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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (2): 158-167.doi: 10.3736/jcim20100211

• Original Experimental Research • Previous Articles     Next Articles

Effects of Yiguanjian Decoction on liver cirrhosis formation： A differential proteomics study in rats

 Ding-zhu Shena,b, Qing Taoa,b, Jin-xing Dua,b, Sai-dan Dinga,b, Gao-feng Chena,b, Yi-yang Rua,b, Ping Liub,c   

  1. a Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    b Key Laboratory for Liver-Kidney Disease Pattern of Ministry of Education, Shanghai 201203, China
    c E-institute of Traditional Chinese Internal Medicine, Shanghai Municipal Education Commission, Shanghai 201203, China
  • Received:2009-10-29 Accepted:2009-12-20 Online:2010-02-20 Published:2010-02-15
  • Contact: Ping LIU E-mail:liuliver@online.sh.cn

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Objective

To investigate the effects of Yiguanjian Decoction, a compound traditional Chinese herbal medicine, on rats with cirrhosis based on the method of differential proteomics.
Methods

Wistar male rats (n=48) were randomly divided into normal control group (n=12) and model-making group (n=36). Rat cirrhosis model was established by intraperitoneal injection of 50% carbon tetrachloride (CCl4) plus olive oil solution (1 mL/kg, twice weekly for 9 weeks). After 3- and 6-week injection, 6 rats each time were sacrificed for dynamic observation before medicine intervention, and the 24 remained rats were randomly divided into untreated group (n=12) and Yiguanjian Decoction group (n=12) at the first day of the 7th week. All animals were sacrificed by the end of the 9th week, and total protein of liver tissue was isolated by two-dimensional gel electrophoresis (2-DE); some differentially expressed protein spots were analyzed and identified by matrix-assisted laser desorption/ionization two-stage time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and database querying. Protein expressions of Cu/Zn superoxide dismutase (Cu/Zn SOD) and DJ-1 were validated by Western blot and immunohistochemical methods.
Results

2-DE maps with high resolution and good repeatability were obtained. In all 50 protein spots identified by MALDI-TOF/TOF-MS and database querying, there were 5 protein spots related to oxidative stress named Cu/Zn SOD, DJ-1, glutathione synthetase, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 respectively. Compared with the normal control group, expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the untreated group were decreased significantly. Expressions of Cu/Zn SOD and aldo-keto reductase family 7, A2 were decreased time-dependently. Compared with the untreated group in 9th week, protein expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the Yiguanjian Decoction groups were increased significantly while expression of glutathione synthetase was decreased notably. Western blot and immunohistochemical results of Cu/Zn SOD and DJ-1 expressions coincided with proteomics results.
Conclusion

Anti-oxidative depression is a key pathological change of cirrhosis induced by CCl4 in rats, and increasing expression of proteins related to anti-oxidative stress may be a major mechanism of Yiguanjian Decoction in treating cirrhosis induced by CCl4 effectively.

Key words: Liver cirrhosis, Carbon tetrachloride, Yiguanjian Decoction, Proteomics, Cu/Zn superoxide dismutase, DJ-1, Rats

Table 1

Liver function and content of Hyp in liver tissues of rats in different groups ($\bar{x}±s$)"

Group n ALT (U/L) AST (U/L) Alb (g/L) GGT (U/L) TBiL
(μmol/L)		 Hyp (μg/g
wet liver)
Normal control 12 33.0±5.5 60.0±17.2 32.60±0.92 21.40±3.65 8.63±0.57 135.29±16.01
Untreated
3rd week 6 106.0±18.2* 107.0±18.3* 30.30±1.13* 31.80±9.77* 10.72±0.92* 284.32±123.76*
6th week 6 156.0±28.3*△ 158.0±23.4*△ 29.10±0.52*△ 43.40±12.18*△ 13.03±1.10*△ 412.13±173.78*△
9th week 12 256.0±23.5*▲ 397.0±48.3*▲ 26.20±1.14*▲ 74.40±18.97*▲ 18.56±2.05*▲ 697.68±190.15*▲
Yiguanjian 12 204.0±21.7□ 320.0±55.0□ 29.80±0.88□ 43.50±8.89□ 13.27±1.67□ 527.93±166.04□
Decoction

Figure 1

Histological changes in liver tissues of rats in different groups observed by light microscopy (HE staining, ×200) A: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group."

