SZ-A ameliorates glucose metabolism, enhances the insulin response, and elevates GLP-1 in glucose levels during oral glucose tolerance tests in diabetic KKAy mice
After a 4-week treatment, the levels of fasting blood glucose (P < 0.01, P < 0.001) and postprandial blood glucose (P < 0.01, P < 0.01) in both SZ-A-treated groups were significantly decreased compared to the DM group (Fig. 1A, B). Hemoglobin A1c (HbA1c) levels in SZ-A-treated groups were lower than those in the DM group after a 6-week treatment (P < 0.05, P < 0.05; Fig. 1C), indicating that SZ-A glycemic control in the KKAy mice during chronic treatment. As shown in Fig. 1D, compared to the DM group, both doses of SZ-A significantly reduced blood glucose levels at 15 min after oral glucose loading (P < 0.05, P < 0.01). We further detected the blood insulin content and active GLP-1 levels as an indication of insulin and GLP-1 secretory function, respectively. As shown in Fig. 1E and 1F, there was no notable increase in blood insulin content and active GLP-1 level in the DM group after oral glucose loading; however, SZ-A-treated groups had increased insulin content and active GLP-1 levels at both baseline and 15 min after glucose stimulation. SZ-A 100 and SZ-A 200 significantly enhanced insulin secretion nearly 1.73-fold and 1.88-fold from baseline at 15 min after glucose stimulation, respectively (P < 0.01, P < 0.05), compared to the DM group (1.11-fold). In addition, both doses of SZ-A elevated active GLP-1 levels nearly 2.7-fold and 2.6-fold at 15 min after glucose stimulation from baseline (P < 0.01, P < 0.05), respectively, compared to the DM group (2.1-fold). Moreover, both doses of SZ-A resulted in decreased blood triglyceride levels after 6 weeks (P < 0.05, P < 0.05), and induced significant weight loss compared to the DM group at the end of treatment (P < 0.01, P < 0.001).
SZ-A modulates gut microbiota profiling and SCFA concentration in feces
The effects of high-dose SZ-A (SZ-A 200, 200 mg/kg) on intestinal microbiota composition were examined by Illumina sequencing-based analysis of bacterial 16S ribosomal RNA in fecal samples collected at the end of 8-week treatment. Compared to the DM group, the operational taxonomic unit (OTU) numbers were reduced in the SZ-A 200 group (Fig. 2A; P < 0.001). The Shannon and Chao indices reflect the diversity and richness of gut microbiota, respectively. As shown in Fig. 2B and 2C, SZ-A diminished the indices of Shannon and Chao (P < 0.05, P < 0.05). Unweighted Unifrac principal coordinate analysis (PCoA) based on OTU levels revealed distinct clustering of microbiota composition in each group (Fig. 2D). Multivariate analysis of variance of PCoA matrix scores revealed that the microbiota community of mice in the SZ-A 200 group differed from that of the DM group (P < 0.001). Additionally, the bacterial community of SZ-A 200-treated mice differed from that of the DM group. Taxonomic profiling at the family level revealed that SZ-A treatments elevated the abundance of Bacteroidaceae, Erysipelotrichaceae, and Verrucomicrobia and reduced that of Rikenellaceae, Desulfovibrionaceae, and Aerococcaceae compared with DM mice (Fig. 2E). Similar results were also observed at the genus level. SZ-A 200 decreased the abundance of Alistipes, Desulfovibrio, and Aerococcus, and increased the abundance of Bacteroides, Faecalibaculum, and Allobaculum compared with the DM group (Fig. 2F). Collectively, these findings indicate that SZ-A 200 modulates the composition of gut microbiota.
Short-chain fatty acids (SCFAs) are generated in the gut by bacterial fermentation of dietary fiber. Fecal SCFA concentrations were quantified to assess the impact of SZ-A on bacterial metabolic activity in KKAy mice. The concentrations of acetic (P < 0.05, P < 0.05) and propionic acids (P < 0.05, P < 0.05) were elevated in both SZ-A-treated groups (Fig. 3A, B), whereas butyric, isobutyric (propionic acid-2-methyl), pentanoic, isopentanoic (butanoic acid-3-methyl), hexanoic, and isohexanoic (pentanoic acid-4-methyl) acids decreased in the SZ-A-treated groups compared to the DM group (Fig. 3C–H). No significant changes were observed in total SCFA concentration between the SZ-A-treated and DM groups (Fig. 3I).
