| Object: To observe the effects of salvianolate on on the proliferation of rats arterial intimal injury and primary cultured rat aortic smooth muscle cells(VSMC)'ability of proliferation,migration and secretion of cytokines.And the same time,to explore the mechanism of lectin like oxidized low density lipoprotein receptor-1(LOX-1)in these processes.Methods:In the animal level,to explore the effects of salvianolate on on the proliferation of rats arterial intimal injurythe and the express the influence,18 SD rats were randomly divided into sham operation group,operation group,salvianolate group,6 rats each group.After postoperative 14 days,IL-6,IL-10,TNF-? and TGF-? expression were detected in serum;carotid artery sections with HE staining under optical microscope morphological observation,using image analysis system to measure carotid artery intimal thickness(IT),media thickness(MT)and IT/MT;Immunofluorescence histochemical method to observe the changes of LOX-1 in artery wall;The LOX-1mRNA and protein expression were measured by RT-PCR and Western Blot.In the cell level,to study the effects of salvianolate on the function of vascular smooth muscle cell and the role of LOX-1,the primary culture of SD rat thoracic aorta vascular smooth muscle cells was done by tissue-piece inoculation.The cultured cells were observed by morphology,and identified by immunohisto chemical staining method.After induced by ox-LDL and intervention by salvianolate,VSMC proliferation and growth activity was detected by CCK-8 assay.Transwell was used to detect the cell migration ability.IL-6,IL-10,TNF-? and TGF-? levels were tested by ELISA.LOX-1expression level was determined by RT-PCR and Western Blot.And to observe the effect of LOX-1 inhibitor of poly inosinic acid on VSMC on proliferation,migration ability and changes of cytokine levels.Results:In the animal level:compared with the sham operation group,carotid artery intimal injury after 14 d visible vascular intimal hyperplasia,lumen stenosis and IT/MT increases,the wall of LOX-1 mRNA and protein expression increased,with statistical difference(P<0.05);serum cytokine IL-6,TNF-? expression increased andIL-10,TGF-? expression decreased,with significant difference(P<0.05);Compared with operation group,salvianolate significantly reduced IT/MT value,decreased serum cytokine IL-6,TNF-? levels,increased IL-10,TGF-? expression,with statistical difference(P < 0.05),and the wall of LOX-1 mRNA and protein expression level decreased significantly,with significant difference(P < 0.05).In the cell level:after 4-6days of primary cultured,showing 70% inoculation survival and surrounding cells began to grow,about 2 weeks can be subcultured.Cell morphology,growth characteristics is not obvious changed at 6 to 8 generations.Under microscope observation of primary cultured vascular smooth muscle cell showed the typical "peak valley" growth and alpha smooth muscle actin(?-SM-actin)immunohistochemical staining showed that the cells were brownish yellow.The ox-LDL+VSMCs group compared with VSMCs group,ox-LDL can induce VSMCs proliferation,and has a statistically significant difference in 48 h and 72h(P<0.05);Enhance the ability of cell migration,cytokines IL-6,TNF-? secretion increased and IL-10,TGF-? secretion decreased,LOX-1 mRNA and protein levels were significantly increased,with statistical difference(P<0.05).Compered with ox-LDL+VSMCs group ox-LDL+VSMCs+salvianolate group and ox-LDL+VSMCs+ polyinosinic acid group,in 48 h and 72 hcell proliferation activity were decreased,with statistical difference(P<0.05)decreased.The ability of cell migration,cytokines IL-6,TNF-? decreased,IL-10,TGF-? increased,with significant difference(P<0.05);the expression of mRNA protein and LOX-1 decreased,with statistical difference(P<0.05).Conclusion:Salvianolate can effectively inhibit the intimal hyperplasia after rat carotid artery injury,inhibit primary cultured rat VSMCs proliferation and migration ability,inhibition of inflammatory response,its mechanism may be related to down regulating the expression of LOX-1. |
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