3.1 Computational analysis of the binding between anthocyanins and CerS6
Docking simulations were used to study two possible anthocyanin binding sites on CerS6. The combination of the model during docking was shown in Fig. 1. Anthocyanins may bind to CerS6, and the binding region was shown in Fig. 1.From the results of molecular docking analysis, the amino acids composing the binding site of CerS6 and delphinidin 3-O-rutinoside are:(LYS-125,PRO-126, GLU-344,LYS-125,THR-128),binding energy of delphinidin 3-O-rutinosid is -5.2 kcal/mol; the binding site of CerS6 and cyaniding3-O-rutinoside are:(GLN-73, GLN-75,GLN-119,ASN-72,GLY-73,PRO-74),binding energy of cyanidin3-O-rutinoside is -8.2 kcal/mol.
3.2 Effects of GA on body weight in HFD mice
GA intervention was given to mice at the same time of intragastric administration of high-fat emulsion. The weight changes during the 8-week experimental period were shown in Table 4. From the initial body weight before death, the average body weight of mice in the CTL group from 31.24 ± 1.05 g to 44.17 ± 1.87 g, HFD group from 30.99 ± 1.14 g to 53.73 ± 2.24 g. There are significant differences in body weight between CTL group, HFD group and GA group,It shows that HFD can effectively promote the weight gain of mice, and GA can alleviate the weight gain of mice. It could be seen in Fig. 2that the weight growth rate of mice in HFD group was 1.76 times that of CTL group, the weight growth rate was significantly increased,the weight growth rate of mice in LGA and HGA groups was significantly lower than that in HFD group.
Table 4
Body weight changes of mice in each group (n = 10,x ± s)The unit is g
Group | | Time(week) | |
0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
CTL | 31.24 ± 1.05 | 34.93 ± 0.97 | 36.17 ± 1.23 | 37.89 ± 1.42 | 40.13 ± 1.53 | 41.03 ± 1.49 | 42.27 ± 1.66 | 43.43 ± 1.75 | 44.17 ± 1.89 |
HFD | 30.99 ± 1.14 | 34.51 ± 1.02 | 38.84 ± 1.81 | 46.57 ± 0.97 | 48.24 ± 2.05 | 45.81 ± 2.06 | 49.13 ± 1.59 | 50.63 ± 1.85 | 53.73 ± 2.24 |
LGA | 30.62 ± 0.87 | 35.40 ± 1.13 | 39.03 ± 1.46 | 42.73 ± 1.67 | 45.56 ± 1.68 | 47.51 ± 2.04 | 49.20 ± 1.89 | 49.97 ± 2.48 | 50.57 ± 2.32 |
HGA | 31.37 ± 0.96 | 34.79 ± 0.79 | 37.92 ± 1.56 | 42.03 ± 1.47 | 44.77 ± 1.36 | 46.43 ± 1.59 | 47.56 ± 2.18 | 48.21 ± 1.91 | 49.02 ± 2.23 |
EGCG | 32.16 ± 0.62 | 35.41 ± 0.86 | 38.43 ± 1.46 | 41.24 ± 1.68 | 44.13 ± 1.81 | 45.33 ± 1.95 | 46.83 ± 1.79 | 47.17 ± 2.06 | 47.82 ± 1.73 |
3.3 Effects of GA on visceral fat and liver index in HFD mice
As shown in Fig. 3 (A), the visceral fat of HFD group was 1.81g, which was significantly higher than that of CTL group (0.74g). The visceral fat of high and low dose GA group decreased compared with HFD group, and the visceral fat value of HGA group decreased to 1.29g, indicating that high fat emulsion could promote the accumulation of adipose tissue in mice, and GA could slow down the accumulation of adipose tissue in HFD mice.
The liver index of CTL group was 3.57% as shown in Fig. 3(B). The liver index of the HFD group was 5.35%, significantly higher than CTL group. Compared with the HFD group, the intervention of EGCG group and HGA group significantly reduced the liver index by 4.75% and 4.69%, and the effect was better than that of the LGA group. It is suggested that high-fat emulsion could increase the liver index of mice, and GA could reduce the liver index of high-fat mice in a dose-dependent manner.