Subsequently, the relationship between fecal SCFAs and intestinal bacterial at the family level was analyzed (Fig. 3J). The results showed that Enterococcaceae and Corynebacteriaceae, Aerococcaceae, Desulfovibrionaceae, and Rikenellaceae were positively correlated with the decreased SCFAs, including butyric, isobutyric, hexanoic, isohexanoic, pentanoic, and isopentanoic acids (Fig. 3J). Enterococcaceae and Corynebacteriaceae abundance was negatively correlated with propionic acid, Corynebacteriaceae was negatively correlated with acetic acid, and Verrucomicrobiaceae was positively correlated and propionic acid was observed (Fig. 3J). Several transport systems play a role in the cellular uptake of SCFAs in the gut, including monocarboxylate transporter-1 (MCT1) and sodium-coupled monocarboxylate transporter 1 (SMCT1) (SLC5A8). As the transporters responsible for the entry and transcellular transfer of these bacterial products in epithelium are critical determinants of gut function, we detected MCT1 and SLC5A8 protein expression levels in the ileum tissue after SZ-A treatment in KKAy mice. The results showed that MCT1 and SLC5A8 protein levels in ileum from SZ-A-treated mice were significantly increased compared with the DM group.
SZ-A alleviates ileal inflammatory injury and pro-inflammatory macrophage infiltration in KKAy mice
Considering microbial SCFAS production (especially acetate, propionate, and butyrate) is essential for gut integrity by regulating the mucus production, providing fuel for epithelial cells and effects on mucosal immune function, the histological alteration of ileum tissue were evaluated by hematoxylin and eosin (H&E) staining. As shown in Fig. 4A, in the DM group, a dense inflammatory cellular infiltrate was present in the mucosa and submucosa and crypts showed typical shortening. Focal crypts were lost and the surface epithelium was damaged (Fig. 4A). Microscopic total score and scores for the three features (inflammation, extent of inflammation, and crypt damage) were given for each group (Fig. 4B–E). Inflammation scores were significantly reduced by both doses of SZ-A (P < 0.05, P < 0.001). Moreover, microscopic total score, crypt damage score, and inflammation score were significantly reduced in the SZ-A 200 group (P < 0.001, P < 0.01, and P < 0.001). Collectively, long-term SZ-A treatment prevented the development of inflammation and restored ileal barrier integrity in diabetic KKAy mice.
The KKAy mice were fed a high-fat diet (HFD) to induce diabetic syndrome. Given the critical role of M1 macrophages in HFD-induced intestinal inflammation, in parallel to those histological changes, macrophage-specific F4/80 and CD11c expression was measured to verify whether SZ-A treatment was able to modulate macrophage infiltration in the ileum tissue. As shown in Fig. 4F, compared to the DM group, fewer pro-inflammatory CD11c-positive macrophages were observed in both doses of SZ-A-treated groups (P < 0.05, P < 0.001). This indicates a reduced inflammatory state after SZ-A treatment, and fully consistent with this result, we found downregulated mRNA expression of F4/80 (P < 0.05, P < 0.05) and multiple pro-inflammatory factors, including MCP1 (P < 0.05, P < 0.01) and TNF-α (P < 0.05, P < 0.05; Fig. 4G), and also reduced CD11c protein expression in the ileum of SZ-A-treated groups (P < 0.05, P < 0.05; Fig. 4H), compared to the DM group. Given that inflammation damages gut permeability and integrity, we also detected the protein expression levels of zonula occludens-1 (ZO-1), an intestinal tight junction component. SZ-A 200 markedly elevated ZO-1 protein levels (P < 0.05) compared to the DM group.
Nuclear factor kappa B (NF-κB) is critically associated with the progression of inflammation and cell proliferation in the intestinal mucosa. Therefore, the effects of SZ-A on NF-κB activity on the ileal mucosa were investigated. The indices of the phosphorylated (p-NF-κB) p65-positive area were markedly reduced with SZ-A treatment (P < 0.01, P < 0.01; Fig. 4I). These findings indicate that SZ-A significantly alleviated ileal inflammatory injury and attenuated the inflammatory state induced by pro-inflammatory macrophage infiltration of the ileum tissue in diabetic KKAy mice.
