| Objective To investigate the effect of micro RNA-499(mi RNA-499)-mediated heat shock protein 90(HSP90)on the expression of inflammatory and immune factors such as interleukin 1(IL-1),IL-6 and Toll-like receptor 2(TLR-2),and the possible signal transduction mechanism during the cardioprotection of ischemic postconditioning(IPO)on rat heart.Background With the increasing incidence of coronary heart disease(CHD),and the treatment of CHD seems to be more and more important.At present,reperfusion therapy is the main treatment for CHD,but reperfusion is often accompanied by the appearance of ischemia reperfusion injury(IRI).IPO is a cardioprotective measure that has been widely demonstrated to reduce IRI,but its mechanism has not yet been fully elucidated.Immune and inflammatory responses are important features of IRI,while mi RNA-499 has been shown to be closely related to the formation of IRI.Our previous studies showed that HSP90 participate in the cardioprotective effects of IPO,and previous studies have confirmed that mi RNA-499 has a regulatory effect on HSP90.Therefore,we speculate that mi RNA-499 regulates HSP90 during the IPO process,thereby regulating myocardial immune and inflammatory responses and mitigating IRI.In order to test and verify this hypothesis,we established a rat model of ischemia reperfusion(IR)and IPO,using geldanamycin(GA)to inhibit HSP90 and adeno-associated virus to interfered mi RNA-499 low-expression and overexpression,to detect the expression of inflammation and immune factors such as IL-1、IL-6 and TLR-2,and the expression of protein kinase C(PKC)and c-Jun N-terminal kinase(JNK),and to investigate the intrinsic link and signal transduction mechanism of the signaling pathway of HSP90 mediated by mi RNA-499 with immune and inflammatory response in IPO.This study will be divided into three chapters.Chapter I The expression of HSP90 in IPO and the effect of TLR-2 mediated by HSP90 on the expression of IL-1 and IL-6Objective To investigate the effect of IPO on the expression of HSP90 in rat models of IR and IPO,and the effect of HSP90 on the expression of TLR-2,IL-1,IL-6 in IPO by using GA.Method Section I,establishment of rat heart ischemia-reperfusion and postcondition model.(1)24 male SD rats were randomly divided into three groups:(1)Sham group: the proximal segment of the anterior descending coronary artery was only threaded and not ligated for 2.5h;(2)IR group,the proximal segment of the anterior descending coronary artery was ligated for 30 minutes and then reperfusion for 2 hours.(3)IPO group: The proximal segment of the anterior descending coronary artery was ligated for 30 minutes,followed by 3 episodes of reperfusions/Ischemia treatment(reperfusion 30 s,ischemia 30s),then reperfusion for 2h.(2)Record the electrocardiograms of rats before and after coronary artery occlusion and use electrocardiographic changes as standard to judge whether the descending anterior descending artery was occlusion or reperfusion.(3)At the end of the experimental treatment,the heart had been clip immediately,and the ventricle was frozen into a hard mass and cut into 2mm thickness slices.Then the the infarct size(IS)was measured by 2,3,5-triphenyhetrazolium chloride(TTC)staining.Section II,effect of IPO on the expression of HSP90 in myocardium.(1)The animal grouping and processing were the same as the section Ⅰ.(2)Serum of the rats was collected after the treatment and use for detecting the content of HSP90 by enzyme linked immunosorbent assay(ELISA).(3)The Left ventricular free wall(the part which close to the interventricular septum)of the rat heart was preserved for detecting the content of HSP90 by Western Blotting(WB).Section III,effect of TLR-2 mediated by HSP90 on the expression of IL-1 and IL-6 in IPO.(1)32 male SD rats were randomly divided into four groups:(1)Sham group,(2)Ischemia/reperfusion(IR)group,and(3)Ischemic postconditioning(IPO) group,and both the same treatment as sectionⅠ.(4)Ischemic postconditioning + geldanamycin(IPO+GA)group: 0.6mg/kg dose of GA solution was injected into the abdominal cavity of rats after anesthesia,then the subsequent treatment was the same as the IPO group.(2)Serum of the rats was collected after the treatment and use for detecting the content of HSP90,IL-1,IL-6 and TLR-2 by ELISA.