Fig. 1(A) Strains screening. (B) The survival rate of three isolates in MRSB broth (MRS [de MAN, ROGOSA and SHARPE] broth containing 0.3% bile salts). (C) Effect of the ratio of Lactobacillus plantarum to Bifidobacterium bifidum on cholesterol-triglyceride reduction. (D) Effect of different polysaccharides on insulin resistance. (E) Effect of Salvia miltiorrhiza Bunge polysaccharide (M) dose on the insulin sensitivity index (ISI). The changes in (F) body weight and (G) food intake for 6 weeks. Food intake was estimated using the following formulas: Food intake=(initial weight of food provided–the final weight of food recovered [g]). MRSB was MRS broth supplemented with 0.3% bile salts after 3 hours at 37℃. The Δlag phase was calculated as the time needed to increase by 0.5 absorbance units at 620 nm in MRSB minus the time requirement in MRS broth according to Ding et al. [37]. C×T=cholesterol reduction rate (%)×triglyceride reduction rate (%). The ISI was calculated according to the following formulas: ISI=Ln [1/(FBG×FINS)], where FBG was the level of fasting blood glucose (mmol/L) and FINS was the level of fasting insulin (mU/L). All measurements were taken in triplicate, and experiments were repeated three times to evaluate the standard deviation. LB, Lactobacillus plantarum X (LpX)-Bifidobacterium bifidum V (BbV); LBM, LpX-BbV+Salvia miltiorrhiza Bunge polysaccharide; L. barbarum, Lycium barbarum; P. linteus, Phellinus linteus; M. charantia L., Momordicacharantia L.; C. militaris, Cordyceps militaris; A. membranaceus, Astragalus membranaceus (Fisch.) Bunge; S. miltiorrhiza, S. miltiorrhiza Bunge. aP<0.05, bP<0.01 indicates statistically significant differences compared with the high-fat diet (HFD) group (control), cP<0.01 indicates statistically significant differences compared with the normal diet (ND) group (control).
Fig. 2Liver (A) total cholesterol (TC) and (B) triglyceride (TG) levels in mice with different treatments. (C) Fecal TC of the four groups at the end of feeding period, mice orally fed high-fat diet (HFD) supplemented with a mixture of L. plantarum-B. bifidum (1:2) and Salvia miltiorrhiza Bunge polysaccharide. (D) Total short-chain fatty acid concentration (the metabolized products of lipids) in cecal content. (E) Serum insulin; (F) fasting blood glucose (FBG). (G) Oral glucose tolerance test (OGTT) at 6 weeks after treatment and (H) the area under the curve (AUC) of OGTT at 6 weeks. (I) The glucose disappearance rate during the insulin tolerance test (KITT). (J) Homeostasis model assessment of insulin resistance (HOMA-IR). Insulin sensitivity index (ISI)=−ln (FPG×fasting insulin [FINS]). HOMA-IR=FINS (mIU/L)×FBG (mmol/L)/22.5. Data are expressed as the mean±standard deviation. ND, normal diet; LB, Lactobacillus plantarum X (LpX)-Bifidobacterium bifidum V (BbV); LBM, LpX-BbV+Salvia miltiorrhiza Bunge polysaccharide. aP<0.05, bP<0.01.
Fig. 3Effect of probiotic combination (LBM) on gut microbiota in the intestines of tested mice. (A) The relative abundance of four groups at the phyla level and (B) the relative abundance of four groups at the genus level. ND, normal diet; HFD, high-fat diet; LB, Lactobacillus plantarum X (LpX)-Bifidobacterium bifidum V (BbV); LBM, LpX-BbV+Salvia miltiorrhiza Bunge polysaccharide.
Fig. 4Effect of probiotic combination LBM on the concentration of short-chain organic acids and endotoxin (lipopolysaccharide [LPS]) in cecal content. (A) Levels of butyrate in cecal content, (B) levels of acetate in cecal content, (C) LPS, (D) mRNA expression of inflammatory genes in the liver of mice. (E, F) Effect of LBM on the expression of the beginning synthesis genes and lipid oxidation genes. Relative mRNA levels are expressed as a ratio relative to β-actin. Values are expressed as the mean±standard deviation. ND, normal diet; HFD, high-fat diet; LB, Lactobacillus plantarum X (LpX)-Bifidobacterium bifidum V (BbV); LBM, LpX-BbV+Salvia miltiorrhiza Bunge polysaccharide; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; IL-6, interleukin 6; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; SREBP-1c, sterol regulatory element binding protein 1c; SCD-1, stearoyl-CoA desaturase-1; PPARα, peroxisome proliferator-activated receptor-α; CPT-1a, carnitine palmitoyltransferase-1a. aP<0.05, bP<0.01.
Table 1Body weight gain, liver weight, and liver index of mice in four groups
Group |
BW gain, g |
Liver weight, mg |
Liver/BW, mg/g |
ND |
7.92±0.42 |
1,030.00±20.1 |
38.66±0.35 |
ND+LB |
7.88±0.35 |
1,029.00±18.5 |
38.76±0.30 |
ND+LBM |
7.82±0.27 |
1,031.00±19.0 |
39.05±0.26 |
HFD |
18.50±0.88 |
2,110.00±35 |
56.72±2.25 |
HFD+LB |
14.00±0.49 |
1,324.00±8.5 |
40.68±0.58 |
HFD+LBM |
10.55±0.43 |
1,128.00±6.5 |
38.64±0.31 |
Table 2Serum lipid levels in the four groups
Group |
TG, mg/dL |
TC, mg/dL |
LDL-C, mg/dL |
HDL-C, mg/dL |
ALTase, U/L |
ASTase, U/L |
ND |
92.9±1.0 |
84.7±0.58 |
25.9±0.15 |
57.5±0.6 |
26.3±0.5 |
55.1±1.2 |
HFD |
115.1±2.1 |
168.5±3.2 |
65.5±0.82 |
25.9±2.1 |
83.5±2.5 |
128.5±12.2 |
HFD+LB |
104.7±1.5 |
114.3±2.5 |
39.8±0.40 |
55.8±1.7 |
34.9±1.2 |
68.2±2.9 |
HFD+LBM |
101.2±1.5 |
100.1±1.2 |
38.2±0.25 |
62.7±2.4 |
23.8±1.1 |
62.5±1.2 |