CXT (20112325) was purchased from Jiangsu Hengrui Pharmaceutical Co., LTD. (Nanjing, China). Lentinus edodes polysaccharide (2011060) was purchased from Hebei Guangren Pharmaceutical Co. LTD. (Shijiazhuang, China). AST (20211115), ALT (20211115), GSH-Px (20210813), MDA (20210813), SOD (20210813), and ROS (20210813) were purchased from Nanjing Jiancheng Technology Co., LTD. (Nanjing, China). TNF-α (20211112) and IL-2 (20211112) were purchased from Suzhou Sizhengbai Biotechnology Co., LTD. (Suzhou, China). The multimode microplate reader used was from Thermo Fisher Scientific, China. We used the Illumina MiSeq/NovaSeq platform (Illumina, China) and the ultra-high-throughput genetic sequencer DNBSEQ-T7 (BGI, China).

Apocynum venetum flowers were collected from Aksu Region, Xinjiang, in July 2015. They were identified by Professor Changqin Li. Dry A. venetum flowers were soaked in petroleum ether and 70% ethanol for 3 days. After drying, distilled water was added according to a solid–liquid ratio of 1:30, and the filtrate was extracted twice at 90°C for 3 h. The filtrate was concentrated and precipitated with 70% ethanol, then deposited with alcohol at 4°C for 24 h, and then filtered and precipitated with anhydrous ethanol, acetone, and petroleum ether 2 times. Protein was removed using the Sevag method, and after re-precipitation with ethanol, the AVFP was freeze-dried to solid (Wang et al., 2019).

Specific pathogen-free (SPF) Kunming (KM) male mice (5–6 weeks, 18–22 g) were obtained from Henan Provincial Laboratory Animal Center (License No. SCXK (Yu) 2019-0002) and then raised for adaptation (temperature 25°C ± 2°C, light 12 h/day, humidity 40%–45%).

After 1 week of adaptation feeding, mice were randomly allocated to six groups (n = 6/group): control group, model control (Mod) group, positive control (LEP) group, AVFP high-dose (AVFP-H) group, AVFP medium-dose (AVFP-M) group, and AVFP low-dose (AVFP-L) group. According to the references, mice in the control and Mod groups were gavaged daily with distilled water, mice in the PC group were gavaged daily with 3 mg/kg Lentinus edodes polysaccharide tablets, and mice in the AVFP-H, AVFP-M, and AVFP-L groups were gavaged daily with 600 mg/kg, 300 mg/kg, and 150 mg/kg AVFP, respectively, for 21 days, and the gavage volume was 0.1 mL/10 g (L. edodes polysaccharide tablets and AVFP were dissolved using distilled water). During the last 3 days of the treatment, an intraperitoneal injection of 80 mg/kg was administered to mice in the treatment group. The above animal experimental designs are briefly listed in Table 1. Mice were weighed every other day. At the end of the experiment, the mice were anesthetized with isoflurane (3% for induced anesthesia, with an oxygen flow rate of 300–500 mL/min; 2% for sustained anesthesia, with an oxygen flow rate of 150–250 mL/min), and the isoflurane concentration was controlled by an anesthetic gas evaporator (Puxin, ZR-02, China). After deep anesthesia, blood was taken from the eyeball. The blood was left at room temperature for 2 h and then centrifuged (3500 rpm/min, 10 min), and the supernatant was taken as serum. Spleen, thymus, liver, and cecum contents were removed and kept at −80°C for subsequent assay analysis (Liang et al., 2021).

the organ index calculation was performed using the following formula: organ index = immune organ weight (mg)/body weight (10 g).

The serum stored at −80°C was taken and placed at room temperature. Blank and sample wells were set up separately. The contents of tumor necrosis factor α (TNF-α), reactive oxygen species (ROS), cytokines, and interleukin 2 (IL-2) were detected by enzyme-linked immunoassay according to the manufacturer’s kit instructions.

