| Aortic dissection(AD)is one of the most common cardiovascular diseases worldwide,without effective drug therapy but high mortality.Therefore,the study of the mechanism of AD can provide a theoretical basis for finding new ways to treat this disease.The first step of our study was using only ?-Aminopropionitrile(BAPN)to establish a Sprague-Dawley(SD)rat aortic dissection model.Base on this model,we found the expression of matrix metalloproteinases-9(MMP-9)is increased after mechanical stretching of the aortic dissection vessels in rats.Ion channels of vascular smooth muscle cells might be activated by mechanical stretching of the dissected vessels,and then mitogen-activated protein kinases(MAPK)signal pathway was activated.Thereby the expression of MMP-9 was regulated at the transcriptional level.At the same time,the secretions of inflammatory cytokines such as IL-1? and TNF-? were increased after mechanical stretching.Inflammatory cytokines also promoted the expression of MMP-9 by activating MAPK signaling pathway.Consequently the occurrence and development of aortic dissection was promoted.Therefore,we believe that: mechanical stretching promote the occurrence and development of aortic dissection by increasing the expression of MMP-9 via MAPK signaling pathway,and promote the release of inflammatory factors at the same time.Part I Establishment of SD rat aortic dissection modelObjectiveDevelop an effective method to establish aortic dissection model in SD rats,which can provide reliable pathological state for the subsequent research.MethodsThe SD rats(3 weeks old,male)were randomly equally divided into four groups: control group,0.6%BAPN group,0.8% BAPN group and 1.0% BAPN group,with 10 rats in each group.The rats in the control group was treated with free drinking diet;0.6%BAPN group was treated with BAPN solution of 0.6% concentration as drinking water for breeding,was fed normal rats diet;group 0.8%BAPN was treated with 0.8% concentration BAPN solution,was fed normal rats diet;group 1.0%BAPN was treated with 1.0% concentration BAPN solution,was also fed normal rats diet.The mean body weight of rats of each group was measured and the general situation was observed.Rat vascular samples of aortic dissection sections were collected,and then samples were treated with HE staining and Masson staining,respectively.ResultsRats in the control group and 0.6%BAPN group were in good condition throughout the experiment,with normal diet and drinking water and gradual weight gain.No deaths occurred during the whole experiment and no aortic dissection was found.No aortic dissection was formed in all rats in the control group.The formation rate of the mezzanine formation was 20.0 %in 0.6%BAPN group.In the experiment,the rats of 0.8% BAPN group were in poor condition,diet intake and water drinking were less,and weight gains were slow.But at the 6th weekend rats were heavier compared to the 1st week(P<0.05).Two rats died of aortic dissection during the whole experiment.The formation rate of the mezzanine formation was 60.0 % in 0.8% BAPN group.In the experiment,the rats of 1.0% BAPN group were worst,diet intake and water drinking were least,and weight gains were very slow.But the 6th week rats were heavier compared to the 1st week(P<0.05).Ten rats died during the whole experiment.The formation rate of the mezzanine formation was 50.0 % in 1.0% BAPN group.ConclusionThe method of 0.8%BAPN feeding alone can induce the formation of aortic dissection in rats.This method is simple and effective.Part II Effect of mechanical stretch on MMP-9 expression via the MAPK signaling pathwayObjectiveTo investigate whether the increase of MMP-9 expression in aortic dissection blood vessels after mechanical stretch is regulated by MAPK signaling pathway,and the correlation between inflammatory cytokines and MAPK signaling pathway.MethodsAortic dissection(AD)vessels collected from 40 SD rats when the AD model was ready and 24 patients with aortic dissection when underwent operation.Both rats and human AD vessels were randomly divided into 8 groups:(1)0g group(vascular ring with mechanical stretch stretching for 0g);(2)3g/5g group(the vascular ring of the rat was used by the mechanical stretch of 3g/ patients with the mechanical stretch of 5g);(3)3g/5g +SB203580(40?mol/L)group(vascular ring was intervened with 40?mol/L SB203580 and then 3g/5g mechanical stretch stretched);(4)3g/5g + SP600125(40?mol/L)group(vascular ring was intervened with 40?mol/L SP600125 and then 3g/5g mechanical stretch stretched);(5)3g/5g + U0126(40?mol/L)group(vascular ring was intervened with 40?mol/L U0126 and then 3g/5g mechanical stretch