Ergosterone ameliorates RRR-induced spleen deficiency by gut microbiota-gut metabolites and P38MAPK signaling pathway

Abstract

Spleen deficiency is an important immune and digestive system change. Ergosterone (ER) is bioactive steroid; however, to date, no relevant studies have explored its potential efficacy in treating spleen deficiency. The aim of the present study was to investigate the therapeutic effects and mechanism of action of ER on spleen deficiency syndrome induced by Rhei Radix et Rhizoma (RRR). RRR was used to induce the development of a spleen deficiency rat model to observe changes in body weight and pathological changes in organ tissues. Additionally, the levels of relevant immune factors and gastrointestinal hormones were measured, as well as the expression of intestinal tight junction proteins and the P38MAPK signaling pathway. Changes in intestinal microbiota and metabolites were measured, and the effect of ER on the RRR-induced spleen deficiency rat model was evaluated. ER notably alleviated the symptoms of RRR-induced spleen deficiency induced in rats and offered protection against organ damage. Ergosterone can increase the expression of immunoglobulins, inhibits the increase in inflammatory factors, improve gastrointestinal hormone disorders, protect the intestinal mucosa, and repair intestinal barrier damage. The ER-treated group exhibited substantial upregulation of claudin and occludin mRNA and protein expression levels in the colonic tissue. Additionally, ER inhibited the P38MAPKsignaling pathway, thereby improving RRR induced spleen deficiency syndrome in rats. ER also influences the metabolic pathways of protein digestion and absorption, biosynthesis of unsaturated fatty acids, and arachidonic acid metabolism. In addition, ER can regulate and enhance the composition of intestinal flora in rats with spleen deficiency, increase the diversity of dominant flora, and inhibit the proliferation of harmful bacteria. ER can treat spleen deficiency syndrome by enhancing immune function, improving gastrointestinal function, repairing the intestinal barrier, and regulating intestinal flora and intestinal metabolites.

Keywords: P38MAPK; gut metabolomic; gut microbiota; intestinal barrier; spleen deficiency.

Figure 1

The effects of ER on spleen deficiency rat. (A) Ergosterone structural formula. (B) Schematic diagram of the animal experiment design. (C) Change trends and final body weight of rats during the experiment. (D) Spleen index and thymus index of rats. (E) The concentrations of IFN-γ, IL-2, IL-6, IgA, IgM, D-xylose AMS, CCK, SS, MTL, GAS, VIP, α-glucosidase, HbA1c, ATL, Trypsin, Pancreatic amylase, Pancreatic lipase (n = 10; ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group).

Figure 2

Effect of ER on the pathology and expression proteins in of spleen deficiency rats. (A) Pathological sections of rats with spleen deficiency, original magnification: 200× (n = 3). (B) Expression of claudin and occludin proteins in colon tissue of spleen deficiency rats. (C) Expression of P38MAPK, P53, and NF-κB proteins in spleen tissue of spleen deficiency rats (n = 3; ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group).

Figure 3

Effect of P38MAPK inhibitor on RRR-induced spleen deficiency rat. (A) Change trends and final body weight of rats during the experiment. (B) Spleen index and thymus index of rats. (C) The concentrations of IFN-γ, IL-2, IL-6, IgA, IgM, D-xylose AMS, CCK, SS, MTL, GAS, VIP, α-glucosidase, HbA1c, ATL, Trypsin, Pancreatic amylase, Pancreatic lipase (n = 10; ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group).

Figure 4

SB203580 has a synergistic effect on ER treatment of spleen deficiency rat. (A) Pathological sections of rats with spleen deficiency, original magnification: 200× (n = 3). (B) Expression of P38MAPK, P53 and NF-κB proteins in spleen tissue of spleen deficiency rats. (n = 3; ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group).

Figure 5

The results of metabolomics data analysis (n = 7). (A) OPLS-DA scores Plot for Colonic Metabolomics (Control_VS_Model,Model_VS_ER–H). (B) Colonic Metabolomics volcano plot indicating upregulated and downregulated metabolites (Control_VS_Model,Model_VS_ER–H). (C) Venn figure of Colonic Metabolomics (Control_VS_Model,Model_VS_ER–H). (D) Heat map expression of differential metabolites in Colonic Metabolomics. (E) Metabolic pathway of differential metabolites in Colonic Metabolomics (top 20, Model_VS_ER–H).

Figure 6

The metabolic pathways with potential markers affected by ER–H in treating spleen deficiency syndrome. (A) Relative abundance of key metabolites significantly affected by ER–H at the metabolic pathways. ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group. (B) The key metabolic pathways significantly affected by ER–H. The metabolites that are marked in red demonstrate a notable increase in expression levels as a result of the treatment with ER–H. In contrast, the metabolites that are identified in green show a significant decrease in their expression.

Figure 7

Results of gut microbiota data analysis (n = 8). (A) Rank abundance. (B) Shannon curve. (C) Simpson curve. (D) Analysis of gut microbiota using PCA was conducted on the OUT data from each sample across the three groups. (E) The relative abundance of phylum. (F) Species significantly influenced by ER–H at the phylum taxonomic level in spleen deficiency rats. ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group.

Figure 8

Effects of ER–H on the composition of gut microbiota at the genus level in rats with spleen deficiency. (A) Bar graph illustrating the relative abundance of species at the genus taxonomic level. (B) The relative abundance of key genus. ^*P < 0.05, vs. control group; ^**P < 0.01, vs. control group; ^#P < 0.05, vs. model group; ^##P < 0.01, vs. model group. (C) LEfSe analysis of dominant biomarker groups among different groups.

Figure 9

To explore the invigorating the spleen effect and mechanism of ergosterone in rats with spleen deficiency through gut microbiota-gut metabolites (By Figdraw).