3 Results
3.1 Effects on body weight and fat
Fig.1a-d shows the body weight (the initial weight, weight of modeling,
and final weight) and body fat percentage in rats. There was no
significant difference in the initial body weight between groups
(Fig.1a) (P > 0.05). During the treatment period, the body
weights of rats in OC, DG, VD12.5, and DG10VD12.5 groups were
significantly increased compared with NO group (Fig.1b) (P <
0.05). However, the increment in body weight was lower in VD and DG
group (278.22, 280.38, P< 0.05), and further lower in
DG10VD12.5 group (260.28, P < 0.05) compared with the OC group
(298.33) (Fig.1c). For the body fat percentage, rats under DG10D12.5
treatment had the lowest value (4.34) and had lower values under VD and
DG treatment (6.03, 5.78) compared with the OC group (9.04, P
< 0.05 for all).(Fig.1d)
3.2 The effects
on
serum lipid levels and metabolism enzyme activity
The effects of DAG and VD on serum lipids are shown in Fig.2a-d. The
obesity groups (OC, DG, VD12.5 and DG10VD12.5 treatments) had
significant increases in the serum levels of TC, TG, and LDL, whereas
reduction in HDL compared with those in NO group (P < 0.05).
The DG group or VD group and DG plus VD group had significant influences
on the serum lipids compared with those in OC group (P< 0.05). For HDL, the highest value was found in rats under
the DG10VD12.5 treatment (3.10), and the VD12.5 and DG10 treatment had
relatively lower value (2.84, 2.73) compared with the OC group (2.02)
(Fig.2c). For TG, TC, and LDL, the rats under the DG10VD12.5 treatment
had the lowest values (1.07, 3.25, and 3.16) compared with the OC group
(1.81, 4.53, and 4.07) (P < 0.05 for all) (Fig.2a, 2b and 2d).
The effects of DAG and VD on the metabolism enzyme activity in the liver
are shown in Fig.3a and 3b. The obesity (OC, DG, VD12.5, and DG10VD12.5
treatments) resulted in significant decreases in the AST and ALT
activity compared with those in the NO group (P < 0.05). There
were significant differences on the AST and ALT activity between OC and
DG10VD12.5 treatments (P< 0.05), however, there were no
significant differences between OC and DG10 or VD12.5 group (P
> 0.05). For AST, the highest value was found in rats under
the DG10VD12.5 treatment (139.14), and the VD12.5 and DG10 treatment had
relatively lower value (133.57, 133.81) compared with the OC group
(129.42) (Fig.3a). For ALT, the highest value was found in rats under
the DG10VD12.5 treatment (49.77) ,and the VD12.5 and DG10 treatment had
relatively lower value (45.79, 46.23) compared with the OC group (43.58)
(P < 0.05 for all) (Fig.3b).
3.3 The effects on FAS/CPT1 mRNA expression
The effects of DAG and VD on the expression of genes involved in the
PPARγ/NF-κBp65 pathway are shown in Fig.4a, Fig.4b. Compared with those
in the NO group, the obesity significantly lowered the CPT1 value (P
< 0.05), but increased the FAS value (P <
0.05). There were no significant differences on FAS and CPT1 between NO
and DG10VD12.5 treatments (P < 0.05). For CPT1 mRNA,
the highest value was found in the rats under the DG10VD12.5 treatment
(0.90) compared with the OC group (0.46) (Fig.4a). For FAS mRNA, the
rats under the DG10VD1.5 treatment had the lower value (1.15) compared
with the OC group (1.61) (Fig.4b).
3.4 The correlation between FAS/CPT1 mRNA expression, lipid metabolism,
and metabolism enzyme activity
The correlation coefficients between the FAS/CPT1 mRNA expression and
the metabolism enzyme activity in the liver, and lipids in serum are
listed in Table 2. The FAS mRNA expression was highly and positively
correlated with the TC, TG, and LDL concentrations in serum (P
< 0.01), but negatively correlated with the ALT and AST
activity (P < 0.01). The CPT1 mRNA was highly and negatively
correlated with the TC, LDL (P < 0.01), and TG concentrations
(P<0.05), and positively correlated with the ALT and AST
activity (P < 0.01).
