The effective fraction isolated from Radix Astragali alleviates glucose intolerance, insulin resistance and hypertriglyceridemia in db/db diabetic mice through its anti-inflammatory activity

Abstract

Background: Macrophage infiltration in adipose tissue together with the aberrant production of pro-inflammatory cytokines has been identified as the key link between obesity and its related metabolic disorders. This study aims to isolate bioactive ingredients from the traditional Chinese herb Radix Astragali (Huangqi) that alleviate obesity-induced metabolic damage through inhibiting inflammation.

Methods: Active fraction (Rx) that inhibits pro-inflammatory cytokine production was identified from Radix Astragali by repeated bioactivity-guided high-throughput screening. Major constituents in Rx were identified by column chromatography followed by high-performance liquid chromatography (HPLC) and mass-spectrometry. Anti-diabetic activity of Rx was evaluated in db/db mice.

Results: Treatment with Rx, which included calycosin-7-β-D-glucoside (0.9%), ononin (1.2%), calycosin (4.53%) and formononetin (1.1%), significantly reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and MCP-1) in human THP-1 macrophages and lipopolysaccharide (LPS)-induced activation of NF-κB in mouse RAW-Blue macrophages in a dose-dependent manner. Chronic administration of Rx in db/db obese mice markedly decreased the levels of both fed and fasting glucose, reduced serum triglyceride, and also alleviated insulin resistance and glucose intolerance when compared to vehicle-treated controls. The mRNA expression levels of inflammatory cell markers CD68 and F4/80, and cytokines MCP-1, TNF-α and IL-6 were significantly reduced in epididymal adipose tissue while the alternatively activated macrophage marker arginase I was markedly increased in the Rx-treated mice.

Conclusion: These findings suggest that suppression of the inflammation pathways in macrophages represents a valid strategy for high-throughput screening of lead compounds with anti-diabetic and insulin sensitizing properties, and further support the etiological role of inflammation in the pathogenesis of obesity-related metabolic disorders.

Figure 1

The schematic diagram of the purification and identification of the active fractions from Radix Astragali.

Figure 2

The chemical structures of the four major constituents in the Rx and HPLC chromatogram of the Rx fraction.

Figure 3

Rx significantly reduced the secretion of cytokines in human THP-1 macrophages. Panels show (a) MCP-1, (b) IL-6 and (c) TNF-α concentrations in human THP-1 macrophage conditioned medium after incubation with Rosiglitazone (10 μg/ml) or Rx (5 μg/ml or 10 μg/ml) for 48 hours. Each bar represents the relative mean fold change ± SEM (n = 6). Data were statistically analyzed using the one-way ANOVA with Dunnette's post hoc test. * P < 0.05; ** P < 0.01.

Figure 4

Rx significantly inhibited LPS-induced NF-κB activity (as indicated by SEAP activity) in mouse RAW-Blue macrophages. Different dosages of Rx (10 μg/ml or 20 μg/ml) or Rosiglitazone (10 μg/ml) together with 100 ng/ml LPS were incubated with RAW-Blue cells for 24 hours or 48 hours. DMSO was used as the vehicle control. The SEAP activities in the conditioned medium of mouse RAW-Blue cells were measured using QUANTI-Blue assay as described in Methods. Each bar represents the relative mean fold change ± SEM (n = 6). Data were statistically analyzed using one-way ANOVA with Dunnette's post hoc test. ** P < 0.01.

Figure 5

Rx decreased both fed and fasting blood glucose levels and alleviated hypertriglyceridemia in db/db diabetic mice. Male db/db diabetic mice were treated with vehicle or Rx (2 g/kg/day) for 12 weeks by oral gavage. (a) The weekly profile of fed glucose levels. (b) The fasting glucose levels at week 2, 6 and 12 measured after 16 hours of starvation. (c) Fasting serum insulin concentration after 12 weeks of Rx treatment. (d) The weekly profile of fed triglyceride level. (e) Serum free fatty acid concentration after 12 weeks of Rx treatment. Data were statistically analyzed using one-way ANOVA with Dunnette's post hoc test. Data are presented as mean ± S.E.M. (n = 5-6). ** P < 0.01.

Figure 6

Rx treatment alleviated glucose intolerance and insulin resistance in db/db diabetic mice. Male db/db diabetic mice were treated with vehicle or Rx (2 g/kg/day) for 12 weeks by oral gavage. (a) Glucose tolerance test (GTT) conducted at week 12 after treatment. (b) Glucose tolerance test expressed as area under curve. (c) Insulin tolerance test (ITT) conducted at week 12 after treatment. (d) Insulin tolerance test expressed as area under curve. All mice were fasted for 16 hours or 6 hours before GTT and ITT were carried out, respectively. Data were statistically analyzed using one-way ANOVA with Dunnette's post hoc test. Data are presented as mean ± S.E.M. (n = 5-6). * P < 0.05; ** P < 0.01.

Figure 7

Rx inhibited the inflammatory state in adipose tissue of db/db diabetic mice. Panels show the relative mRNA levels of the inflammatory cell markers (a) CD68 and (b) F4/80, pro-inflammatory cytokines (c) TNF-α and (d) MCP-1 and alternatively activated macrophage marker (e) arginase I in adipose tissue of vehicle- or Rx-treated db/db diabetic mice and lean control. Data were statistically analyzed using one-way ANOVA with Dunnette's test. Data are presented as relative mean fold change ± S.E.M. (n = 5-6). * P < 0.05; ** P < 0.01.

Figure 8

Effects of Rx on the basic metabolic parameters of db/db diabetic mice. Panels show (a) daily food intake, (b) body weight and (c) liver weight of the treated db/db diabetic mice. Data were statistically analyzed using one-way ANOVA with Dunnette's test. Data are presented as mean ± S.E.M. (n = 5-6). * P < 0.05; ** P < 0.01.

Figure 9

Rx treatment increased the adipocyte size of db/db diabetic mice. Panels show the representative images of hemotoxylin and eosin-staining of epididymal adipose tissue of (a) db/db mice treated with vehicle and (b) db/db mice treated with Rx (2 g/kg/day). (c) Quantification of the adipocyte size. Data were statistically analyzed using one-way ANOVA with Dunnette's post hoc test. Data are presented as mean ± S.E.M. (n = 5-6). * P < 0.05.

Figure 10

Rx treatment did not affect the hepatic lipid profile of db/db diabetic mice. Panels show the hepatic (a) free fatty acid (FFA), (b) cholesterol and (c) triglyceride concentrations of db/db diabetic mice. Data were statistically analyzed using one-way ANOVA with Dunnette's test. Data are presented as mean ± S.E.M. (n = 5-6).