Proteomics study on the effect of silybin on cardiomyopathy in obese mice

Abstract

Due to the increase in the number of obese individuals, the incidence of obesity-related complications such as cardiovascular disease and type 2 diabetes is higher. The aim of the present study was to explore the effects of silybin on protein expression in obese mice. Firstly, serum was collected, and it was used to detect serum lipids and other serological indicators. Secondly, total protein from epididymal adipose tissue was extracted for differential expression analysis by quantitative tandem mass tag (TMT) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by bioinformatics and protein-protein interaction (PPI) network analyses of these proteins. Lastly, real-time polymerase chain reaction (RT-PCR) and parallel reaction monitoring (PRM) were used to further validate the expression of identified differentially expressed proteins (DEPs) at the mRNA and protein level, respectively. The results revealed that silybin could improve abnormal lipid metabolism caused by the high fat diet in obese mice. A total of 341, 538 and 243 DEPs were found in the high fat/control (WF/WC), silybin/high fat (WS/WF) and WS/WC groups, respectively. These DEPs mainly participated in lipid metabolism and energy metabolism. Notably, tropomyosin 1 (TPM1), myosin light chain 2 (MYL2), myosin heavy chain 11 (MYH11) and other DEPs were involved in hypertrophic cardiomyopathy, dilated cardiomyopathy and other pathways. Silybin could protect cardiac function by inducing the protein expression of TPM1, MYL2 and MYH11 in the adipose tissue of obese mice.

Figure 1

Concentrations of (A) total cholesterol, (B) triglycerides, (C) low-density lipoprotein-cholesterol and (D) high-density lipoprotein-cholesterol in the WC, WF and WS groups. *P < 0.05 WF vs. WC, ^#P < 0.05 WS vs. WF, ^&P < 0.05 WS vs. WC.

Figure 2

Volcano plots of differentially expressed proteins in the (A) WF/WC, (B) WS/WF and (C) WS/WC groups. The horizontal axis is the relative quantitative value of protein after log2 conversion, and the vertical axis is the value of the P-value after − log10 conversion. Red and blue dots indicates significantly up-regulated and down-regulated proteins, respectively.

Figure 3

Histogram of the distribution of differentially expressed proteins in the WF/WC, WS/WF and WS/WC groups. Red and blue indicate significantly up-regulated and down-regulated proteins, respectively.

Figure 4

Gene Ontology functional analysis of differentially expressed proteins in the (A) WF/WC, (B) WS/WF and (C) WS/WC groups. Green, red and purple represent biological process, cellular component and molecular function, respectively.

Figure 5

Enriched bubble charts of Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially expressed proteins in the (A) WF/WC, (B) WS/WF and (C) WS/WC groups.

Figure 6

Signaling pathways of hypertrophic cardiomyopathy, dilated cardiomyopathy and cardiac muscle contraction. Diagrams created by Kanehisa Laboratories and used with permission.

Figure 7

Protein–protein interaction network of differentially expressed proteins in the (A) WF/WC, (B) WS/WF and (C) WS/WC groups. Red circle represents the interaction network between tropomyosin 1 (TPM1), myosin light chain 2 (MYL2) and myosin heavy chain 11 (MYH11).

Figure. 8

Reverse transcription-quantitative PCR validation of myosin heavy chain 11 (MYH11), tropomyosin 1 (TPM1) and myosin light chain 2 (MYL2) in adipose tissue samples of WC, WF and WS groups. (A) The mRNA expression of MYH11 in the group of WC, WF and WS; (B) the mRNA expression of TPM1 in the group of WC, WF and WS; (C) the mRNA expression of MYL2 in the group of WC, WF and WS. *P < 0.05 WF vs. WC, ^#P < 0.05 WS vs. WF, ^&P < 0.05 WS vs. WC.

Figure 9

Electronic verification of differentially expressed proteins at the mRNA level. (A) The mRNA expression result of myosin heavy chain 11 (MYH11); (B) The mRNA expression result of tropomyosin 1 (TPM1). Control and case groups represent the standard group and the high-fat and high-energy feed groups, respectively. P < 0.05 was considered to indicate a statistically significant different.

Figure 10

Proteomics verification of the distribution of ion peak area of myosin heavy chain 11 (MYH11) protein in the WC, WF and WS groups. Different colors represented different ion peak areas. In the WF group, the protein levels of MYH11 were significantly decreased. When silybin was added, the protein levels of MYH11 in the WS group increased.