mice and non-alcoholic fatty liver disease in rats ( Song QB et al., 2022 Song QB et al., 2022 ; Zhong et al., 2022 Zhong et al., 2022 ). Given the vital roles of gut microbiota and the reported prebiotic effects of APS, we evaluated the regulatory effects of APS on the gut microbiota and TLR4/NF‍‍-‍κB pathway in rats with DSSD syndrome. We found that APS increased the richness and diversity and notably changed the classification of the gut microbiota of rats with DSSD syndrome, which agrees with the finding of modulation effect of APS on gut microbiota. Moreover, we found that APS decreased the expression levels of TLR4, NF-κB p65, and p-IκBα, and increased the IκBα expression level, which suggests that APS inhibited the activation of TLR4/NF-‍κB signaling pathway and reduced the production of inflammatory factors. Firmicutes and Bacteroidetes are two main phyla in the gut microbiota of both rats and humans. Most studies found that a reduction in Bacteroidetes and an increase in Firmicutes were associated with obesity, type 2 diabetes mellitus (T2DM), and other metabolic diseases ( Turnbaugh et al., 2006 Turnbaugh et al., 2006 ; Crovesy et al., 2020 Crovesy et al., 2020 ; Zhu et al., 2020 Zhu et al., 2020 ), which is consistent with our result. We found that APS slightly increased the reduction in Bacteroidetes and decreased the increase in Firmicutes of the DSSD rats, which did induce some critical changes in bacterial abundance. Furthermore, the relative abundance of Proteobacteria was increased in the model group and APS administration groups. The predominance of Proteobacteria in the gut of patients with IBD, obesity, and T2DM has been known ( Massier et al., 2020 Massier et al., 2020 ; Oddi et al., 2020 Oddi et al., 2020 ; Putignani et al., 2021 Putignani et al., 2021 ); however, there are complex interactions among different genera of gut microbiota, and the role of different bacteria from the same phylum or genus might be different; the diversity of disease course and presentation may represent different levels of microbial involvement in different individuals. An increase in the relative abundance of Proteobacteria in the APS administration groups may be due to a decrease in the relative abundance of other unprofitable strains. We found that APS reversed the increase in the relative abundance of Pseudofalvonifractor and the decrease in the relative abundance of Parasutterella . Moreover, Pseudofalvonifractor was negatively correlated with the body weight gain and the spleen index, whereas Parasutterella was positively correlated with the body weight gain and the spleen index. Pseudofalvonifractor belongs to the family Ruminococcaceae and phylum Firmicutes. Studies found that a higher abundance of Pseudoflavonifractor contributes to losing weight ( Louis et al., 2016 Louis et al., 2016 ), and the downregulation of Pseudoflavonifractor was associated with increase in the proportion of T cells in tumor-infiltrating lymphocytes ( Messaoudene et al., 2022 Messaoudene et al., 2022 ). These findings are consistent with our results. Research showed that Parasutterella from the phylum Proteobacteria is the member of the core gut microbiota and contributes to lipid metabolic functionalities ( Ju et al., 2019 Ju et al., 2019 ); moreover, Parasutterella was associated with the intestinal inflammatory response ( Wu et al., 2022 Wu et al., 2022 ). Polysaccharide-rich extracts could reverse the microbiota dysbiosis to improve inflammatory factor levels by increasing the abundance of Parasutterella ( Li MX et al., 2020 Li MX et al., 2020 ). Our results agreed with the above findings. Therefore, changes in the abundance of Pseudoflavonifractor and Parasutterella might be among the reasons for abnormal immune response and weight loss, and are also regarded as two kinds of bacterial indexes in estimating the pathological status of rats with DSSD syndrome and two of the target bacteria during APS treatment. We also found that APS intervention slightly reversed the reduction in the abundance of Barnesiella , Lactobacillus , and Corynebacterium genera in model rats, which were enriched in normal rats. Furthermore, the correlation analysis suggested that Barnesiella of Bacteroidetes was positively correlated with body weight gain. Barnesiella exerts the effect of competitive inhibition of pathogens, immune regulation, and anti-inflammatory protection ( Ubeda et al., 2013 Ubeda et al., 2013 ; Weiss et al., 2014 Weiss et al., 2014 ), which helps to produce butyric acid, isobutyric acid, and a smaller amount of propionic, acetic, and succinic acids ( Sakamoto et al., 2009 Sakamoto et al., 2009 ). The genus Lactobacillus is a diverse group and includes many species considered as probiotics ( Goldstein et al., 2015 Goldstein et al., 2015 ). The reported effects of these bacteria are consistent with our results. Besides Parasutterella , eight genera including Corynebacterium , Staphylococcus , Dorea , Butyricicoccus , Oscillibacter , Gemella , Parabacteroides , and Clostridium XIVb were enriched in the APS intervention groups in this study, which are gut microbiome associated with immune or inflammatory processes ( Bernard, 2012 Bernard, 2012 ; Paharik and Horswill, 2016 Paharik and Horswill, 2016 ; Zaidi et al., 2018 Zaidi et al., 2018 ). The genus Butyricicoccus of the family Ruminococcaceae and phylum Firmicutes is a butyrogenic bacterium, and butyrate is involved in the regulation of inflammatory cytokines ( Shang et al., 2016 Shang et al., 2016 ; Zhang et al., 2017 Zhang et al., 2017 ). In addition, the levels of Butyricicoccus and Oscillibacter were related to intestinal inflammatory response ( Devriese et al., 2017 Devriese et al., 2017 ; Wu MN et al., 2019 Wu MN et al., 2019 ). Parabacteroides species include a variety of probiotics, such as Parabacteroides goldsteinii and Parabacteroides distasonis , which were negatively correlated with obesity, metabolic dysfunctions, and inflammation ( Wang et al., 2019 Wang et al., 2019 ; Wu TR et al., 2019 Wu TR et al., 2019 ). Parabacteroides and Clostridium XIVb are also SCFA-producing bacteria ( Wang et al., 2019 Wang et al., 2019 ; Lei et al., 2021 Lei et al., 2021 ). These bacteria or their metabolites might play positive roles in activating immune function and metabolism in the therapeutic process of APS. In this study, the abundance of enriched Paraprevotella in normal rats was decreased in the APSM and APSH groups, which was negatively correlated with the immune organ index. Hibberd et al. (2019) Hibberd et al. (2019) reported that Paraprevotella was highly abundant in overweight and obese human adults and also positively correlated to body fat mass, which suggests that its increased abundance may be detrimental to health. Yuan et al. (2021) Yuan et al. (2021) showed that dysbiosis of gut microbiota was presented in the metabolically healthy obese children, and the abundance of Paraprevotella was positively correlated with serum IL-6. Therefore, a high abundance of Paraprevotella may be a remedial response to poor health, and APS could reverse this change. SCFAs as metabolites of intestinal microbiota play a beneficial role in modulating host immune response, providing energy for colonic epithelium, mitigating chronic inflammation and metabolism as signaling molecules, and suppressing the production of pro-inflammatory cytokines ( Koh et al., 2016 Koh et al., 2016 ; Parada Venegas et al., 2019). Ming et al. (2022) Ming et al. (2022) found that APS treatment inhibited LPS-induced inflammation in mice, which was partly caused by increased abundance of SCFA-producing genera and the serum concentrations of butyrate and propionate. Song QB et al. (2022) Song QB et al. (2022) reported that A. membranaceus polysaccharides increased the production of fecal SCFAs in db/db mice. In our study, APS increased the contents of butyric acid, valeric acid, caproic acid, isobutyric acid, and iso-valeric acid in the model group, which was consistent with these earlier reports. The promoting effects of APS on SCFA production were supported by the observation of enriched Barnesiella , Roseburia , Ruminococcus , Parabacteroides , Butyricicoccus , and Clostridium XIVb . The genus Roseburia has butyrate-producing capacity and is an anti-inflammatory factor that can decrease the risk of metabolic and inflammatory diseases ( Louis and Flint, 2009 Louis and Flint, 2009 ; Tamanai-Shacoori et al., 2017 Tamanai-Shacoori et al., 2017 ). Ruminococcus , Clostridium XIVb , and Roseburia all belong to the family Lachnospiraceae, and many bacteria from this family are known as potent SCFA producers ( Wei et al., 2018 Wei et al., 2018 ; Kim et al., 2020 Kim et al., 2020 ). Therefore, our data illustrated that these SCFAs-producing bacteria might play a significantly positive role in the process of APS to relieve the DSSD syndrome. 5 Conclusions 1 Overall, the results of this study indicated that APS could ameliorate the immune disorders in rats with DSSD syndrome induced by HFLP diet plus exhaustive swimming via modulating the gut microbiota and their metabolites, as well as the TLR4/NF-‍κB pathway. We supposed that the effect of APS on DSSD rats was closely related to some bacteria, which were associated with the production of SCFAs and immune and inflammatory responses. Therefore, we suggest that modifying the gut microbiota might be one of the mechanisms to attenuate the DSSD syndrome. 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Lipids Health Dis , 19 : 20 . https://doi.org/10.1186/s12944-019-1167-4 https://doi.org/10.1186/s12944-019-1167-4 Dietary flaxseed oil rich in omega-3 suppresses severity of type 2 diabetes mellitus via anti-inflammation and modulating gut microbiota in rats Wenxiao ZHAO conceptualized and designed the study, wrote the initial draft, and provided financial support. Chenchen DUAN, Guangying LU, Qin LYU, Xiumei LIU, Jun ZHENG, and Xuelian ZHAO performed the experiment and analyzed the data. Yanli LIU, Shijun WANG, and Haijun ZHAO coordinated the research activity execution. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. 81903947) and the Key Research and Development Project of Shandong Province (No. 2019GSF108209), China. 10.1631/jzus.B2200491 1673-1581（2023）07-0650-13 赵文晓,段晨晨,刘艳丽等.黄芪多糖通过肠道菌群和TLR4/NF-κB途径对脾虚水湿不化大鼠免疫功能紊乱的调节作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):650-662. 赵文晓,段晨晨,刘艳丽等.黄芪多糖通过肠道菌群和TLR4/NF-κB途径对脾虚水湿不化大鼠免疫功能紊乱的调节作用[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(07):650-662. This work was supported by the National Natural Science Foundation of China (No. 81903947) and the Key Research and Development Project of Shandong Province (No. 2019GSF108209), China. 2022-10-06 2023-03-28 2023-07-15 2023-07-14 2023-06-12 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 39916516 Wenxiao ZHAO, Chenchen DUAN, Yanli LIU, Guangying LU, Qin LYU, Xiumei LIU, Jun ZHENG, Xuelian ZHAO, Shijun WANG, and Haijun ZHAO declare that they have no conflict of interest. All experimental procedures in this study were approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (No. SDUTCM20210315009). All institutional and national guidelines for the care and use of laboratory animals were followed. Compliance with ethics guidelines Supplementary information 1 Fig. S1; Table S1 浙江大学学报（英文版）（B辑：生物医学和生物技术） 7 24