Beneficial Effects of Potentilla discolor Bunge Water Extract on Inflammatory Cytokines Release and Gut Microbiota in High-Fat Diet and Streptozotocin-Induced Type 2 Diabetic Mice

Abstract

Potentilla discolor Bunge (PDB), a perennial herb, has been used as a traditional Chinese medicine in the therapy of many diseases. The aim of the current study was to investigate the effect of PDB water extract on systemic inflammation and gut microbiota in type 2 diabetic (T2D) mice induced by high-fat diet (HFD) and streptozotocin (STZ) injection. C57BL/6J mice were randomly divided into a normal diet (ND) group, T2D group, and PDB group (diabetic mice treated with PDB water extract at a dose of 400 mg/kg body weight). Results showed that PDB significantly decreased the levels of lipopolysaccharide (LPS) and pro-inflammatory cytokines in serum. Further investigation showed that PDB significantly reduced the ratio of Firmicutes/Bacteroidetes and the relative abundance of Proteobacteria in fecal samples of diabetic mice. In addition, PDB notably alleviated intestinal inflammation as evidenced by decreased expression of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), and inflammatory cytokines. PDB also reversed the decreased expression of intestinal mucosal tight junction proteins including Claudin3, ZO-1, and Occludin. Meanwhile, the levels of fecal acetic acid and butyric acid and their specific receptors including G-protein-coupled receptor (GPR) 41 and 43 expression in the colon were also increased after PDB treatment. Our results indicated that PDB might serve as a potential functional ingredient against diabetes and related inflammation.

Keywords: Potentilla discolor Bunge water extract; diabetes; gut microbiota; inflammation; intestinal mucosal barrier function; short chain fatty acids.

Figure 1

Outline of study design. After four weeks high-fat diet (HFD) administration, male C57BL/6J mice were daily injected with streptozotocin (STZ) at a dose of 40 mg/kg body weight for five days. At seven days following STZ treatment, the fasting blood glucose (FBG) was measured and the mice with FBG levels > 11.1 mmol/L were subjected daily to oral gavage of Potentilla discolor Bunge (PDB) (400 mg/kg body weight). After eight weeks administration, the effects of PDB on gut microbiota and inflammation of diabetic mice were evaluated.

Figure 2

Effects of PDB on systemic endotoxemia and inflammation of type 2 diabetic (T2D) mice. The fecal lipopolysaccharide (LPS) (a), serum LPS (b), serum TNF-α (c), serum IL-1β (d), and IL-6 (e) were determined by ELISA kits. Data are presented as mean ± SD, n=6. ^a p < 0.05, ^aa p < 0.01 versus the normal diet (ND) group. ^b p < 0.05, ^bb p < 0.01 versus the T2D group.

Figure 3

Effects of PDB on the diversity and structure of the gut microbiota in HFD and STZ-treated mice. Rarefaction curves of observed species from fecal samples including sobs index (a) and Shannon index (b). The α-diversity including Chao index (c) and Shannon index (d). The β-diversity presenting as principal component analysis PCA (e,f). There were six mice in each group.

Figure 4

PDB changed gut microbiota composition of diabetic mice induced by HFD and STZ. Taxonomic distribution of bacterial communities of diabetic mice fecal samples at phylum level (a), family level (d) and genus level (g). The relative abundance of Firmicutes, Bacteroidetes, and Proteobacteria (b); The ratio of Firmicutes to Bacteroidetes (c); The relative abundance of Bacteroidales_S24-7_group (e), Helicobacteraceae (f), norank_f_Bacteroidales_S24-7_group (h), and Helicobacter (i). Data are presented as mean ± SD, n = 6. ^a p < 0.05, ^aa p < 0.01 versus the ND group. ^b p < 0.05, ^bb p < 0.01 versus the T2D group.

Figure 5

PDB intervention modulated taxonomic diversity of gut microbiota in diabetic mice. The cladogram showed gut bacterial taxa with a linear discriminant analysis (LDA) score > 3.0. There were six mice in each group.

Figure 6

Correlation analysis between T2D-related biochemical parameters and the relative abundance of gut microbiota at genus level. A positive correlation was represented by red while a negative correlation was shown in green in the heap map. Statistical significance was calculated by Spearman’s rho non-parametric correlation analysis (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

Effects of PDB on intestinal inflammation and intestinal mucosal barrier function of diabetic mice. The protein expression of TLR4, MyD88, NF-κB (a,b), Claudin3, ZO-1, and Occludin (f,g) in colon tissue was determined by western blot. TNF-α (c), IL-6 (d), and IL-1β (e) mRNA expression was measured by qRT-PCR. Data are presented as mean ± SD, n = 6. ^a p < 0.05, ^aa p < 0.01 versus the ND group. ^b p < 0.05, ^bb p < 0.01 versus the T2D group.

Figure 8

Effects of PDB supplementation on the SCFAs levels and protein expression of GPR41 and GPR43 in diabetic mice. Acetic acid (a), propionic acid (b), and butyric acid (c) content of feces samples were detected by GC-MS. The protein expression of GPR41 and GPR43 in colon tissue was determined by western blot (d,e). Data are presented as mean ± SD, ^a p < 0.05, ^aa p < 0.01 versus the ND group. ^b p < 0.05, ^bb p < 0.01 versus the T2D group.