Cationic Polystyrene Resolves Nonalcoholic Steatohepatitis, Obesity, and Metabolic Disorders by Promoting Eubiosis of Gut Microbiota and Decreasing Endotoxemia

Abstract

A pandemic of metabolic diseases, consisting of type 2 diabetes, nonalcoholic fatty liver disease, and obesity, has imposed critical challenges for societies worldwide, prompting investigation of underlying mechanisms and exploration of low-cost and effective treatment. In this report, we demonstrate that metabolic disorders in mice generated by feeding with a high-fat diet without dietary vitamin D can be prevented by oral administration of polycationic amine resin. Oral administration of cholestyramine, but not the control uncharged polystyrene, was able to sequester negatively charged bacterial endotoxin in the gut, leading to 1) reduced plasma endotoxin levels, 2) resolved systemic inflammation and hepatic steatohepatitis, and 3) improved insulin sensitivity. Gut dysbiosis, characterized as an increase of the phylum Firmicutes and a decrease of Bacteroidetes and Akkermansia muciniphila, was fully corrected by cholestyramine, indicating that the negatively charged components in the gut are critical for the dysbiosis. Furthermore, fecal bacteria transplant, derived from cholestyramine-treated animals, was sufficient to antagonize the metabolic disorders of the recipient mice. These results indicate that the negatively charged components produced by dysbiosis are critical for biogenesis of metabolic disorders and also show a potential application of cationic polystyrene to treat metabolic disorders through promoting gut eubiosis.

Figure 1

Metabolic disorders in the mice fed with HD are resolved by oral administration of cholestyramine. C57/B6 mice were fed with control chow (Ctrl, AIN93, containing vitamin D₃ 1,000 IU/kg) or HD (60% calories from fat without vitamin D supplement) for 22 weeks, as a negative and positive control, respectively. For the treatment groups, the mice were initially fed with HD for 10 weeks. Then cholestyramine or polystyrene at 3% (weight for weight) was added in the HD diet, as HD+CHOL and HD+PS, respectively (n = 10). A: Time course of changing body mass (n = 10). B: Visceral fat coefficient, the percentage of visceral fat mass over the body mass. C: Fasting blood glucose (12 h), n = 10. D: IPGTT and area under the curve (AUC) for the mice after 20 weeks of feeding, n = 6. E: HOMA-IR after 20 weeks feeding. F: Serum total cholesterol, serum total triglyceride, serum nonesterified fatty acids (NEFA), and serum LDL-C. G: The levels of plasma endotoxin (LPS), n = 5. EU, endotoxin units. H: The levels of serum TNF-α, n = 5. I: In vitro sequestration of plasma LPS levels by cholestyramine (CHOL), polylysine cellulose resin (Poly-Lys), and polystyrene (PS). Each experiment was repeated three times. * or #, P < 0.05; ** or ##, P < 0.01; ***P < 0.001. * and **Comparisons were made against the HD group. For panels A–H, comparisons were also made between the HD-CHOL and HD-PS groups. # and ##Comparisons were made against the HD+CHOL group. For panel I, the in vitro endotoxin depleting experiment, the comparisons were made for sequestration efficacy within each type of resin. Data were mean ± SEM.

Figure 2

NASH features are antagonized by cholestyramine treatment. The liver tissues and serum ALT levels of the mice described in Fig. 1 were assessed for NASH characteristics. A: Hematoxylin-eosin (H&E) staining. B: Hepatic steatosis and lesions were assessed for NASH based on H&E staining. C: Masson’s trichrome staining for liver fibrosis. D: Hepatic gene expression of Coll-α1, n = 6–7. E and F: Immunohistochemical staining of T-cell marker CD3 of liver tissue. CD3⁺ cell number counting on liver tissue stained with CD3 (×400-fold). Liver inflammation as measured by gene expression of TNF-α (G), arginase 1 (H), and iNOS (I), n = 4–6. J: Serum ALT levels, n = 8–10. *P < 0.05; **P < 0.01; ***P < 0.001. * and **Comparisons were made against the HD group. # and ##Comparisons were made against the HD+CHOL group. Data were mean ± SEM. Ctrl, control.

Figure 3

Gut dysbiosis is rebalanced through cationic polystyrene treatment. The microbiota in the ileum and feces of the mice described in Fig. 1 was examined. A: The abundances of phyla Firmicutes (Firm) and Bacteroidetes (Bact), species A. muciniphila (Akk), and class Gammaproteobacteria (γ-proteo) in the feces were detected by 16S rDNA-qPCR (n = 6 for each group). B: The bacteria population in the lumen of ileum was determined by high-throughput sequencing analysis (n = 5 for each group). UPGMA (unweighted pair-group method with arithmetic mean) clustering based on weighted unifrac distance of the top 10 relative abundant phyla. C: NMDS (non-metric multidimensional scaling) plot; every point shows one mouse, different colors signify different groups, and the stress = 0.127. D: The relative abundance of bacteria at phylum levels in the ileal lumen. E: The relative abundance of A. muciniphila in the ileal content. Ctrl, control.

Figure 4

FBT demonstrates that the microbes from the cholestyramine treatment are sufficient to antagonize the metabolic disorders of the recipient mice. The mice fed with HD or the control chow for 20 weeks were used as two types of recipients, which were subjected to gavage transplant from the donor microbes derived from cholestyramine-treated (F-CHOL) or HD (F-HD) mice (n = 10). A: Percent visceral fat deposition among the body mass, of the mice at the end of FBT treatment. B: IPGTT and area under the curve (AUC) of the mice at the end of FBT treatment. C: Fasting blood glucose of the mice at the end of FBT treatment. D: Plasma cholesterol. E: Plasma LDL-C. F: Serum ALT levels for liver injury. G: Hematoxylin-eosin (H&E) staining of liver tissues for hepatic steatosis; Masson’s trichrome staining of liver tissues for fibrosis; immunohistological staining of CD3⁺ lymphocytes for liver inflammation of the mice at the end of FBT. H: Liver inflammation was assessed by expression of TNF-α by the mice at the end of FBT. I: Liver fibrosis was assessed by expression of type I collagen. * or #, P < 0.05; ** or ##, P < 0.01. Comparisons were made against the F-HD group. Data were mean ± SEM.