Bacillus subtilis-Fermented Amomum xanthioides Ameliorates Metabolic-Syndrome-Like Pathological Conditions in Long-Term HFHFD-Fed Mice

Abstract

In modern society, numerous metabolic disorders are widespread globally. The present study aimed to demonstrate whether Bacillus subtilis-fermented Amomum xanthioides (BSAX) exerts anti-metabolic disturbance effects compared with the ethyl acetate fraction of Amomum xanthioides (EFAX), a previously verified functional fraction. Mice fed with a high-fat, high-fructose diet (HFHFD) for 10 wk presented a typical model of metabolic dysfunction, and BSAX significantly attenuated a string of metabolic-syndrome-related pathological parameters, such as body, fat, organ mass, lipid markers (TGs, TC, free fatty acids), and glucose metabolism (glucose, insulin), without influencing appetite. Further, BSAX markedly lowered malondialdehyde (MDA) and ROS in the blood and restored antioxidative parameters (SOD, GSH, and CAT in liver tissue, and total bilirubin in serum) by elevating Nrf2 and HO-1. Moreover, BSAX noticeably restored gut microbiota diversity and normalized lipid-metabolism-associated proteins, including SREBP-1, p-AMPK, and PPAR-α. Generally, most metabolic parameters were improved by BSAX to a greater extent than EFAX, except for liver weight and hepatic TC. In conclusion, BSAX alleviates metabolic dysfunction by enhancing lipid metabolism and antioxidative capacity and is more effective than EFAX. Therefore, the application of high-yield, effective BSAX might be a promising approach for curing and preventing metabolic disorders.

Keywords: Amomum xanthioides; Bacillus subtilis; fermentation; gut microbiota; metabolic diseases; oxidative stress.

Figure 1

Procedure for obtaining the herbal extract, HPLC fingerprinting, and the experimental scheme. (A) Flowchart of the Bacillus subtilis-fermented Amomum xanthioides extraction (BSAX) process. (B,C) HPLC fingerprinting of BSAX and the ethyl acetate fraction of Amomum xanthioides (EFAX). (D) Animal experiment schedule.

Figure 2

BSAX mitigated HFHFD-induced metabolic disturbance. (A,B) Final body weight (the body weight gain during the drug treatment is shown in yellow) and total white adipose tissue (WAT) weight were recorded on the last experimental day. (C,D) Serum triglycerides (TGs) and total cholesterol (TC) were determined using a Chemistry Auto Analyzer (Chiron, Emeryville, CA, USA). (E) After 18 h of fasting, fasting blood glucose (FBG) was detected using a drop of blood from the tail end. (F) Serum insulin levels were measured with the mouse insulin ELISA kit (Invitrogen, Carlsbad, CA, USA). ^# p < 0.05, ^## p < 0.01, compared with the normal group; * p < 0.05, ** p < 0.01, compared with the HFHFD control.

Figure 3

BSAX ameliorated hepatic steatosis by inhibiting lipogenesis. (A) Hematoxylin and eosin (H&E) staining and Oil Red O (ORO) staining were carried out on the liver tissues. (B) Nonalcoholic fatty liver disease activity scores (NASs) were calculated for comparison. (C)The area of red in the ORO staining was analyzed using ImageJ v1.8.0 (NIH, Bethesda, MD, USA). (D) The livers were removed and weighed immediately at the end of the experimental period. (E,F) The hepatic triglyceride (TG) and total cholesterol (TC) levels were measured with a Chemistry Auto Analyzer (Chiron, Emeryville, CA, USA). (G) The levels of hepatic lipid-metabolism-associated proteins were assessed by Western blotting. (H) ImageJ v1.8.0 was used to quantify the relative intensities of the bands. ^# p < 0.05, ^## p < 0.01, compared with the normal group; * p < 0.05, ** p < 0.01, compared with the HFHFD control.

Figure 4

BSAX attenuated systemic and hepatic oxidative stress through the restoration of antioxidant capacity. (A) The levels of ROS, TAC, and total bilirubin in sera were determined according to the appropriate methods. (B,C) The levels of hepatic MDA, ROS, TAC, and the main antioxidative parameters (SOD, GSH, CAT) were also evaluated for comparison. (D) The levels of oxidative stress regulator-related proteins in hepatic tissue were detected by Western blotting. (E,F) ImageJ v1.8.0 was used for quantification of the relative intensities of the bands. ^# p < 0.05, ^## p < 0.01, compared with the normal group; * p < 0.05, ** p < 0.01, compared with the HFHFD control. ROS, reactive oxygen species; TAC, total antioxidant capacity; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; CAT, catalase.

Figure 5

The gut microbiota was significantly resilient and was recovered by BSAX. (A–C) Alpha diversity indices (observed OTU, ACE, and Chao1 indices) for the gut microbiota analysis. (D) PcoA analysis of the fecal 16s rDNA sequencing data was conducted and diagramed to evaluate the similarities among the groups. (E) The Venn diagram illustrates the number of each group’s shared and unique species in the gut microbiota. ^## p < 0.01, compared with the normal group; ** p < 0.01, compared with the HFHFD control.