3.4 Effect of GA onserum biochemical indexes in HFD mice
As shown in Fig. 4, the contents of TC and TG in the serum of mice in HFD group were higher than those in CTL group. The contents of TC and TG in serum of HGA group and LGA group were decreased in varying degrees, which indicated that GA could effectively reduce the content of TC and TG in serum of mice. The content of LDL-C in serum of mice in HFD group was higher than that in CTL group, and the content of HDL-C in serum in HFD group was lower than that in CTL group. The content of LDL-C in serum of HGA and LGA group showed a decreasing trend compared with that of HFD group. Compared with HFD group, the content of HDL-C in serum of HGA and LGA group showed an upward trend. The results showed that GA could reduce the content of LDL-C and increase the content of HDL-C in serum of HFD mice.
3.5 Effects of GA on oxidative stress related enzyme activity in HFD mice
As shown in Fig. 5, ,the levels of SOD and GSH-Px are related to oxidative stress. The decrease of SOD and GSH-Px levels indicates the decrease of antioxidant capacity, which will lead to the increase of ROS and oxidative stress. The levels of SOD and GSH-Px in CTL group were 106.69U/mL and 136.44U/mL. Oxidative damage occurred in the liver of mice in HFD group, and the levels of SOD and GSH-Px in HFD group mice decreased significantly to 69.49U/mL and 96.74 U/Ml. HGA group returned to 105.86U/mL and 117.07U/mL, which were significantly higher than those in HFD group. The results showed that GA could significantly increase the levels of SOD and GSH-Px in hyperlipidemic mice, and the oxidative stress indexes of HGA group and EGCG group were close to those of CTL group. The results showed that GA could alleviate oxidative stress in mice in a dose-dependent manner.
3.6 Effects of GA on insulin sensitivity and insulin resistance index in HFD mice
As shown in Fig. 6, after 8 weeks of GA intervention, the level of ISI in HFD group was significantly lower than that in CTL group. With the increase of GA concentration, the level of ISI increased, and the level of ISI in HGA group increased to-4.54, which increased significantly. It is proved that the intervention of GA can effectively improve the insulin sensitivity of mice. At the same time, the HOMA-IR level of CTLmice was 2.47, and the HOMA-IR level of HFD mice was 6.57, which was significantly higher than that of CTL group, which proved that HFD mice produced IR successfully. With the increase of GA concentration, the IR index decreased significantly, and the HOMA-IR level of HGA group decreased to 5.75. The results showed that GA could alleviate IR in mice.
3.7 Effect of GA on liver morphology in HFD mice
As shown in Fig. 7, the hepatocytes in the CTL group arranged regularly, there were almost no fat vacuoles and steatosis in the visual field, the structure was normal, and there were no obvious pathological changes; in the HFD group, the hepatocytes were accompanied by a certain degree of steatosis, irregular structural arrangement, steatosis, enlarged hepatocytes, obvious intracellular fat infiltration and fat vacuoles of different sizes, resulting in hepatocyte degeneration. After intervention of GA, the liver cells of the intervention group were significantly improved compared with HFD group, and the degree of steatosis of liver cells was alleviated, which was close to the level of the CTL group. The results showed that GA could improve the fat accumulation in liver of HFD mice.
3.8 Effects of GA on the expression of inflammatory cytokines in HFD mice
It could be seen from Fig. 8, the mRNA expression of IL-6 and TNFα in liver tissue of HFD group was higher than that of CTL group. Compared with HFD group, the mRNA expression of IL-6 and TNFα in EGCG and GA groups showed a downward trend, indicating that GA can effectively down-regulate the mRNA expression of IL-6 and TNFα in liver tissue of HFD mice, reduce inflammation in HFD mice
3.9 GA on S1P/Cer signaling pathway protein in liver tissue of HFD mice
S1P-SP-Cer metabolisminfluence insulin signaling via Akt phosphorylation andCD36(Fig. 9).As shown in Fig. 10,compared with the CTL group, the expression of CerS6 protein and the downstream protein PKCζ in the liver of the HFD group were significantly increased, and the phosphorylation of the downstream protein Akt and p-Akt/Akt were significantly inhibited. Compared with the HFD group, GA intervention could significantly promote the phosphorylation of Akt and significantly increase the expression of p-Akt/Akt, and significantly inhibit the expression of CerS6 and PKCζ proteins.
It could be seen from Fig. 11,compared with the CTL group, the expression of SphK2 protein in the liver of the HFD group was significantly decreased, and the expression of PPARγ and its downstream proteins CD36 was significantly increased; the expression level of insulin receptor(ISR) was significantly down-regulated. Compared with the HFD group, GA intervention could significantly up-regulate the expression of SphK2 protein and significantly inhibit the expression levels of PPARγ and CD36 proteins, while GA intervention could significantly up-regulate the expression of ISR.