| Objective: Myocardial ischemic/reperfusion injury (MI/RI) refers to asyndrome which was after having ischemia for some time, myocardium notonly did not reduce the degree of injury after the blood flow restored, but alsoaggravated compared with ischemic. MI/RI was characterized by local andsystemic inflammatory response. If the inflammation expanded, myocardiumwould be injured and seriously affected the left ventricular function. More andmore studies had shown that MI/RI was a group of chronic inflammatorydiseases. Nowadays, scientists discovery Toll-like receptors (TLRs) play aimportant role at inflammation reaction. The relationship between TLRs andinflammation becomes more and more closely. To explore the role of TLR2and TLR4in myocardial ischemia/reperfusion injury (MI/RI) by observing thedynamic expression changes at mRNA and protein levels early aftermyocardial ischemia/reperfusion.Methods: Choose42male wistar rats with similar weight, and randomlydivided them into7groups (reperfusion for1h,2h,4h,6h,12h,24h, and7d),each group had6rats. Anesthetized each rat with Pentobarbital sodium,and then fixed it on a operating board. With the help of animal’s ventilator,opening the chest, supplanting the heart, ligation of left anterior descendingcoronary (LAD), finishing the ischemic model. After ischemic for1h, openthe chest again, shear the ligature wire with microsurgical scissor, finishingthe reperfusion model. After reperfusion reaches desired time (1,2,4,6,12,24hours and7days), anesthetized the rat again, fixed on the board, separatedthe skin and subcutaneous tissue of neck, exposing the trachea, and incubated.Then isolated the right carotid and incubated. Before cutting off the heart,ligation of the cardiovascular again in situ, injected about1~1.5mlphthalocyanine blue solution into the carotid artery, it can be seen that the heart partly turned blue immediately. Cut the heart, and put it in clean icesaline, rinsing clean, and placed it in refrigerator using a clean Petri dishes forabout8min. Taken the heart out and cut it into slices of about2mm, followedby TTC staining. Finally, using a scanner to scan slices and save the picturesfor analysis the area at risk and infracted area with image analysis software,and according to the calculation results, assessing ischemic size and infarctsize.Elected84male Wistar rats, which had the same weight range with aboverats. They were randomly divided into I/R and sham group, each group has42rats, I/R group and sham group randomly divided into seven groups accordingto different reperfusion time (1h,2h,4h,6h,12h,24h, and7d). After finishingthe reperfusion model, anesthetized the rat again at the predetermined timepoint, thoracic and separate pericardium, cut the heart off the level of theaortic arch, rinsing the heart clean with ice saline and put it on a clean Petridishes, then put them in refrigerator for about8min. When get the heart out,discarded the apical and the bottom, then cut it to thickness about2mmmyocardial slice, of which one put into the embedding box and embedded inparaffin, then the paraffin-embedded sections were stained with differentstains, including HE stain and immunohistochemistry stains. After removingthe right ventricular, other myocardial slices were separated by ischemicinfarct area and ventricular septal, and loaded them respectively into differentcryogenic tubes, placed them in liquid nitrogen for1h and then stored in therefrigerator. These samples are stored for RNA extraction and PCR. ThemRNA and protein levels of TLR2and TLR4were detected using real-timePCR (RT-PCR), and immunohistochemistry respectively. Myocardial TNF-αwas determined by immunohistochemistry, too. MCP-1and IL-6mRNAlevels were measured by reverse transcriptase-polymerase chain reaction(rt-PCR).Results:(1) The results of double stain with phthalocyanine blue andTTC: With time going, the risk size (RS) had no difference to each other, butmyocardial infarct size (IS) increased smoothly, and reached the plateau at2h, then stayed in the platform. After reperfusion for7d, the left ventricular wallwas significantly thinner than before, the septum was significantly thicker, theventricular remodeled finally.(2)HE stain: At the beginning of reperfusion, myocardial structureshowed no significant change in sham group, but had different degrees ofinjury in I/R group. Although the myocardium got supply of blood again, theinjury was still continuing. In the group of reperfusion for7d could visible theleft ventricular remodeling, the thickness of left ventricular wall wassignificantly thinner than before, large number of fibroblasts replaced themyocardial cells.(3) Immunohistochemical Results: Compared with sham group, TLR2and TLR4protein levels were elevated, reaching a peak at reperfusion for4h,then decreased gradually, but increasing again at reperfusion for7d, althoughthe difference had no statistically significant with the24h group. TNF-αprotein increased since reperfusion for1h, and had a slight decline afterpeaked at reperfusion for6h. TLR2and TLR4protein levels were positivelycorrelated, the correlation coefficient r=0.935, P <0.01. TLR4protein levelswas correlated with TNF-α, and the correlation coefficient r=0.818(P <0.05),but TLR2protein was not correlated with TNF-α.(4)The results of RT-PCR: Compared to sham group，TIR2, TLR4mRNA levels were increased in myocardium in I/R group. TLR4mRNA hadpeaked at4h of reperfusion (vs. sham group，2.71±0.30，P<0.01), and thendecreased gradually, and rose again at reperfusion for7days. TLR2mRNAlevel also peaked at reperfusion for4h (vs. sham group,3.95±0.58, P<0.01),and declining with the prolongation of reperfusion time. TLR2and TLR4mRNA expression levels were positively correlated, the correlation coefficientr=0.844, P <0.01.(5)rt-PCR: IL-6mRNA peaked at4h (3.36±0.41, P<0.01), followed bya gradually decrease, and reached the level of sham group at reperfusion for24h, and did not elevated again when reperfusion for7days. MCP-1mRNAlevel in I/R group remained fairly with sham group at the beginning of reperfusion, and markedly elevated at7d (1.97±0.51，P<0.01). TLR2andIL-6mRNA expression levels were positively correlate, the correlationcoefficient r=0.813, P<0.01, TLR4and IL-6mRNA expression levels werealso positively correlated, the correlation coefficient r=0.791, P <0.01.(6) The relationship of TLRs and IS: From reperfusion for2h toreperfusion for24h, TLR2and TLR4mRNA both positively correlated withIS, the correlation coefficient r=0.936(P<0.05), r=0.907(P<0.05).Conclusion: At the early stage of myocardial ischemia/reperfusion, thelevel of TLR2and TLR4mRNA and protein increased rapidly, and promotedthe production of downstream inflammatory cytokines, such as IL-6andTNF-α, which caused early injury of myocardium. At the late stage ofreperfusion, TLRs levels rose again and so as the inflammatory factors, likeMCP-1, which may probably affected the remodeling of ventricular, andinjured myocardial structure and function. There was a positive correlationbetween TLR2and TLR4, these results may concluded that there may beexisting some kind of linkages between them, and promoting the expression ofeach one of them, thus they worked as a interacting whole in the inflammatoryprocess. |
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