Influence of paclitaxel-hirudin stent coating compound on TLR4-MyD88 signaling pathway during HCASMC inflammatory activation

Li Hongmei; Wang Xian

doi:10.3969/j.issn.1674-4055.2019.01.24

Abstract：To investigate the influence of paclitaxel-hirudin stent coating compound (PHSCC) on TLR4-MyD88 signaling pathway during inflammatory activation of human coronary artery smooth muscle cells (HCASMC) induced by lipopolysaccharide (LPS). Methods HCASMC of 4 to 6 generations were selected, and a low dose and a high dose of PHSCC without cytotoxicity for intervening HCASMC were screened by using MTT colorimetric method for maintaining cell viability above 90%. All cells were divided into blank control group, LPS modeling group, high-dose LPS+PHSCC group and low-dose LPS+PHSCC group. The high-dose LPS+PHSCC group and low-dose LPS+PHSCC group were pretreated with PHSCC 4 h before LPS modeling. TLR4 mRNA and MyD88 mRNA were detected by using Q-PCR, and expressions of corresponding proteins were detected by using Western blotting assay. Results The growth of HCASMC was significantly inhibited in group of 1 μmol/L paclitaxel+0.8 mg/mL hirudin, and there was significant difference in cell viability compared with blank control group (P<0.05). The cell viability was reduced to 78.7% group of 1 μmol/L paclitaxel+0.4 mg/mL hirudin, but was kept above 90% in other groups. After pretreatment with PHSCC for 4 h, the results of Q-PCR showed that the expressions of TLR4 mRNA and MyD88 mRNA were activated at the same time in LPS modeling group compared with blank control group (P<0.05). The inflammatory model of HCASMC was successfully established. The expression of TLR4 mRNA decreased significantly (P<0.05) and expression of MyD88 mRNA was not influenced (P>0.05) in all treatment groups compared with LPS modeling group. The results of Western blotting assay showed that protein expressions of TLR4 and MyD88 increased significantly in LPS modeling group compared with blank control group (P<0.05), and a stable inflammatory model of HCASMC was successfully established. After treatment with PHSCC, the protein expressions of TLR4 and MyD88 decreased significantly in all treatment groups compared with LPS modeling group (P<0.05), which was more significant in high-dose LPS+PHSCC group. Conclusion PHSCC has significant inhibitory effect on activation of TLR4-MyD88 pathway during LPS-induced inflammatory activation of HCASMC. This inhibition is not produced by affecting the expression of MyD88 gene, but may be related to the regulation of MyD88 protein level.