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Journal of Chinese Integrative Medicine ›› 2010, Vol. 8 ›› Issue (12): 1165-1173.doi: 10.3736/jcim20101209

• Original Experimental Research • Previous Articles     Next Articles

Effects of Chinese herbal medicine Yiqi Huoxue Formula on TGF-p/smad signal transduction pathway and connective tissue growth factor in rats with renal interstitial fibrosis

Yong-mei LiuaRui-hua Liua,Wen-jun Liua, Li Liub, Zhi-kui Wua,Yu-ying Chena   

  1. Department of Molecular Biology, Guang’nmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
    b Department of Anatomy and Histology, Capital Medical University, Beijing 100069, China
  • Received:2010-05-18 Accepted:2010-08-17 Online:2010-12-20 Published:2010-12-15
  • Contact: Wen-jun Liu E-mail:liuwenjunlyzh@263.net

Objective

 To observe the effects of Yiqi Huoxue Formula (YQHXF), a compound Chinese herbal medicine, on transforming growth factor-β (TGF-β)/smad signal transduction pathway and connective tissue growth factor (CTGF) in rats with renal interstitial fibrosis.

Methods

Unilateral ureteral obstruction (UUO) rat model was established and the rats were randomly divided into 5 groups: untreated group, high-, medium-, and low-dose YQHXF groups and fosinopril sodium group. Another group with sham operation was set as control. All rats were administered with corresponding drugs for 3 weeks. After the last administration, each rat was sacrificed and weighed and the serum was separated for creatinine (Cr) and blood urea nitrogen (BUN) detection. Kidneys of the rats were taken out, and mRNA and protein expressions of TGF-β, smad2, smad7 and CTGF were measured with real-time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting respectively; fibrosis of the kidney tissue was observed with hematoxylin-eosin (HE) staining and Masson trichrome staining.

Results

Compared with sham-operation group, Cr and BUN in serum of UUO groups were increased, while high-dose YQHXF treatment decreased the UUO-induced increase of Cr and BUN levels. HE staining and Masson staining results showed that the renal tubular epithelial cells in untreated group got atrophied; lumens of renal tubules expanded; fibroplastic proliferation and inflammatory cell infiltration were observed in renal interstitium; the number of glomerulus decreased and collagen increased significantly compared with sham-operation group. In the high- and medium-dose YQHXF groups and fosinopril sodium group, the histopathological changes of inflammatory cell infiltration, fibroplastic proliferation, expansion of lumens of renal tubules was improved as compared with the untreated group. The mRNA and protein expressions of TGF-β, smad2 and CTGF in untreated group were higher than those in sham-operation group (P<0.05), and the mRNA and protein expressions of smad7 in untreated group were lower than those in the sham-operation group (P<0.05). Compared with untreated group, high- and medium-dose of YQHXF significantly down-regulated the mRNA and protein expressions of TGF-β, smad2 and CTGF (P<0.01, P<0.05), and up-regulated the mRNA and protein expressions of smad7 (P<0.01, P<0.05).

Conclusions

The mRNA expression of CTGF in UUO rats may be regulated by TGF-β/smad signaling transduction pathway. YQHXF might inhibit the expression of CTGF through down-regulation of TGF-β and smad2 and up-regulation of smad7, thus inhibiting the progression of renal interstitial fibrosis.

Key words: Compounds (TCD), Transforming growth factor-beta, Smad proteins, Connective tissue growth factor, Renal interstitial fibrosis, Rats

Table 1

Forward and reverse primers used for real-time fluorescent quantitative reverse transcription PCR"

Table 2

Results of body weight, Cr and BUN of rats $\overline{x}$±s"

Group n Body weight (g) Cr (jumol/L) BUN (mmol/L)
Sham-opreation 6 332. 20±38. 64 61. 00±6. 14 9. 72±1. 14
Untreated 6 342. 00±31.68 68. 13±10. 40 11.07±1.07
High-dose YQHXF 6 351. 10±23. 15 62. 64±4. 57 10. 27±0. 76
Medium-dose YQHXF 6 354. 00±12. 65 73.46±12. 86 11. 77±2. 14
Low-dose YQHXF 6 346. 33±34. 85 71.52±12. 82 11. 50±1. 69
Fosinopril sodium 6 356. 10±23. 81 68. 31±12. 10 11.46±1.82

Figure 1

Pathological changes of kidney in different groups observed by HE staining (Light microscopy, ×200)A: Sham-operation group; B: Untreated group; C: Fosinopril sodium group; D: High-dose YQHXF group; E: Medium-dose YQHXF group; F: Low-dose YQHXF group."

Figure 2

Pathological changes of kidney in different groups observed by Masson staining (Light microscopy, ×200) A: Sham-operation group; B: Untreated group; C: Fosinopril sodium group; D: High-dose YQHXF group; E: Medium-dose YQHXF group; F: Low-dose YQHXF group."

Table 3

Expressions of TGF-β, smad2, smad7 and CTGF mRNAs in kidney tissue of rats in different groups"

$\overline{x}$±s
Group n TGF-p smad2 smad7 CTGF
Sham-opreation 6 5, IS土0, 72 7, 90 土 1,71 3, 70 土 1,54 18,23 士 7,48
Untreated 6 8,35土3,01 ? 15, 69 土 7,38 ? 1,45 土 0,95 ? 26,28土2,02?
High-dose YQHXF 6 3,54±1,34AA 9,85 土 4,29 3,35±0,48AA 19,91±5,00A
Medium-dose YQHXF 6 3,02±0,47AA 7,40土0,93心 2,06士2,58心 H,18 士 4,37AA
Low-dose YQHXF 6 4,03土1,4私 7,33土2,55么 1,13 士 0,92 U,82±7,32AA
Fosinopril sodium 6 5,〇5±2,〇5A 15,89 土 6,55 2, 61 土0,的 19,10±S,17A

Figure 3

Dissociation curves for β-actin PCR reactions (A) and agarose electrophoresis analysis (B) 1: DNA DL 2 000 Maker; 2: Sham-operation group; 3: Untreated group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group; 7: Fosinopril sodium group."

Figure 4

Dissociation curves for TGF-β PCR reactions (A) and agarose electrophoresis analysis (B) 1: DNA DL 2 000 Maker; 2: Sham-operation group; 3: Untreated group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group; 7: Fosinopril sodium group."

Figure 5

Dissociation curves for smad2 PCR reactions (A) and agarose electrophoresis analysis (B) 1: DNA DL 2 000 Maker; 2: Sham-operation group; 3: Untreated group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group; 7: Fosinopril sodium group."

Figure 6

Dissociation curves for smad7 PCR reactions (A) and agarose electrophoresis analysis (B) 1: DNA DL 2 000 Maker; 2: Sham-operation group; 3: Untreated group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group; 7: Fosinopril sodium group."

Figure 7

Dissociation curves for CTGF PCR reactions (A) and agarose electrophoresis analysis (B) 1: DNA DL 2 000 Maker; 2: Sham-operation group; 3: Untreated group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group; 7: Fosinopril sodium group."

Table 4

Expressions of CTGF, TGF-β, smad2 and smad7 proteins in kidney tissue of rats in different groups"

Figure 8

Protein expressions of CTGF, TGF-β, smad2 and smad7 in renal tissues of rats in different groups 1: Sham-operation group; 2: Untreated group; 3: Fosinopril sodium group; 4: High-dose YQHXF group; 5: Medium-dose YQHXF group; 6: Low-dose YQHXF group."

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