SZ-A attenuates endotoxin content, pro-inflammatory cytokine, and chemokine levels in serum of diabetic KKAy mice
The literature suggests that gut dysbiosis not only leads to increased intestinal permeability, but it also results in the translocation of bacteria or bacterial products into circulation, inducing a state of chronic low-grade inflammation, such as LPS in HFD-induced diabetes. Considering the effects of SZ-A on modulating gut microbiota profiling and alleviating ileal inflammatory injury, serum endotoxin content, and levels of cytokines and chemokines were determined after SZ-A treatment of diabetic KKAy mice.
Compared with the DM group, both doses of SZ-A markedly diminished serum levels of the endotoxin content (P < 0.01, P < 0.001; Fig. 5A), inflammatory cytokines and also chemokines, such as interleukin 1β (IL-1β, P < 0.01, P < 0.05; Fig. 5E), IL-6 (P < 0.001, P < 0.001; Fig. 5H), chemokine ligand 4 (CCL4) (P < 0.05, P < 0.01; Fig. 5K), and CCL5 (P < 0.05, P < 0.05; Fig. 5L). Additionally, serum levels of IL-12β (P < 0.05; Fig. 5F), CCL11 (P < 0.05; Fig. 5I), and CXCl1 (P < 0.05; Fig. 5J) were also significantly reduced in the SZ-A 200-treated group. However, serum levels of anti-inflammatory cytokines IL-10 (P < 0.05, P < 0.05; Fig. 5C) and IL-13 (P < 0.05, P < 0.05; Fig. 5D) serum were markedly elevated (Fig. 3B).
Functional enrichment analysis of differentially abundant proteins in the ileum after SZ-A treatment
Proteomics were used to determine the molecular characteristics of the ileum in the high-dose SZ-A-treated group (SZA) and DM group in KKAy mice. Liquid chromatography tandem mass spectrometry identified 208,219 secondary spectra. A total of 42,677 matched effective spectra were obtained. Using a false discovery rate (FDR) < 1% at the peptide and protein levels, 25,816 of the 25,060 peptides were identified as specific, and 5043 of the 4352 proteins were quantifiable (Fig. S1A). A total of 34 proteins were differentially expressed (fold change > 1.2, P < 0.05; Fig. 6A) between the DM group and SZA group, of which 24 proteins were upregulated and 10 were downregulated. To determine the characteristics of the differentially expressed proteins, we annotated the subcellular localization, Clusters of Orthologous Group, and Gene Ontology (GO) of the 34 proteins. Annotation of the subcellular localization showed that 44.12% of all identified differentially expressed proteins were localized in the cytoplasm, 14.71% in the plasma membrane, 14.71% in the mitochondria, 11.76% in the nucleus, 8.82% in the extracellular space, and 5.88% in the endoplasmic reticulum (Fig. S1B). Most differentially abundant proteins participated in and regulated the cellular and metabolic processes (Fig. S1C). COG functional classification revealed that most of these differentially abundant proteins played a role in posttranslational modification, protein turnover, and chaperones (Fig. S1D).
Bioinformatics analysis was performed to identify the main biological pathways and functional categories of the differentially abundant proteins (fold change > 1.2; P < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the most significantly altered pathways were involved in primary bile acid biosynthesis, peroxisome proliferator-activated receptor (PPAR) signaling pathway, synthesis and degradation of ketone bodies, and terpenoid backbone biosynthesis (Fig. 6B). We identified 34 abundant proteins that were mainly involved in the above-mentioned pathways (Fig. 6C). These proteins included downregulation of cluster of differentiation 36 (CD36), a protein related to the PPAR pathway; CYP27a1, the representative differentially abundant protein related to primary bile acid biosynthesis; and histocompatibility 2 class II antigen E beta, which is critical in the antigen processing and presentation pathway and is also described as major histocompatibility complex class II (MHC II). The expression level of these key regulators identified via proteomics was also confirmed by Western blotting. The results showed the level of CD36 (SZ-A 200 group; P < 0.05) and MHC II (P < 0.05, P < 0.01) were significantly reduced in SZ-A-treated groups compared to the DM group (Fig. 6D), which is consistent with proteomics analysis.