Baicalin inhibit endothelial injury induced neointimal formation and its mechanism
|School||Hebei Medical University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Baicalin Vascular smooth muscle cells Proliferation Balloon injury Neointimal|
Objective: Vascular smooth muscle cells (vascular smooth muscle cell, VSMC) proliferation of vascular restenosis main pathology. Baicalin (baicalin) is separated from the herbs skullcap out of a flavonoid. Previous studies have found that baicalin on tumor cell proliferation was significantly inhibited. But whether they affect vascular endothelial injury induced intimal hyperplasia is unclear. In this study, the following aspects discussed baicalin on neointimal formation and its mechanism. Method: 1 VSMC culture: isolated and cultured according to attached method VSMC, take 3 ~ 6 cells for experiments. VSMC subcultured grown to 80% to 90% confluence, different concentrations of baicalin (5,10,20,40 μmol / L) Pretreatment. 2 Western blot analysis: take the same amount of protein extract samples for SDS-PAGE. And transferred to a PVDF membrane. Then with the corresponding primary antibody and secondary antibody, antigen detection by chemiluminescence antibody binding zone. With a digital imaging analysis system software for quantitative analysis of the results. 3 balloon injury model of replication and grouping SD rats (250 ~ 300g) disinfection routine anesthesia, incision along the midline of the neck to expose the left external carotid artery, the external carotid artery cut a wedge-shaped incision, the balloon catheter from incision into the abdominal aorta to the distal end, filling the balloon pull back after repeated three times, quit, ligation of the external carotid artery, close the incision. Experimental animals of the sham group, balloon injury group, baicalin group. 4 specimens after 14 days of treatment in blood from the femoral artery, rats were sacrificed isolated carotid artery biopsy preparation. Results: 1 baicalin inhibited PDGF-induced VSMC proliferation MTT assay results showed that baicalin in a concentration (5,10,20,40 μmol / L) inhibited VSMC proliferation-dependent manner, with a significant amount - effect relationship. 2 baicalin on VSMC proliferation marker gene expression of PCNA immunocytochemistry and Western blot analysis showed that normal cells showed a low expression of PCNA, PDGF (10 ng / ml) stimulated a significant increase in the level of PCNA, while drugs were pre-incubated then the PCNA levels. Tip baicalin inhibit PDGF-induced VSMC proliferation. 3 baicalin inhibited PDGF signaling pathways activated PDGFR/MEK/ERK1/2 Western blot analysis showed, PDGF stimulates cell 15 min, PDGFR phosphorylation levels were significantly increased; while after baicalin pretreatment VSMC, PDGFR phosphorylation in a concentration reduce dependency; thus its downstream MEK, ERK1 / 2 activity were analyzed and found MEK and ERK1 / 2 phosphorylation also increased with the drug concentration decreases; however phosphorylated JNK, p38, Akt without drugs Treatment. Tip baicalin by specific inhibition MEK/ERK1/2 pathway to inhibit the PDGF-induced proliferation of VSMC. 4 baicalin after balloon injury inhibited intimal hyperplasia after balloon injury 14 days, baicalin group intimal hyperplasia was significantly reduced compared with balloon injury group, two groups of vascular intima / media (I / M) thickness ratios were 0.43 ± 0.04 and 2.36 ± 0.15, with a significant difference (P lt; 0.01). Showed that baicalin can inhibit balloon injury-induced intimal hyperplasia. 5 baicalin inhibited neointimal morphological analysis of PCNA expression: normal intimal PCNA expression in only a small amount, 14 days after balloon injury, blood vessels of PCNA-positive cells was significantly increased; while baicalin can inhibit PCNA in expression of PCNA-positive compared with the model group, the number of cells decreased 53.4%, with a significant difference (P lt; 0.01). Tip baicalin inhibit vascular cell proliferation activity. 6 baicalin balloon injury-induced inhibition of ICAM-1 and VCAM-1 expression by immunohistochemical staining showed: normal intima visible in very small amounts of ICAM-1 and VCAM-1 colored particles. 14 days after balloon injury, two proteins positive staining cells and single-cell staining intensity was significantly increased compared with the normal group. The baicalin could significantly inhibit balloon injury-induced ICAM-1 and VCAM-1 expression, compared with model group positive staining cells were reduced 55.8 ± 6.9% and 57.2 ± 5.5%, with significant difference (P lt; 0.01, P lt; 0.01). Conclusion: Baicalin inhibited PDGF-induced VSMC proliferation. 2 baicalin inhibited VSMC proliferation mechanisms and blocking PDGFR/MEK/ERK1/2 signaling pathway. 3 baicalin can prevent endothelial injury induced intimal hyperplasia and stenosis. 4 baicalin inhibit vascular intimal hyperplasia and its inhibition of vascular cell proliferation and adhesion molecule expression.