AST catalyzes the transamination of α-ketoglutarate and aspartate to form glutamate and oxaloacetate. It appears red–brown in alkaline solutions, and the change in absorbance is detected using a multimode microplate reader. ALT acts on a substrate composed of alanine and α-ketoglutarate under conditions of 37°C and pH 7.4 to form pyruvate and glutamate. A series of reactions ensue, resulting in the formation of a red–brown color, and the change in absorbance is measured. The specific experimental operation details are based on the manufacturer’s kit instructions.

The mouse liver tissues were accurately weighed, rinsed with ice-cold saline to remove blood, and wiped dry with filter paper. Tissue homogenates were prepared at 1:9 on the basis of the instructions provided. The supernatant was collected to detect the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in mouse liver tissue.

To fix approximately 1 cm of spleen tissue, it was immersed in a 4% paraformaldehyde solution for 24 h. Following fixation, trimming, dehydration, embedding, sectioning, and staining, the tissue was mounted. The Eclipse Ci-L camera microscope was used to capture images at ×200 magnification. During imaging, it was ensured that the tissue filled the entire field of view, and consistent backlighting was maintained for each photograph (Liang et al., 2022).

Transcriptomic analysis was performed on nine spleen samples from the control, Mod, and AVFP-M groups. First, we took 60 mg of tissue and pulverized it into powder in liquid nitrogen. The sample was centrifuged after homogenizing. We added 0.3 mL of isoamyl alcohol to the supernatant and centrifuged the sample. After that, 1 mL of 75% ethanol was added to wash twice, and the sample was centrifuged. In the end, DEPC-treated water was added to dissolve RNA, followed by the identification and quantification of total RNA using a NanoDrop spectrophotometer and the Agilent 2100 Bioanalyzer. Quality control was carried out on the data obtained by sequencing. After passing the quality control, gene quantitative analysis and gene screening based on the gene expression level were carried out. According to the differential genes, GO functional significance enrichment analysis, pathway significance enrichment analysis, and other further mining analyses were carried out.

Three cecal contents were selected from each group for DNA extraction, PCR amplification, product purification, library preparation, and detection, and multiple libraries were mixed for Illumina MiSeq/NovaSeq sequencing. The 16S rDNA amplification sequencing for this study was completed by Suzhou Genomics Co., Ltd. (Jiang et al., 2020).

A total of 0.3 g of cecal contents were taken from each mouse, 1 mL of deionized water was added, and centrifugation was performed at 12,000 rpm/min to obtain the supernatant, and then HCL and ether were added sequentially for extraction. The mixture was passed through a 0.22-um filter membrane. SCFAs in cecal contents were detected through high-performance liquid chromatography (HPLC) (Liang et al., 2022).

The data were evaluated using the Shapiro–Wilk test to confirm the normality of the distribution. All data were shown as the means ± standard error. SPSS 26.0 software was used to carry out data statistics. A one-way ANOVA and Dunnett’s post hoc multiple comparison tests were conducted. p < 0.05 was considered significant.

Body weight is closely related to the number and function of immune cells, and the thymus and spleen are essential immune organs for lymphocyte differentiation, maturation, and immune responses. To evaluate the effect of AVFP on immunosuppressed mice, indicators such as the body weight, immune organ index, and cytokine secretion capacity of the mice were measured. We monitored the weight of the mice every other day. AVFP ameliorated the loss of body weight in CTX-induced mice (Figure 1A). In addition, the thymus index and spleen index were extremely reduced in CTX-induced mice. After the AVFP gavage, the thymus index in the AVFP-M and AVFP-L groups was particularly increased, and the spleen index in the AVFP-H group was dramatically increased (Figures 1B, C). In Figures 1D–F, the levels of TNF-α and ROS in the Mod group were higher than those in the control group, suggesting that CTX could induce immunosuppression in mice. The levels of TNF-α and ROS were reduced by AVFP gavage, so the levels in the AVFP-H, AVFP-M, and AVFP-L groups were significantly reduced. On the contrary, the levels of IL-2 were remarkably reduced in the Mod group, illustrating that CTX could restrain the immune capacity in mice. After treatment, the levels of IL-2 significantly increased in the AVFP-L group. These findings demonstrated that AVFP could improve weight loss in immunosuppressed mice by CTX induction, improve the protective ability of immune organs, ameliorate the level of cytokines, and increase the immune capacity of the body.

