| objective:1.To observe the function of JAK-STAT pathway on myocardial ischemia reperfusion injury of blood stasis syndrome of rats.2. Through the tests of in vitro and in vivo,to observe the role of Xintongshu preconditioning on JAK-STAT pathway and mediated cardiomyocyte apoptosis,to play a myocardial ischemia reperfusion injury in effect mechanism.Methods:1.The models of myocardial ischemia reperfusion injury of blood stasis syndrome of rats was used. Thirty two male SD rats were randomly divided into four groups namely sham group、blood-stasis- syndrome group. ischemia-reperfusion(I/R) group and blood-stasis- syndrome & I/R group. Each group had eight rats. Electrocadiogram was used to observe the change of cardiac electricity, HE Stain Technology to bserve the morphological change of myocardiun, TUNEL to bserve the apoptosis of cardiomyocyte, Western-blot to detect the protein expression of p-JAK2、p-STAT1、p-STAT3.2.The models of myocardial IRI of blood stasis syndrome of rats were used. Forty eight male SD rats were randomly divided into six groups namely sham group、blood-stasis- syndrome & sham group、blood-stasis- syndrome & I/R 30 min group、blood- stasis- syndrome & I/R 60 min group、blood- stasis-syndrome & I/R 30 min group & xintongshu group、blood-stasis- syndrome & I/R 60 min group & xintongshu group. HE Stain Technology was used to bserve the morphol- ogical change of myocardiun, immunohistochemical staining to locate the protein expression of the gene-related apoptosis Bcl-2、Bax and Caspase-3; RT-PCR to test the expression of mRNA of the gene-related apoptosis Bcl-2、Bax and Caspase-3; Western-blot to detect the protein expression of p-JAK2、p-STAT1、p-STAT3.3.The models of hypoxia/reoxygenation(H/R) of baby rats cardiomyocyte were used. Baby rats cardiomyocytes were randomly divided into four groups namely normal serum control group、H/R group、AG490 group、serum containing xintongshu group. LuoDanming B stain technology was used to observe the morphology of baby rat cardiomyocyte; AO/EB double fluores- cent staining and flow cytometry to detect the apoptosis of cardiomyocyte; RT-PCR to test the expression of mRNA of the gene-related apoptosis Bcl-2、Bax and Caspase-3; Western-blot to detect the protein expression of p-JAK2、p-STAT1、p-STAT3.Results:1. There were some changes in the blood stasis syndrome of rat myocardium IRI that QRS waves heightening and broadening,the point J raising up, cardio-myocyte becoming dropsical, cardiac muscle fibers getting fractured, many focal hemorrhage and many inflammatory cells Soakaging in myocardium interstitium, which could alleviated by xintongshu pretreatment.Compared with sham group, the apoptosis of cardiomyocyte in blood-stasis- syndrome group、I/R group and blood-stasis- syndrome & I/R group all increased and the differences were notable(P<0.01); Compared with blood-stasis- syndrome group, I/R group and blood-stasis- syndrome & I/R group conduced much more apoptosis of cardiomyocyte than simplex blood stasis(P<0.01).2.By comparison with sham group, the protein expression of phosphorylate JAK2、STAT1、STAT3 in blood-stasis- syndrome group. I/R group and blood-stasis-syndrome & I/R group obviously rosed, and statistically the differences are significant (P<0.01); compared with blood-stasis- syndrome group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in I/R group and blood-stasis- syndrome & I/R group obviously rosed, and statistically the differences are significant (P<0.05); in comparison with I/R group, the protein expression of phosphorylate JAK2、STATland STAT3 in blood-stasis-syndrome & I/R group obviously rosed, and statistically the differences are significant (P< 0.05).3.Compared with blood-stasis- syndrome & sham group, the protein and mRNA expression of Bcl-2、Bax and Caspase-3 in blood-stasis- syndrome & I/R 30 min group and blood-stasis- syndrome & I/R 60 min group all increased,and the ratio of Bcl-2/Bax reduced, the differences of which were statistical (P<0.01); by comparison with blood-stasis- syndrome & I/R 30 min group, the protein and mRNA expression of