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Journal of Chinese Integrative Medicine ›› 2009, Vol. 7 ›› Issue (1): 65-69.doi: 10.3736/jcim20090110

• Original Experimental Research • Previous Articles     Next Articles

Effects of ligustrazine injection on high glucose-induced type Ⅰ collagen, matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expressions in human peritoneal mesothelial cells in vitro

 Gui-song Zhu, Jin-song He   

  1. Department of Nephrology, the Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu Province 210008, China
  • Received:2008-06-18 Accepted:2008-07-22 Online:2009-01-20 Published:2009-01-15

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Objective

To investigate the effects of ligustrazine injection on type Ⅰ collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expressions in human peritoneal mesothelial cells (HPMCs) cultured in high glucose conditions.
Methods

 HPMCs were isolated from human omenta by trypsin digestion method and subcultured. Then, the HPMCs were divided into normal control group, high glucose group and high glucose plus low-, medium- and high-dose ligustrazine (10, 20 and 40 mg/L ligustrazine respectively) groups. Semi-quantitative reverse transcription-polymerase chain reaction was used to detect the expressions of type Ⅰ collagen, MMP-1 and TIMP-1 mRNAs in HPMCs. Proteins of type Ⅰ collagen, MMP-1 and TIMP-1 in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Cell protein concentration was measured by trace bicinchoninic acid method to correct the ELISA assay results.
Results

Ligustrazine injection could significantly decrease high glucose-induced type Ⅰ collagen and TIMP-1 expressions in a dose-dependent manner both in protein and gene levels (P<0.05, P<0.01). In addition, medium- and high-dose ligustrazine injection could significantly increase MMP-1 expression which was inhibited by high glucose concentrations (P<0.05).
Conclusion

 Ligustrazine injection does not only decrease type Ⅰ collagen synthesis, but also promote its degradation by modulating unbalanced MMP-1/TIMP-1 expression in HPMCs cultured in high glucose conditions.

Key words: Ligustrazine injection, Human peritoneal mesothelial cells, High glucose, Type I collagen, Matrix metalloproteinase-1, Tissue inhibitor of metalloproteinase-1, In vitro

Table 1

The primer sequences and PCR conditions of type Ⅰ collagen, MMP-1, TIMP-1 and β-actin"

Target gene Primer sequence Fragment size (bp) PCR condition
Type Ⅰ collagen forward 5'-TCC AAA GGA GAG AGC GGT AA-3' 693 95 ℃ 2 min, 94 ℃ 30 s, 56 ℃ 30 s, 72 ℃ 1 min, 72 ℃ 10 min, 30 cycles
reverse 5'-GAC CAG GGA GAC CAA ACT CA-3'
MMP-1 forward 5'-CAT CCA AGC CAT ATA TGG ACG TTC C-3' 611 95 ℃ 1 min, 94 ℃ 30 s, 60 ℃ 1 min, 72 ℃ 1.5 min, 72 ℃ 10 min, 32 cycles
reverse 5'-TCT GGA GAG TCA AAA TTC TCT TCG T-3'
TIMP-1 forward 5'-TGC ACC TGT GTC CCA CCC CAC CCA CAG ACG-3' 551 94 ℃ 3 min, 94 ℃ 50 s, 58 ℃ 50 s, 72 ℃ 1 min, 72 ℃ 10 min, 35 cycles
reverse 5'-GGC TAT CTG GGA CCG CAG GGA CTG CCA GGT-3'
β-actin forward 5'-TCG ACA ACG GCT CCG GCA T-3' 241 95 ℃ 3 min, 94 ℃ 40 s, 56 ℃ 30 s, 72 ℃ 1 min, 72 ℃ 10 min, 28 cycles
reverse 5'-AAG GTG TGG TGC CAG ATT TTC-3'

Table 2

Effects of ligustrazine on type Ⅰ collagen, MMP-1 and TIMP-1 protein levels in culture supernatants (x±s, ng/mg protein)"

Group n Type Ⅰ collagen MMP-1 TIMP-1
Normal control 9 1.15±0.40 12.16±0.35 0.73±0.03
High glucose 9 6.63±0.08** 7.75±0.21** 6.69±0.27**
10 mg/L ligustrazine 9 5.99±0.26△ 7.90±0.42 4.98±0.06△△
20 mg/L ligustrazine 9 4.46±0.28△△ 8.95±0.42△△ 3.97±0.09△△
40 mg/L ligustrazine 9 3.40±0.24△△ 10.39±0.35△△ 3.24±0.03△△

Figure 1

Expressions of type Ⅰ collagen, MMP-1 and TIMP-1 mRNAs in different groups A: Normal control group; B: High glucose group; C: 10 mg/L ligustrazine group; D: 20 mg/L ligustrazine group; E: 40 mg/L ligustrazine group."

Figure 2

Semi-quantification of type Ⅰ collagen, MMP-1 and TIMP-1 mRNAs △P<0.05, △△P<0.01, vs high glucose group."

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