CTX causes immunosuppression and also generates free radicals in the body, leading to oxidative stress and damaging liver function (Jing et al., 2018). When inflammation occurs in liver cells, they become damaged, and transaminases are released into the blood, resulting in an increase in serum transaminase levels. We tested the antioxidant stress capacity and transaminase activity of mice. In Figures 2A, B, AST, and ALT activities in the Mod group were significantly improved compared with those in the control group. After AVFP gavage, AST activity in the LEP, AVFP-H, and AVFP-M groups was significantly reduced, and ALT activity in both LEP and AVFP gavage groups was remarkably decreased, especially in the polysaccharide-administered groups. As illustrated in Figures 2C, D, CTX could significantly reduce the activities of GSH-Px and SOD, increase the activity of MDA, and reduce the ability to resist oxidative stress. However, after AVFP gavage, GSH-Px and SOD activities were significantly increased. However, the content of MDA was extremely decreased in the LEP and AVFP-L groups, as shown in Figure 2E. Thus, AVFP could decrease AST and ALT activities and enhance the body’s ability to resist oxidative stress.

The spleen is an important immune organ, and the state of CTX-induced immunocompromised mice can be judged by observing and photographing the histological morphology of the spleen (Li et al., 2017). As shown in Figure 3, the pathological section observation showed that the spleen of mice in the control group had normal morphology and neatly arranged cells. After CTX modeling, the area of the white pulp was smaller, the proportion of the red pulp was larger, and the boundary between the white pulp and the red pulp was not obvious. After the administration of AVFP, the pathological recovery degree of the white pulp in the AVFP-H group was similar to that in the LEP group, but there was no significant change in the AVFP-M and AVFP-L groups. Our experimental results showed that AVFP could reduce CTX-induced spleen damage, protect immune organs, and improve the body’s immune stress ability.

By sequencing the RNA of different samples, transcriptomics allows for the comparison of gene expression levels and changes between study samples, thereby further exploring the mechanisms underlying the occurrence of disease. A total of nine samples were detected via the RNA-Seq platform. The average comparison rate of the sample comparison genome was 92.30%, and the average comparison rate of the comparison gene set was 71.27%. A total of 16,623 genes were detected. Differential genes were screened with |log2FC| >1 and p < 0.05. A total of 193 differential genes were identified in the control and Mod groups, and 99 differential genes were identified in the Mod and AVFP-M groups. Compared with the control group, 82 differential genes were upregulated, and 111 differential genes were downregulated in the Mod group. Compared with the Mod group, 28 differential genes were upregulated, and 71 differential genes were downregulated in the AVFP-M group, as illustrated in Figures 4A, B. Combined with the literature, the 26 differential genes shared by the three groups were further screened (Figure 4C), and finally, 6 differential genes with greater immune-related and antioxidant correlation were obtained, namely, Bcl3, Hp, Lbp, Cebpd, Gstp2, and Lcn2. The expression levels derived from transcriptomics are shown in Figure 4D.

GO and KEGG enrichment analyses play significant roles in bioinformatics research. They can assist researchers gain a deeper understanding of the functions, pathways, and biological processes of gene sets under specific conditions. In Figure 5A, 99 differential genes between the Mod group and AVFP-M group are annotated into 53 GO items: 26 biological processes, 17 cellular components, and 10 molecular functions, among which the immune system process, antioxidant activity, and detoxification were related to immunity. The KEGG enrichment analysis is helpful to further research the biological function of the genes. A total of 26 DEGs into 43 KEGG pathways were enriched, of which the first 20 KEGG pathways are shown in Figure 5B. The abscissa is obtained by counting the number of differential genes in the pathway. In Figure 5B, the higher the rich factor value, the higher the relative percentage of differential genes in the pathway. Differential genes were enriched by immune-related signaling pathways, such as the NF-κB signaling pathway (p = 0.013).