Figure 2

Histological changes in liver tissues of rats in different groups observed by light microscopy (Sirius red staining, ×100)"

Figure 3

2-DE maps of protein expressions in liver tissues in different groups A: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group.Spots of the differential expressions of five proteins related to oxidative stress are labeled with arrows. 1: Cu/Zn SOD; 2: DJ-1; 3: Glutathione synthetase; 4: Glutathione S-transferase Yb-1 subunit; 5: Aldo-keto reductase family 7, member A2."

Figure 4

Histograms of corrected optical density value and close-up images for spots of the differential expressions of five proteins related to oxidative stress in different groupsA: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group. The values are represented as x±s. Expressions of Cu/Zn SOD protein in different (A, B, C, D, E) groups were 0.158±0.022, 0.089±0.013, 0.078±0.014, 0.071±0.009, and 0.160±0.027 respectively. Expressions of DJ-1 protein in different (A, B, C, D, E) groups were 0.202±0.019, 0.148±0.016, 0.145±0.018, 0.141±0.013 and 0.223±0.021 respectively. Expressions of glutathione synthetase protein in different (A, B, C, D, E) groups were 0.061±0.008, 0.097±0.011, 0.098±0.013, 0.104±0.012 and 0.059±0.007 respectively. Expressions of glutathione S-transferase Yb-1 subunit protein in different (A, B, C, D, E) groups were 0.142±0.012, 0.101±0.008, 0.097±0.009, 0.095±0.009 and 0.124±0.013 respectively. Expressions of aldo-keto reductase family 7, member A2 protein in different (A, B, C, D, E) groups were 0.086±0.007, 0.047±0.006, 0.037±0.004, 0.033±0.004 and 0.079±0.008 respectively. *P<0.05, vs normal control group;□P<0.01, vs 9th week of untreated group."

Figure 5

Expressions of Cu/Zn SOD and DJ-1 proteins in liver tissues in different groups observed by Western blottingA: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group. Quantitative data were normalized to the intensity for GAPDH. The values are represented as x±s, n=6. Expressions of Cu/Zn SOD protein in different (A, B, C, D, E) groups were 0.32±0.08, 0.21±0.07, 0.18±0.05, 0.13±0.04, and 0.46±0.06 respectively. Expressions of DJ-1 protein in different (A, B, C, D, E) groups were 1.35±0.14, 0.79±0.11, 0.56±0.07, 0.38±0.08 and 0.95±0.08 respectively. *P<0.05, vs normal control group; △P<0.05, vs 3rd week of untreated group; ▲P<0.05, vs 6th week of untreated group; □P<0.05, vs 9th week of untreated group."

Figure 6

Expression of Cu/Zn SOD in liver tissues in different groups observed by immunohistochemical method (Light microscopy, ×200)A: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group."

Figure 7

Expression of DJ-1 in liver tissues in different groups observed by immunohistochemical method (Light microscopy, ×200)A: Normal control group; B: Untreated group after 3-week injection of CCl4 plus olive oil; C: Untreated group after 6-week injection of CCl4 plus olive oil; D: Untreated group after 9-week injection of CCl4 plus olive oil; E: Yiguanjian Decoction group."

Table 2

Semi-quantitation of expressions of Cu/Zn SOD and DJ-1 in liver tissues of rats in different groups ($\bar{x}±s$)"

Group n Cu/Zn SOD DJ-1
Normal control 6 9.58±0.52 7.31±0.62
Untreated
3rd week 6 7.46±0.47* 2.72±0.58*
6th week 6 5.73±0.49△ 2.65±0.46*
9th week 6 2.78±0.38▲ 2.63±0.32*
Yiguanjian Decoction 6 4.85±0.34□ 3.85±0.52□
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