Bcl-2、Bax and Caspase-3 in blood-stasis- syndrome & I/R 60 min group all ascended and the differences were statistically(P< 0.01); in blood-stasis- syndrome & I/R 30 min & XTS group, the protein and mRNA expression of Bcl-2 Bcl-2/Bax significantly increased, but that of Bax and Caspase-3 lowed,the differences of which were statistical (P<0.01); in comparison with blood-stasis- syndrome & I/R 60 min group, in blood-stasis-syndrome & I/R 60 min & XTS group, the protein and mRNA expression of Bcl-2 and Bcl-2/Bax significantly increased, but that of Bax and Caspase-3 lowed, the differences of which were statistical (P< 0.01).4.By comparison with sham group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in blood-stasis- syndrome & sham group% blood-stasis- syndrome & I/R 30 min group and blood-stasis- syndrome & I/R 60 min group obviously rosed, and statistically the differences are significant (P< 0.01).Compared with blood- stasis-syndrome & sham group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in blood-stasis-syndrome & I/R 30 min group and blood-stasis-syndrome & I/R 60 min group obviously rosed,and statistically the differences are significant (P< 0.01). Compared with blood-stasis-syndrome & I/R 30 min group, the protein expression of phosphorylate JAK2 increased and that of STAT1 and STAT3 decreased,but in blood-stasis-syndrome & I/R 30 min & XTS group that all decreased and the differences were statistical(P<0.05). In comparison with blood-stasis- syndrome & I/R 30 min group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in blood-stasis- syndrome & I/R 60 min & XTS group all decreased and the differences were statistical(P<0.05).5.Compared with normal serum control group,the ratio of apoptosis of cardiomyocyte in H/R group. AG490 group and serum containing xintongshu group significantly increased and statistically the differences are significant (P <0.01). Compared with H/R group, the ratio of apoptosis of cardiomyocyte in AG490 group and serum containing xintongshu group all decreased and statistically the differences are significant (P<0.05). By comparison with AG490 group, the ratio of apoptosis of cardiomyocyte in serum containing xintongshu group decreased and statistically the differences are significant (P< 0.05).6.By comparison with normal serum control group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in H/R group increased and statistically the differences are significant (P<0.01). Compared with H/R group, the protein expression of phosphorylate JAK2、STAT1 and STAT3 in AG490 group and serum containing xintongshu group obviously decreased and statistically the differences are significant (P<0.01). In comparison with AG490 group, the protein expression of phosphorylate JAK2 was no statistical difference(P>0.05),but that of phosphorylate STAT1 decreased and phosphorylate STAT3 increased and statistically the differences are significant (P<0.05).7.By comparison with normal serum control group,in H/R group the mRNA expression of Bcl-2 decreased but that of Bax and Caspase-3 increased,and statistically the differences are significant (P<0.01). Compared with H/R group, the mRNA expression of Bcl-2 increased but that of Bax and Caspase-3 decreased,and statistically the differences are significant (P<0.01) in AG490 group and serum containing xintongshu group. Compared with AG490 group, the mRNA expression of Bcl-2 increased but that of Bax and Caspase-3 decreased,and statistically the differences are significant (P<0.05) in serum containing xintongshu group.Conclusion:1. It played a significant role in myocardium IRI in rats of blood stasis syndrom that JAK-STAT pathway activated and promoted the myocardial apoptosis.2.The pretreatment of Xintongshu can protect rat IRI myocardium of blood stasis syndrom.The mechanism is related to blocking JAK-STAT pathway、down-regulating bid pro-apoptosis gene bid bax and reducing the activity of enzyme Caspase-3% upregulating the apoptosis gene repressor Bcl-2 and restraining the apoptosis of cardiomyocyte. |
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