Lixu Xu1,2, Xue Li3, E Zhang1,2, Hao Liang1,2, Weiting Li1,2, Shangyi Wang1,2, Shuliang Song1,2, Aiguo Ji1,2,3. 1. a Marine College , Shandong University , Weihai , China. 2. b Weihai International Biotechnology Research and Development Centre , Shandong University , Weihai , China. 3. c School of Pharmaceutical Sciences , Shandong University , Jinan , China.
Abstract
OBJECTIVE: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity. METHODS: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6). RESULTS: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 μg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 μg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively. CONCLUSIONS: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.
OBJECTIVE: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity. METHODS: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6). RESULTS: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 μg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 μg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively. CONCLUSIONS: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.