The Study Of Natural Hirudin’s Inhibitory Effect And Its Mechanism On Fibroblast Cell Of Tendon

Objective In the process of using medicine to prevent and cure the tendon’s adhesion, the traditional Chinese medicine as well as its extractive gain lots of attention and studies because of their distinct effect. As a kind of traditional Chinese medicine, the hirudin can promote blood circulation, so people broadly study the hirudin to inherent the proliferation of all kinds of fibroblast. Especially when the hirudin is used in healing hypertrophic scar after burn, it has the distinct curative effect. We study the inhibitory effect and mechanism of the hirudin on the fibroblast in order to provide with the accurate and objective evidence.then bring the light to the mechanism about hirudin’s curative function on tendon adhesion. We also provide with the animal experiment basis for the study of the inhibitory effect of hirudin on the fibroblast, which is beneficial to the cure of tendon’s postoperative adhesion using Chinese medicine and its preparation in clinical.Methods The tendon fibroblast of rabbit were obtained through primary culture. The tendon fibroblasts of rabbit were cultured in vitro with different concentration hirudin:0U/ml,1U/ml,10U/ml,100U/ml. Then we observed the morphological changes of the fibroblast. We detected the proliferation activity of the cell by MTT method and observed the inhibitory effect of different concentration hirudin to tendon fibroblast’s proliferation. We also detected the expression of transforming growth factor beta 1(TGFβ1), basic fibroblast growth factor(bFGF) and matrix metalloproteinase 2(MMP-2) by enzyme-linked immunosorbent assay(ELISA), in order to study the mechanism of the hirudin’s inhibitory effect on tendon fibroblast.Results1. When the concentration of hirudin was 1U/ml, 10U/ml, 100U/ml, the absorbance (A) of tendon fibroblast detected by MTT method was 0.42±0.05,0.41±0.28,0.35±0.26 respectively, all of which were lower than the control group (OU/ml):0.52±0.17. The difference was statistically significant comparing to control group (P<0.05).2. When the concentration of hirudin was 1U/ml,10U/ml,100U/ml, the concentration of TGF(31 in tendon fibroblast measured by ELISA method was 289.50±25.60pg/ml, 269.56±29.34 pg/ml,173.25±56.47 pg/ml respectively, all of which were lower than the control group (OU/ml):398.89±46.70pg/ml. The difference was statistically significant comparing to control group (P<0.05).3. When the concentration of hirudin was 1U/ml, 10U/ml,100U/ml, the concentration of bFGF in tendon fibroblast measured by ELISA method was 114.50±84.95pg/ml, 117.17±79.12 pg/ml,273.83± 142.88 pg/ml respectively, all of which were higher than the control group (OU/ml):80.40±59.96 pg/ml. The difference was statistically significant comparing to control group (P<0.05).4. When the concentration of hirudin was 1U/ml,10U/ml,100U/ml, the concentration of MMP-2 in tendon fibroblast measured by ELISA method was2500.75±566.13pg/ml, 2580.57±534.90 pg/ml,4956.00±792.35 pg/ml respectively, all of which were higher than the control group (OU/ml):1780.50±328.40 pg/ml. However, these hirudin with lower concentration (1U/ml,10U/ml) had weaker promote effect to MMP-2, so these two groups had no statistically significance comparing to control group(P>0.05). But the hirudin with higher concentration(100U/ml) had stronger promote effect to MMP-2, so this group had statistically significance comparing to control group (P<0.05).Conclusion1. The natural hirudin can inhibit the proliferation of the rabbit’s tendon fibroblast cultured in vitro, With increasing the concentration of hirudin, the inhibitory effect was stronger.2.The natural hirudin can inhibit the expression of the TGFβ1 in rabbit’s tendon fibroblast and can promote the expression of the bFGF and MMP-2; With increasing the concentration of hirudin, the inhibitory effect to TGFβ1 and the promote effect to bFGF were both stronger; As for the promote effect to MMP-2, the hirudin with high concentration (100U/ml) was more obvious, But the promote effect to MMP-2 of the hirudin with low concentration (1U/ml, 10U/ml) was weaker.3. the natural hirudin can inhibit tendon fibroblasts proliferation significantly, so it has broad prospects in the prevention of tendon adhesion in clinical.