(3)The Left ventricular free wall of the rat heart was preserved for detecting the content of HSP90,IL-1,IL-6 and TLR-2 by WB.Result Section I,(1)The ECG of the rats after ligation appears to be gradually elevate in the ST segments of multiple leads(V3、V5、I、II、III、a VL and a VF)and gradually merges with QRS wave;when treat with postcondition,about 10 seconds after releasing the suture line,the ST segment will gradually fall back,and the QRS wave become narrow as before;when ligation again the ST segment will elevate once again.(2)The slices of myocardium of Sham group were totally brick-red;in the IR and IPO groups,most of the slices of myocardium were brick-red,but all appeared gray areas with different degrees,and IS [(5.02±0.81)%] in the IPO group was significantly lower than that in the IR group [(9.80±1.30)%](P<0.01).Section II,(1)Results of ELISA: Sham group(190.5±7.8)pg/m L,IR group(277.3±9.7)pg/m L,IPO group(299.8±11.3)pg/m L.Compared with Sham group,the content of HSP90 in IR group and IPO group increased significantly(P<0.05).Compared with IR group,the content of HSP90 in IPO group increased significantly(P<0.05).(2)Results of WB: Compared with Sham group(0.44±0.03),the expression of HSP90 in IR group(0.76±0.06)and IPO group(0.86±0.05)increased significantly(P < 0.05);Compared with IR group,the expression of HSP90 in IPO group increased significantly(P < 0.05).Section III,(1)Results of ELISA: Comparison of HSP90: Compared with Sham group,the content of HSP90 in IR group,IPO group and IPO+GA group increased significantly.Compared with IR group,the content of HSP90 in IPO group increased significantly.Compared with the IPO group,the content of HSP90 in IPO+GA group decreased significantly.Comparison of IL-1,IL-6 and TLR-2: Compared with Sham group,the content of IL-1,IL-6 and TLR-2 were significantly increased in IR group,IPO group and IPO+GA group;Compared with IR group,the content of IL-1,IL-6 and TLR-2 in IPO group decreased significantly.Compared with IPO group,the content of IL-1,IL-6 and TLR-2 in IPO+GA group increased significantly.(2)Results of WB were similar to ELISA.Conclusion(1)IPO can reduce IS after IRI.(2)Both IR and IPO could increase the expression of HSP90 in rat myocardium;IPO could increase the expression of HSP90 in rat myocardium on the basis of IR.(3)IPO could increase the expression of HSP90 in rat myocardium and at the same time reduce the production of inflammation and immune factors such as IL-1,IL-6 and TLR-2;In IPO,GA could specifically reduce the expression of HSP90 and at the same time increase the production of IL-1,IL-6 and TLR-2 in rat myocardium.Chapter II Changes of the expression of mi RNA-499 in IPO and its effect on the expression of HSP90Objective To investigate the effect of IPO on the expression of mi RNA-499 in rat models of IR and IPO,and the effect of mi RNA-499 on the expression of HSP90 by using the mi RNA-499 low-expression and over-expression adeno-associated virus during IPO.Method Section I,effect of IPO on the expression of mi RNA-499 in myocardium.(1)The animal grouping and processing were the same as the section I in chapter I.(2)The Left ventricular free wall of the rat heart was preserved to detect the content of mi RNA-499 by Real-time fluorescence quantitative polymerase chain reaction(q PCR).Section II,construction,packaging,and validation of mi RNA-499 over-expression and low-expression adeno-associated virus for rat’s myocardium.(1)Construction of mi RNA-499 over-expression and low-expression genes,and using adeno-associated virus as a vector,integrated into the viral gene.(2)The constructed viral vector was transfected into AAV-293 cells(virus packaging),virus purified and tested for viral titer.(3)Four male SD rats were randomly divided into four groups:(1)Control(C)group: routinely reared for 2 weeks without any treatment;(2)Negative control(NC)group: A negative adeno-associated virus was injected through the tail vein and then routinely reared for 2 weeks;(3)Over-expression(mi R499+)group: mi RNA-499 over-expression adeno-associated virus was injected via the tail vein then routine rearing for 2 weeks;(4)Low-expression(mi R499-)group: mi RNA-499 low-expression adeno-associated virus was injected via the tail vein then routine rearing for 2 weeks.After 2 weeks,the ventricle tissue of the rat heart was used for detecting the content of mi RNA-499 by q PCR.Section III,effect of the intervention of mi RNA-499 expression on the expression of HSP90 in IPO.