stretched);(6)0g+ SB203580(40 mol/L)group(vascular ring was intervened with 40?mol/L SB203580 and then 0g mechanical stretch stretched);(7)0g+ SP600125(40?mol/L)group(vascular ring was intervened with 40?mol/L SP600125 and then 0g mechanical stretch stretched);(8)0g+ U0126(40?mol/L)group(vascular ring was intervened with 40?mol/L U0126 and then 0g mechanical stretch stretched).The pathological changes of each group were detected by HE staining and Masson staining.The expressions of ERK1/2,p38,JNK1 and MMP-9 and the phosphorylation of ERK1/2,p38 and JNK1 were detected by immunohistochemistry.The expressions of m RNA of IL-1??TNF-? and MMP-9 were detected by RT-PCR.The expressions of IL-1??TNF-? and MMP-9 and the phosphorylation of ERK1/2,p38 and JNK1 were detected by Western blot.ResultsCompared with 0g group,the expressions of IL-1?,TNF-?,p-ERK1/2,p-p38,p-JNK1 and MMP-9 decreased after adding inhibitor,but the expressions of ERK1/2,p38 and JNK1 did not change significantly.While the expressions of IL-1??TNF-??p-ERK1/2?p-p38?p-JNK1 and MMP-9 of the 3g group were significantly increased,and the expressions of IL-1?,TNF-?,p-ERK1/2,p-p38,p-JNK1 and MMP-9 were also significantly decreased after treated with inhibitor(p<0.05).Compared with 0g group,the expressions of p-ERK1/2,p-p38,p-JNK1 and MMP-9 of the 5g group were significantly increased,but the expressions of ERK1/2,p38 and JNK1 did not change significantly.The expressions of p-ERK1/2,p-p38,p-JNK1 and MMP-9 of the 5g group decreased significantly after adding inhibitor(P<0.05),but the expressions of ERK1/2,p38 and JNK1 did not change significantly.Conclusions1.Mechanical stretch induced the expression of MMP-9 in the aortic dissection.The mechanism may be the stretch of activated vascular channels on vascular smooth muscle cells and the enhancement of ERK1/2,p38 and JNK1 signaling pathways activity,which regulate the expression of MMP-9 at transcriptional level.2.The secretion of IL-1? and TNF-? increased after mechanical stretching in the aortic dissection.Part III Molecular mechanism of inflammatory factors IL-1? and TNF-? affecting the expression of MMP-9 through the MAPK signaling pathwayObjectiveTo investigate the effect of inflammatory factors such as IL-1?,and TNF-? on the expression of MMP-9 in aortic dissection after mechanical stretch and whether it plays a role in activating MAPK signaling pathway.MethodsThe vascular rings were used to induce mechanical stretching on the blood vessels of the aortic dissection model SD rats(the same as the second part),along with the addition of inflammatory cytokines.Groups of the experiment one were set as follows:(1)3g group;(2)3g + IL-1?(160pg/ml)group;(3)3g + TNF-?(150pg/ml)group;(4)0g group;(5)0g + IL-1?(160pg/ml)group;(6)0g+TNF-?(150pg/ml)group.Groups of the experiment two were set as follows:(1)3g + IL-1?(160 pg/ml)group;(2)3g+ IL-1?(160 pg/ml)+ SB203580(40 ?mol/L)group;(3)3g + IL-1?(160 pg/ml)+ SP600125(40 ?mol/L)group;(4)3g+IL-1?(160 pg/ml)+ U0126(40?mol/L)group;(5)3g + TNF-?(150pg/ml)group;(6)3g + TNF-?(150pg/ml)+ SB203580(40?mol/L)group;(7)3g + TNF-?(150pg/ml)+ SP600125(40?mol/L)group;(8)3g + TNF-?(150pg/ml)+ U0126(40?mol/L)group.The expressions and phosphorylation of ERK1/2,p38 and JNK1,and the expression of MMP-9 of each group were detected by immunohistochemistry.The expression of IL-1??TNF-? and MMP-9 were detected by PCR and Western blot.The phosphorylation of ERK1/2,p38 and JNK1 were detected by Western blot.ResultsCompared with 3g group and 0g group,the expression of IL-1? significantly increased after adding IL-1?,and the expression of TNF-? significantly increased after adding TNF-?(P <0.05).Compared with 3g + IL-1? group and 3 g + TNF-? group,the expression of IL-1? and TNF-? significantly decreased(P <0.05)in the groups treated with inhibitors.Compared with 3g group and 0g group,the expression of p-ERK1/2,p-p38,p-JNK1 and MMP-9 were significantly increased after IL-1? and TNF-? added,but ERK1/2,p38 and JNK1 did not change significantly.Compared with 3g+ IL-1? group and 3g+ TNF-? group,the expression of p-ERK1/2,p-p38,p-JNK1 and MMP-9 were significantly decreased(P <0.05)in the groups treated with inhibitors,while the expression of ERK1/2,p38 and JNK1 did not change significantly.ConclusionsThe secretion inflammatory factors such as IL-1? and TNF-? increased after the mechanical stretch was acted on the aortic dissection.Meanwhile,inflammatory cytokines promote the expression of MMP-9 via activation of ERK1/2,p38,JNK1 signaling pathway and eventually promote the occurrence and development of aortic dissection. |
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