Effect Of Sulodexide On Rat Peritoneal Mesothelial Cells Fibrosis Induced By High Glucose

[Objective]This study is aimed to investigate the effect and the mechanism of sulodexide on rat peritoneal mesothelial cells fibrosis induced by high glucose in vitro.[Methods]The primary culture of rat peritoneal mesothelial cells were derived from rat peritoneum by trypsin digestion, identification of cells was carried on by morphological identification and scan electron microscopy,transmission electron microscopy. MTT test was carried out to determine the safe dose of SLX. Passage 2 of RPMCs were used for experiments. Briefly, RPMCs were placed in various culture media, including control medium, high glucose medium(4.25% glucose), high glucose plus low dose SLX medium (4.25% glucose + SLX 50μg/ml), high glucose plus moderate dose SLX medium(4.25% glucose + SLX 400μg/ml), high glucose plus high dose SLX medium(4.25% glucose + SLX 800μg/ml). TGF-β1,Smad2,CTGF andα-SMA mRNA levels were semi-quantified by RT-PCR. The protein ofα-SMA in RPMCs was tested by immuocytochemistry and semi-quantified by IOD measurement. All the experiments were repeated for 3 times at least.[Results]1. RPMCs were characterized by polygonal shape and showed cobblestone morphological appearance under inverted phase contrast microscope. Numerous microvilli was on the cell surface under scanning electron microscope. There were a large number of rough endoplasmic reticulum, mitochondria and Golgi apparatus in cytoplasm under transmission electron microscope, and weibel palade bodies could not be found.2. MTT test: Different doses of SLX (0μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 400μg/ml, 800μg/ml) were placed in culture media on rat peritoneal mesothelial cells for 48h. There was no significant difference between different doses of SLX in the rate of cell death. So we considered these doses of SLX were safe to rat peritoneal mesothelial cells.3. Compared with control group, the TGF-β1 and Smad2 mRNA expressions were significantly increased in high glucose group(P<0.05), and SLX reduced the increasing TGF-β1,and Smad2 mRNA expressions(P<0.05). There was significant difference between low dose of SLX and moderate or high dose of SLX(P<0.05). But there was no significant difference between moderate dose of SLX and high dose of SLX(P>0.05).4. As were shown in RT-PCR, CTGF andα-SMA mRNA expressions were significantly increased in high glucose group(P<0.05), and SLX reduced increasing CTGF andα-SMA mRNA expressions(P<0.05). There was significant difference between low dose of SLX and moderate or high dose of SLX(P<0.05). But there was no significant difference between moderate dose of SLX and high dose of SLX(P> 0.05).5. As was shown in immuocytochemistry, the expression ofα-SMA protein was significantly increased in high glucose group(P<0.05), and SLX reduced the increasingα-SMA protein expression (P<0.05). There was significant difference between low dose of SLX and moderate or high dose of SLX(P<0.05). But there was no significant difference between moderate dose of SLX and high dose of SLX(P>0.05).[Conclusions]1. SLX inhibits the high expressions of TGF-β1,Smad2,CTGF induced by high glucose on rat peritoneal mesothelial cells in vitro.2. SLX reduces the transformation of rat peritoneal mesothelial cells into fibroblasts induced by high glucose to inhibit peritoneal fibrosis in vitro. These results suggest that SLX could inhibit high glucose-inducing peritoneal fibrosis of the patients with PD.