(1)32 male SD rats were randomly divided into four groups:(1)Control(C)group: routinely reared for 2 weeks without any treatment.Then treat with ischemia and postconditioning like the modle above after 2 weeks;(2)Negative control(NC)group:negative adeno-associated virus was injected via the tail vein,then routinely reared,finally treat with ischemia and postconditioning 2 weeks later;(3)Low-expression group(mi R499-)group: the mi RNA-499 low-expression adeno-associated virus was injected via the tail vein,then routinely reared,finally treat with ischemia and postconditioning 2 weeks later; (4)Over-expression group(mi R499+): mi RNA-499 over-expression adeno-associated virus was injected via the tail vein,then routinely reared,finally treat with ischemia and postconditioning 2 weeks later.(2)Serum of the rats was collected after the treatment and use for detecting the content of HSP90.(3)The Left ventricular free wall of the rat heart was preserved for detecting the content of HSP90 by WB.Result Section I,results of q PCR: Compared with Sham group(1.02±0.07),the expression of mi RNA-499 in the IR group(0.68±0.07)decreased significantly(P < 0.05).Compared with Sham group,the expression of mi RNA-499 in the IPO group(1.22 ± 0.09)increased significantly.Section II,(1)Successfully integrated mi RNA-499 over-expression and low-expression genes into adeno-associated virus vector.(2)Viruses were successfully packaged and purified.The titer of mi RNA-499 over-expressing adeno-associated virus(HBAAV2/9-rno-mir-499-GFP)and mi RNA-499 low-expressing adeno-associated virus(HBAAV2/9-rno-mi R-499-5p-sponge-GFP)both were 1.3 x 1012 vg/m L,and the titer of negative control virus(HBAAV2/9-GFP)was 1.6 x 1012 vg/m L.(3)QPCR results: The expression of mi RNA-499 in each group: C group: 1.00,NC group: 1.08,over-expression group: 4.17,low-expression group: 0.56.Section III,(1)Results of ELISA: There was no significant difference between C group and NC group on the content of HSP90;Compared with NC group,the content of HSP90 in mi R499-group decreased significantly;Compared with NC group,the content of HSP90 increased significantly in mi R499+ group.(2)Results of WB were similar to ELISA.Conclusion(1)IR could reduce the expression of mi RNA-499 in rat myocardium,while IPO could increase its expression.(2)Low-expression the mi RNA-499 of myocardium could reduce the expression of HSP90 during IPO;While over-expression the mi RNA-499 of myocardium could increase the expression of HSP90 during IPO.Chapter III The mechanism of HSP90-TLR-2 signaling pathway mediated by mi RNA-499 in IPOObjective To investigate the effect of mi RNA-499 on the expression of TLR-2,IL-1,IL-6 and PKC,JNK by using the mi RNA-499 low-expression and over-expression adeno-associated virus during IPO in rat models of IR and IPO.Method Section I,effect of the HSP90-TLR-2 signaling pathway mediated by mi RNA-499 on the expression of IL-1 and IL-6 in IPO.(1)The animal grouping and processing were the same as Section III of Chapter II.(2)Serum of the rats was collected after the treatment and use for detecting the content of IL-1 and IL-6 and TLR-2 by ELISA.(3)The Left ventricular free wall of the rat heart was preserved for detecting the content of IL-1,IL-6 and TLR-2 by WB.Section II,effect of the HSP90-TLR-2 signaling pathway mediated by mi RNA-499 on the expression of PKC and JNK in IPO.(1)The animal grouping and processing were the same as Section III of Chapter II.(2)Serum of the rats were collected after the treatment and use for detecting the content of PKC and JNK by ELISA.(3)The Left ventricular free wall of the rat heart was preserved for detecting the content of PKC and JNK by WB.Result Section I,(1)Results of ELISA: There were no significant difference between C group and NC group on the content of IL-1,IL-6 and TLR-2;Compared with NC group,the content of IL-1,IL-6 and TLR-2 in mi R499-group increased significantly;Compared with NC group,the content of IL-1,IL-6 and TLR-2 in mi R499+ group decreased significantly.(2)Results of WB were similar to ELISA.Section II,(1)Results of ELISA: There was no significant difference between C group and NC group on the content of PKC and JNK;Compared with NC group,the content of PKC and JNK in mi R499-group increased significantly;Compared with NC group,the content of PKC and JNK in mi R499+ group decreased significantly.(2)Results of WB were similar to ELISA.Conclusion(1)Low-expression the mi RNA-499 of myocardium could increased the production of IL-1,IL-6 and TLR-2 during IPO;While over-expression the mi RNA-499 of myocardium could reduce the production of IL-1,IL-6 and TLR-2 during IPO.(2)Low-expression the mi RNA-499 of myocardium could increase the expression of PKC and JNK during IPO;While over-expression the mi RNA-499 of myocardium could reduce the expression of PKC and JNK during IPO. |
PDF Full Text Request