Plant polysaccharides also have the effect of regulating the structure of the gut microbiota and improving intestinal microecology. We used 16S rDNA technology to evaluate the impact of AVFP on the gut microbiota of immunocompromised mice. In Figure 6A, the Chao1 index of the LEP, AVFP-H, and AVFP-L groups was higher than that of the Mod group, suggesting that AVFP could elevate the diversity of gut microbiota in CTX-induced mice. In Figure 6B, the Shannon index was considerably improved in the AVFP-H, AVFP-M, AVFP-L, and LEP groups, suggesting that AVFP could enhance the diversity of intestinal flora in mice. β-diversity is used to analyze the diversity difference between different samples, and PCA can analyze the distribution of sample colonies based on the distance of the samples. In Figures 6C, D, it can be seen that the distance between the control group and the LEP group was close, and the difference was small. Likewise, mice treated with AVFP were remarkably different from CXT-induced mice in terms of gut microbiota distribution and similar to the control group, indicating that AVFP could regulate the structural composition of intestinal microbes in CTX-induced mice.

In Figure 6E, at the phylum level, AVFP could ameliorate the CTX-induced increase of Bacteroidota abundance and decrease of Firmicutes and Proteobacteria abundance. At the genus level (Figure 6F), compared to the control group, the abundance of Helicobacter, Alloprevotella, Muribaculaceae, Parabacteroides, Lachnoclostridium, Blautia, Prevotellaceae_Ga6A1_group, and Lactobacillus was remarkably enriched in CTX-induced mice. However, the CTX-increased abundance of Alloprevotella, Muribaculaceae, Parabacteroides, Lachnoclostridium, Prevotellaceae_Ga6A1_group, and Lactobacillus was reduced by AVFP gavage. Similarly, CTX decreased the abundance of Lachnospiraceae_NK4A136_group, Roseburia, Lachnospiraceae_UCG-001, Corynebacterium, and AVFP could reverse these changes. LEfSe analysis also verified the microbiota abundance at the genus level (Figure 6G).

Collectively, the CTX-induced gut dysbiosis was restored by AVFP gavage.

Likewise, the COG pathway was used to evaluate the potential function of the gut microbiota. AVFP mainly reversed 10 metabolic pathways, namely, chromatin structure and dynamics, amino acid transport and metabolism, nucleotide transport, and metabolism, coenzyme transport and metabolism, transcription, cell wall/membrane/envelope, cell motility, and secondary metabolite biosynthesis, transport, and catabolism (Figure 7A). The results indicated that AVFP could enhance the immunity of the body by regulating the metabolic function of intestinal microorganisms.

In Figures 7B–D, CTX could evidently reduce the levels of acetic acid and butyric acid but had no apparent effect on propionic acid. After the administration of AVFP, the levels of acetic acid and butyric acid were elevated in the AVFP-H and AVFP-M groups, but the levels of butyric acid in the AVFP-L group did not change significantly. However, AVFP also had no overt effect on the levels of propionic acid. This implied that AVFP could regulate the gut microbiota by improving the levels of acetic acid and butyric acid and enhancing the effect of immunity.

To further explore the potential relationship among gut microbiota, biochemical markers, and immunity via the Spearman correlation analysis (Figure 8), fecal SCFA levels, Lachnospiraceae_NK4A136_group, Corynebacterium, Lachnospiraceae_UCG-001, and Roseburia were negatively connected with serum TNF-α, serum ROS, serum ALT, serum AST, and liver MDA, and positively related with liver SOD, liver GSH-Px, serum IL-2, and the thymus and spleen indexes. Conversely, the abundance of Muribaculaceae, Alloprevotella, Prevotellaceae_Ga6A1_group, Parabacteroides, and Lactobacillus was positively associated with liver MDA, serum ALT, serum AST, and serum TNF-α, and negatively associated with liver SOD, liver GSH-Px, serum IL-2, and the thymus and spleen indexes. These results demonstrated that the gut microbiota could modulate immune-related markers to ameliorate CTX-induced immunodeficiency.