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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (11): 1286-1292.doi: 10.3736/jcim20121113

• Original Experimental Research • Previous Articles     Next Articles

Effects of Chinese herbal medicine Xiaopi Pill in preventing rats from dimethylnitrosamine-induced liver fibrosis

Xiao Zhang1, Bing-bing Ning1, Shuang Ren1, Li-jun Zhang1, Wen-meng Zhang2, Jia-mei Chen1, Gao-feng Chen1, Hua Zhang1, Yong-ping Mu1, Ping Liu1,3()   

  1. 1. Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    2. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province, China
    3. E-Institute of Shanghai Municipal Education Commission, Shanghai Municipal Education Commission, Shanghai 201203, China
  • Received:2012-06-01 Accepted:2012-06-28 Online:2012-11-20 Published:2018-11-15

Objective: To explore the intervention effects of Xiaopi Pill (XPW), a compound traditional Chinese herbal medicine, on the development progress of dimethylnitrosamine (DMN)-induced liver fibrosis in rats.

Methods: Liver fibrosis model was established by intraperitoneal injection of 0.5% DMN 2 mL/kg thrice a week for 4 weeks. Rats were divided into control group given saline and treatment group given XPW during the 3rd week of DMN injections. Rats were sacrificed at the end of the experiment, and then liver histological changes, liver function and mRNA expression of the liver fibrosis-associated markers were observed.

Results: (1) At the end of the 2nd and 4th weeks of DMN injection, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increased significantly in rats (P<0.01 orP<0.05); content of total bilirubin (TBil) increased significantly compared with the normal group until the end of the 4th week (P<0.05); compared with the model group after 4 weeks of DMN injection, the serum levels of ALT, AST, ALP and TBil were decreased remarkably in the XPW-treated group (P<0.01 or P<0.05). (2) The hepatic inflammation and collagen deposition in hepatic tissues increased by different degrees in experimental rats. Parts of pathological changes in the rat liver were found at the end of the 4th week, including a complete round structure of false flocculus round, meantime, the hydroxyproline content of hepatic tissue was increased significantly at the end of the 2nd and 4th weeks (P<0.05). Compared with the 4-week model group, the hepatic inflammation, collagen deposition and hydroxyproline content in hepatic tissues were alleviated dramatically (P<0.05). (3) Compared with the normal and 2nd week groups, protein expression of alpha-smooth muscle actin (α-SMA) was gradually increased, and that of the 4th week group were aggrandized significantly (P<0.01). Compared with the normal group, the mRNA expression of α-SMA, transforming growth factor-β1 (TGF-β1), tissue inhibitor of melalloproteinase-1 (TIMP-1), and heme oxygenase-1 (HO-1) was gradually increased. Further changes in above-mentioned abnormalities were found in the model rats at the end of the 4th week (P<0.01); while compared to the 4th week group, protein and mRNA levels of α-SMA and mRNA levels of TGF-β1, TIMP-1, and HO-1 were decreased significantly in the XPW group (P<0.01 or P<0.05).

Conclusion: Progressive DMN-induced liver fibrosis in rats can be suppressed by XPW; the mechanism may be associated with inhibition of the activated hepatic stellate cells.

Key words: drugs,Chinese herbal, liver cirrhosis, experimental, dimethylnitrosamine, liver function tests, hydroxyproline, rats

Table 1

Primer sequences of real-time polymerase chain reaction"

Primer name Sequence Note
α-SMA Forward 5′-CGAGAGGACGTTGTTAGCATAGAG-3′ SYBR Green
Reverse 5′-GGGCATCCACGAAACCA-3′
TGF-β1 Forward 5′-GCCTGAGTGGCTGTCTTTTG-3′ SYBR Green
Reverse 5′-ACCTCGACGTTTGGGACTG-3′
HO-1 Forward 5′-CGTGGCAGTGGGAATTTATG-3′ SYBR Green
Reverse 5′-AGGCTACATGAGACAGAGTTCACA-3′
GAPDH Forward 5′-CCTCTATGCCAACACAGT-3′ SYBR Green
Reverse 5′-AGCCACCAATCCACACAG-3′
TNF-α Forward 5′-GCTCCCTCTCATCAGTTCCATG-3′ SYBR Green
Reverse 5′-TACGGGCTTGTCACTCGAGTTTTG-3′

Figure 1

Pathological changes of liver tissues (a): Photographs of hematoxylin-eosin staining (Light microscopy, ×200); (b): Photographs of Sirius red staining (Light microscopy, ×100). A: Normal group; B: 2-week model group; C: 4-week model group; D: Xiaopi Pill group."

Table 2

Liver function of rats in different groups (Mean±standard deviation)"

Group n ALT (IU/L) AST (IU/L) ALP (IU/L) TBil (mmol/L)
Normal 10 42.00±5.03**△△ 112.70±15.41**△△ 136.10±23.56*△△ 7.45±1.37
Two-week model 6 101.17±13.35 160.50±26.97 248.50±49.91 8.25±3.34
Four-week model 13 120.38±31.58 156.15±38.8 377.15±127.31**△△ 12.56±7.80
Xiaopi Pill 13 80.54±15.65 121.85±16.66△△ 248.38±66.81△△ 6.90±2.11

Table 3

Hydroxyproline content of liver tissues in different groups (Mean±standard deviation)"

Group n Hydroxyproline content (μg/g)
Normal 10 167.51±25.87**△△
Two-week model 6 217.76±36.79
Four-week model 13 390.74±51.34**
Xiaopi Pill 13 262.57±49.22

Table 4

Degree of collagen fiber distribution of liver fibrosis of rats in each group"

Group n Distribution of staging of liver fibrosis (n)
0 Ridit value
Normal 10 10 0 0 0 0 0.05△△
Two-week model 6 0 5 1 0 0 0.33△△
Four-week model 13 0 0 10 3 0 0.73
Xiaopi Pill 13 0 10 3 0 0 0.35△△

Figure 2

Protein expression of α-SMA in liver tissues (a) Expression of α-SMA protein observed by immunohistochemical method (Light microscopy, ×200). (b) Expression of α-SMA protein observed by Western blotting. (c) The gray-level score of Western blotting results. △△P<0.01, vs four-week model group. A: Normal group; B: 2-week model group; C: 4-week model group; D: Xiaopi Pill group; α-SMA: alpha-smooth muscle actin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase."

Figure 3

mRNA expressions of α-SMA, TGF-β1, TIMP-1 and HO-1 in liver tissues △P<0.05, △△P<0.01, vs four-week model group. A: Normal group; B: 2-week model group; C: 4-week model group; D: Xiaopi Pill group. α-SMA: alpha-smooth muscle actin; TGF-β1: transforming growth factor-β1; TIMP-1: tissue inhibitor of metalloproteinase-1; HO-1: heme oxygenase-1."

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