3.5
Effects
on microbiota in the caecum
The diversity indices of microbiota, such as operational taxonomic units
(OUTs), ACE index, Chao 1 index, Simpson index and Shannon index, in the
caecal content are shown in
Fig.
5a - 5e. The OC group had significant lower values of the diversity
indices compared with those for the other four groups (P <
0.05). For OTUs, the high-fat diet reduced OTUs compared with the NO
group, and the DG10VD12.5 treatment (459.33) had highest values compared
with the OC group (233.00) (Fig.5a). For ACE index, the high-fat diet
reduced ACE index compared with the NO group, and the DG10VD12.5
treatment (421.46), the DG10 treatment (393.81) and the VD12.5 treatment
(372.71) had higher values compared with the OC group (263.73) (Fig.5b).
For Chao 1 index, the high-fat diet reduced Chao1 index compared with
the NO group, and the DG10VD12.5 treatment (423.38) had highest values
compared with the OC group (264.79) (Fig.5c). No significant differences
in Simpson (Fig.5d) and Shannon (Fig.5e) indices for the microbiota were
found between the NO group and the DG10VD12.5 treatment (P
> 0.05).
The difference of the microbiota composition was explored using the NMDS
analysis. Fig. 6 is the result of the NMDS analysis. The Stress value
lesser than 0.2 indicates that the NMDS analysis has certain
reliability. The distance between the OC group and the NO group was the
largest, indicating a significant difference in microbial composition.
The distance between DG10VD12.5 treatment and the OC group was closer to
that between VD12.5 or DG10 treatment and the OC group.
The microbiota taxonomic distribution at the genus level is shown in
Fig. 7a - Fig. 7f. Obesity
significantly reduced the abundances of Bacteroides andLactobacillus, whereas increased Allobaculum ,Desulfovibrio , Lachnospiraceae , and Alloprevotellagenera compared with those for the NO group (P < 0.05).
In general, the additions of VD or DAG or VD plus DAG increased the
abundances of Bacteroides and Lactobacillus , while reduced
the abundances ofLachnospiraceae ,Alloprevotella , Desulfovibrio , and Allobaculum . ForLactobacillus , the highest value was found in rats under the
VD12.5 treatment (0.159) ,and the DG10VD12.5 treatment had relatively
lower value (0.107) compared with the OC group (0.023) (P <
0.05 for all) (Fig.7d). For the other five bacterial genera, the best
effects were found in rats under the DG10VD12.5 treatment (0.051, 0.066,
0.014, 0.034, and 0.01) compared with the OC group (0.110, 0.013, 0.032,
0.051, and 0.066) (Fig.7a, 7b, 7c, 7e and 7f).
The changes in the microbiota composition were also explored using the
Line Discriminant Analysis Effect Size (LEfSe) analysis. Fig. 8a and
Fig. 8b are the results of the LDA analysis and evolutionary branch
diagram. With the LDA threshold set at 4, a total of twenty groups of
biomarker with statistical differences were detected. The bacterial taxa
with significantly higher abundance in the DG10VD12.5 group wereAcidaminococcaeae and Phascolarctobacterium , while for the
DG10 treatment were Rikenellaceae- RC9-gut, Collinsella ,
and Faecalibacterium . In contrast, Campylobacterales ,Helicobacteraceae , Helicobacter , andEpsilonproteobacteria were higher in the OC group, whereasPrevotellaceae -UCG-003, Prevotella -2, Treponema -2,Runminococcaceae -UCG-005, Selenomonadales ,Veillonellaceae , Negativicutes, Spirochaetales andRoseburia were higher in the NO group.