| Objective: The goal of thwas study is to modifie traditional animal model ofin-stent restenoswas with establwashing vascular atherosclerotic lesions beforestent implantation, and to observe the Periostin and Rho kinase expressionchanges and the relationship between the two in the acute phase after carotidstenting for adminwastrating fasudil (a kind of Rho kinase inhibitor).Methods: Sixteen healthy aged male beagles were selected, began withhigh-fat diet after basal diet for one week, and switched to the basal diet afterstent implantation until the end. All animals were randomly divided into twogroups first, the common carotid artery was only exposed in control group(group A, n=4), and atheroscleroswas model was establwashed in experimentalgroup (group E, n=12). Then group E was randomly divided into B, C and Dthree groups with equal numbers of beagles, group B will not accept anytreatment, group C and group D underwent carotid artery stenting, which ingroup D intravenous drip of fasudil injection was given6mg/kg daily, dividedtwo times to input in the morning and evening. All experimental animals wereaccepted carotid artery angiography before stenting,10minutes and8weeksafter stenting. The angiography images were used for measuring percentagediameter stenoswas, percentage area stenoswas, obstruction diameter andreference diameter to assess in-stent restenoswas of model and the effect of fasudil. Carotid arteries were harvested at eighth week after stent implantation,a part of specimens were fixed with formaldehyde to HE staining forhwastological evaluation, the other part were preserved in liquid nitrogen,detected Periostin and Rho kinase mRNA in target twassue by quantitativeRT-PCR, and detected Periostin and Rho kinase protein by Western Blot.Moreover, peripheral vein blood was collected at the six time points includingthe time before atheroscleroswas modeling, before stenting,2days,7days,4weeks and8weeks after stenting to detect the expression levels of serumPeriostin by ELISA method.Results:1. Carotid artery angiography showed8weeks after atheroscleroswas model,the minimum diameter in group E was significantly reduced than in group A,while stenoswas rate of carotid artery increased significantly. The phenomenonof unobstructed Lumen, fine support of stick wall and expedite lumen withrelieving restenoswas was exhibited in group C and D at10minutes point afterstenting. On the eighth week after stenting, narrow diameter in group C wassignificantly lower than in group D, while percent diameter stenoswas wassignificantly higher，suggesting that restenoswas rate in fasudil group waslower than in stent group.2. Histological analys was showed intimal thickness, area and stenoswas rate ingroup B and C was significantly higher than that of group A and D, suggestingthat neointimal thickening was obvious in group B and C; stenoswas rate of group D was significantly higher than that of group A, but was significantlylower than that of group B and C, suggesting that fasudil have the effects ofinhibition on neointimal proliferation.3. Quantitative RT-PCR assay indicated that Periostin and Rho kinase mRNAexpression increased after modeling in group B and C, but significantly ingroup C; compared with group C, the Periostin and Rho kinase mRNAexpression decreased in the group D, but only Rho kinase mRNA expressiondecreased significantly, suggesting that fasudil can significantly inhibit Rhokinase mRNA expression, also may have the inhibition trend of PeriostinmRNA expression. Pearson correlation coefficient of Periostin and Rho kinasemRNA showed that r=0.595，p=0.015, suggesting that there was a positivecorrelation between the two.4. Western Blot quantitative analyswas results show that Periostin andP-MYPT1-Thr696(On behalf of Rho kinase activity) protein expressionincreased after modeling in group B and C, but significantly in group C;compared with C group, the expression of Periostin and Thr696proteindecreased in group D, but only Thr696protein expression decreasedsignificantly, suggesting that fasudil of can significantly inhibit Thr696proteinexpression, also may have the inhibition trend of Pearson correlation coefficientof Periostin and Thr696protein showed that r=0.810, p=0.000, suggesting thatthere was a high positive correlation between the two.5. ELISA results were analyzed by general linear model-repeated measures, and showed the effects of time factors on serum Periostin expression had asignificant meaning, suggesting that Periostin serum concentration of differenttime point detection was different in at least two time point; Interaction of timeand group also significantly influenced on serum Periostin concentration. Iftime factors was fixed, analyswas of serum Periostin concentration in differentgroups can explain that the concentrations of Periostin increased significantly ingroup B, C and D, Periostin concentration in group D significantly reduced thanin group C, indicated that serum Periostin expression increased throughatherosclerosis modeling or stent implantation, and fasudil can inhibit serumPeriostin expression. However, the analysis of contrast between differentgroups in each time point found at2d and7d points statistical p values betweengroup C and D were seperately0.028and0.036, indicated that serum Periostinconcentration significantly reduced in group D; statistical p values betweengroup C and D were seperately0.759and0.420at4w and8w points, indicatedthat serum Periostin concentration had no significant difference in group C andD. This suggested that serum Periostin expression reduced significantly duringthe period of fasudil treatment, while rised back once stopping the drug.Conclusion:1. Based on the traditional in-stent restenosis animal model, target vesselatherosclerotic animal model before stent implantation was establwashed.According to guideline of carotid artery stenting, antiplatelet drugs was given inperi-operation period, thus traditional in-stent restenosis animal model was modified scientifically and reasonably, to provid more valuable reference forresearching in-stent restenosis pathophysiological me-chanwasm.2. Neointima was formed in vessel lumen, and lumen became narrowedgradually after stenting. However, postoperative short-term fasudil (Rho kinaseinhibitor) probably inhibited neointimal formation to prevent luminalstenoswas.3. Fasudil inhibited the expression of serum periostin during the treatment, butrised back after stopping fasudil, and periostin expression in tissue wasconsistent with the results in serum at the eighth week point after stenting,indicating that fasudil could suppress periostin upregulation, but this may notbe the only factor or the key of neointima inhibition by fasudil.4. Whether atherosclerosis molding or stenting caused vascular injury, Periostin(an extracellular matrix protein) and Rho kinases may participate in the process,and their expression in tissues were high positive correlated, suggesting thatperiostin may involve in the process of in-stent restenosis was through theRho/ROCK signals pathway. |
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