Integrating transcriptomics, metabolomics, and network pharmacology to investigate multi-target effects of sporoderm-broken spores of Ganoderma lucidum on improving HFD-induced diabetic nephropathy rats

Abstract

Diabetes mellitus (DM) is a major metabolic disease endangering global health, with diabetic nephropathy (DN) as a primary complication lacking curative therapy. Sporoderm-broken spores of Ganoderma lucidum (GLP), an herbal medicine, has been used for the treatment of metabolic disorders. In this study, DN was induced in Sprague-Dawley rats using streptozotocin (STZ) and a high-fat diet (HFD), and the protective mechanisms of GLP were investigated through transcriptomic, metabolomic, and network pharmacology (NP) analyses. Our results demonstrated that GLP intervention ameliorated renal damage and inflammation levels in DN rats. Integrative metabolomic and transcriptomic analysis revealed that GLP treatment modulated glucose and cellular energy metabolisms by regulating relevant genes. GLP significantly suppressed the inflammations by impacting glucose and energy metabolism-related gene expression (Igfbp1 and Angptl4) and enhanced metabolic biomarkers of 4-Aminocatechol. In addition, NP analysis further indicated that GLP may efficiently alleviate DN via immune-related pathways. In conclusion, this study provides supportive evidence of the anti-inflammatory effects of GLP supplements, highlighting their potential for promising clinical applications in treating DN.

Keywords: Diabetic nephropathy; Metabolome; Network pharmacology; Sporoderm-broken spores of Ganoderma lucidum; Transcriptome.

Graphical abstract

Fig. 1

Experimental design workflow and amelioration of sporoderm-broken spores of Ganoderma lucidum (GLP) in this study. (A) Schematic of the experimental design. (B) Body weight. (C) Fasting blood glucose. (D) Kidney/body weight ratio. (E) Urine creatinine. (F–L) Inflammatory cytokines including interleukin (IL)-6 (F), IL-1β (G), tumor necrosis factor-alpha (TNF-α) (H), matrix metallopeptidase 3 (MMP3) (I), IL-10 (J), monocyte chemoattractant protein-1 (MCP-1) (K) and C-reactive protein (CRP) (L). Values are presented as mean ± standard error of the mean (SEM) (n = 5 in each group). Student's t-test was used for comparing Control vs. diabetic nephropathy (DN) and DN vs. GLP-treated high-fat diet (HFD)-fed group (DN + GLP). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗∗P < 0.0001. mRNA: messenger RNA; STZ: streptozotocin; H&E: hematoxylin-eosin; qPCR: quantitative polymerase chain reaction; LC-MS: liquid chromatography-tandem mass spectrometry.

Fig. 2

Amelioration of sporoderm-broken spores of Ganoderma lucidum (GLP) on high-fat diet (HFD)-induced diabetic nephropathy (DN) rats. (A, B) Representative histology of glomerulus and renal tubule were assessed by hematoxylin-eosin (H&E) staining (A) and Masson staining (B), respectively. (C–E) Glomerular diameter (C), glomerular sclerosis ratio (D), and tubular injury scores (E) were measured to observe glomerular hypertrophy and renal injury. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. DN + GLP: GLP-treated HFD-fed group.

Fig. 3

Effects of sporoderm-broken spores of Ganoderma lucidum (GLP) treatment on kidney transcriptome. (A) Principal component analysis (PCA) plot. (B–D) Volcano plot of significant differentially expressed genes (DEGs) of diabetic nephropathy (DN) vs. Control (B), GLP-treated HFD-fed group (DN + GLP) vs. DN (C), and DN + GLP vs. Control (D). Red dots, upregulated; blue dots, downregulated. (E) The heatmap analysis of DEGs in Control, DN and DN + GLP groups. Red, upregulated differential genes; green, downregulated differential genes. (F) Venn diagram of the 18 expression signals consistently upregulated in DN vs. Control group, downregulated in DN + GLP vs. DN group, and no changes in DN + GLP vs. Control group. (G) Quantitative polymerase chain reaction (qPCR) results of glucose and energy-related top-ranking genes of Igfbp1 and Angptl4.^∗P < 0.05. PC: principal component.

Fig. 4

Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs). (A–C) Bubble diagram of GO enrichment analysis in diabetic nephropathy (DN) vs. Control (A), GLP-treated high-fat diet (HFD)-fed group (DN + GLP) vs. DN (B), and DN + GLP vs. Control (C). (D–E) Bubble diagram of KEGG enrichment analysis in DN vs. Control (D), DN + GLP vs. DN (E), and DN + GLP vs. Control (F). Size of dots represents genes number in each GO term and KEGG term.

Fig. 5

Metabolomic analysis under the positive and negative ion mode (n = 3). (A–D) Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) score plots depicting the comparison of metabolomic profiles among Control, diabetic nephropathy (DN), and sporoderm-broken spores of Ganoderma lucidum (GLP)-treated high-fat diet (HFD)-fed (DN + GLP) group. PCA score plot under positive ion mode (A), PCA score plot under negative ion mode (B), OPLS-DA score plot under positive ion mode (C), OPLS-DA score plot under negative ion mode (D). (E–G) Volcano plot of differentially accumulated metabolites in three groups including DN vs. Control (E), DN + GLP vs. DN (F), DN + GLP vs. Control (G). (H–J) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis of differentially accumulated metabolites in three groups including DN vs. Control (H), DN + GLP vs. DN (I), and DN + GLP vs. Control (J). Red dots, upregulated; blue dots, downregulated.FC: fold change; VIP: variable importance; ABC: ATP-binding cassette; PPAR: peroxisome proliferators-activated receptor; GnRH: gonadotropin-releasing hormone; TRP: transient receptor potential; RI: refractive index; CoA: coenzyme A; Th17: IL-17-producing T helper; FoxO: orkhead box transcription factors class O.

Fig. 6

Correlation analysis amongst differentially expressed genes (DEGs) and differentially accumulated metabolites. (A, B) Spearman's correlation of metabolome and transcriptome analysis in diabetic nephropathy (DN) vs. Control (A), and sporoderm-broken spores of Ganoderma lucidum (GLP)-treated high-fat diet (HFD)-fed (DN + GLP) vs. DN (B). ^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Fig. 7

Network pharmacology (NP) of intersecting targets of sporoderm-broken spores of Ganoderma lucidum (GLP) towards diabetic nephropathy (DN). (A) Screening of GLP targets. (B) Screening of DN-related targets. (C) Venn diagram showed the common targets of DN and GLP. (D) Protein–protein interaction (PPI) network analysis of drug-disease intersection targets. (E) Frequency of the top 10 most frequent target genes based on 11 algorithms. (F) Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of key targets (P < 0.05). Key targets and pathways are marked with red rectangles. (G) Visualization of KEGG pathways and selected key targets through Cytoscape. DN + GLP: GLP-treated high-fat diet (HFD)-fed group; TTD, therapeutic target database; IL: interleukin; CXCL: C-X-C motif chemokine ligand; TLR4: toll-like receptor 4; CCL: C-C motif chemokine ligand; FOS: FBJ murine osteosarcoma viral oncogene homolog; MAPK: mitogen activated protein kinase.

Fig. 8

Proposed mechanism of sporoderm-broken spores of Ganoderma lucidum (GLP) treatment on ameliorating diabetic nephropathy (DN) rats. IL: interleukin; TNF-α: tumor necrosis factor-alpha; MCP-1: monocyte chemoattractant protein-1; CRP: C-reactive protein；CX3CR1: C-X3-C motif chemokine receptor 1; CXCL: C-X-C motif chemokine ligand.