| IntroductionCytoskeleton is an impotant strucure and substance basis in maintaining shape, integrity and normal functions of cells. There are three types of protein filaments: microfilament (5-8 nm in diameter) , intermediate filaments ( IFs, 10 — 12 nm) and micro tubule ( about 25 nm). Moreover, the changes of IFs were directly related with some diseases of mankind. Now there are six IFs, including keratin, vimentin(Vim) , desmin(Des) , glial fibrillary acidic protein, neural filaments, nestin, etc. In myocardium, mainly they are Des and Vim. Recent studies showed that disturbance of Des structure arrangement in myocardium impacted on the motion of sarcomeres, causing the drop of myocardial contractility. Therefore, research on change pattern and action mechanism towards these two IFs would help us to understand the processes of generation and development about some cardiovascular diseases, in order to further search new clinical drugs and therapeutic targets.Reperfusion — injury manifested reperfused arrhythmia, myocardial stunning, structural damage, intramyocardial bleeding, etc. The mechanism mainly depended on two factors, oxygen free radicals and intracellular calcium overload. Recent reports indicated that when cytoplasm Ca2+ was at a high level in early reperfusion period, cardiac myocyte excess contraction would happened. While the deformation of cytoskeleton components exceed to normal contraction, irreversible injury in the skeleton structure took place, resulting in intercellular destroy mutually. Also, during myocardial ischemia, sundry cytoskeleton components participated in processes related to release of cytoplasmic creatine ki-nase and irreversible injury of cells induced by anoxia/reoxidation. Up to present, theres no concrete related researches reported.In our previous studies, dl - Praeruptorum A ( Pd - la) , one of active components isolated from Bai - Hua Qian - Hu ( Peucedanum praeruptorum Dunn) , a Chinese traditional medicine, as a Ca + - influx blocker and K+ -channel opener, displayed myocardial protection from myocardial ischemic reperfusion injury) through mechanisms such as inhibition of expression of apop-tosis related protein Fas and reduction of interleukin - 6 level. So we speculate that Pd - la may produce favorable effects to IFs Des and Vim in ischemic reperfusion myocardium ( MIRI). In the present study, we overall investigated the variation regulation of Des and Vim in MIRI of rats for the first time, in order to further explore the activity characteristics and mechamism of Ifs and Pd - la.Materials and MethodsForty - two healthy Wistar rats (250 - 350 g) of both sexes, were randomly divided into seven groups: Group I, sham operation control, Group II, operation control, Group III, solvent control (polyethylene glycol 400, PEG;1 ml/kg) , nifedipine (Nif, 5 mg/kg) , and Pd - la at lower, medium and higher dosage (PLO. 5, PM 1.0 and PH 2.0 mg/kg, respectively).MIRI models: Animal were firstly prepared under anesthesis, open - chested and artificial ventilation, and administered drugs after 15 min -stabilization, then ligated the left anterior descending coronary artery. Thirty minutes ischemia followed by 120 min reperfusion was produced. Mean carotid arterial pressure ( MAP) and heart rates were measured at 30, 60 and 120 min after reperfusion. Partial myocardium from area subjected to MIRI was scissored snap - frozen, and sliced by freezing microtome or stored in 10% formalin. Des and Vim determination were performed by immunohistochemistry, computer image analysis system and Western bloting. Brown staining in epicyte or cytoplasm was evaluated as positive expression, which was examined by calculating the ratio of mean optic density and area in positive stain, namely, positive expressive index ( PEI) . Pathological observation was examined by Hematoxylin - Eosin stainingunder optical microscope. All data was expressed as mean SD. Comparison of parameters before and after administration of drugs was performed using one -way ANOVA analysis of variance. P <0.05 was considered to be significant.Results1. Variation characteristics of Des and effects of Pd - la Immunihistochemistry: There was more intense positive expression of Desin both myocardial normal cross striation and intercalated disk. However, an obvious weaken of Des positive expression, or even turned into negative was found in MIRI area and around myocardium.Western Bloting: A protein strap was located in molecular weight about 53 kDa. There was more intense positive expression of Des in normal myocardial cells, an marked deletion in MIRI cells. Intergrated density valuae (IDV) was decreased from 265335 24651 to 69062 12968, P <0. 01, vs sham).Pd -la alleviated the deletion degree of Des, the IDV of PL, PM and PH were ascended from 690621 ±12968 to 177408 ± 10395^19585784 ±20057 and 209587 ± 17459, respectively ( P <0.01, vs Sol) . Also Nif relieved the deletion of Des, the IDV was increased from 69062 ± 12968 to 201134 ± 14879, P <0. 01, vs MIRI).2. Variation characteristic of Vim and effects of Pd - la Immunihistochemistry;There was positive expression of Vim in both myocardial normal cross striation and around conjunctive tissues. An evident attenuation of Vim positive expression, or even turned into negative was found in MIRI area and around myocardium.Western Bloting: A protein strap was located in molecular weight about 57 kDa, an marked deletion of Vim was found in MIRI cells, and decreased from 218790 19857 to 64870 10872( P <0. 01, vs sham) . Pd - la alleviated the deletion degree of Des, the IDV of PL and PM were rised from 59189 ± 19853 to 164781 ± 19543 and 185696 ± 20957, respectively ( P < 0. 01, vs Sol = . PH and Nif on Vim expression, were non significant in statistics(P >0.05).3. Pathological diagnosisMyocardial fiber in sham operation group arranged orderly without degeneration and necrosis. In MIRI group, we discovered more pathological changes, such as Acidophil change of myocyte, augment of infitration of neutrophile gran-ulocyte, vacuolar degeneration, disturbance of myocardial fibers, formation of contractile band, hemorrhage of inter - muscle fibers, focus necrosis, etc. In Both Pd - la and Nif groups, we all can view orderly arrangement of myocardial fibers, infitration of neutrophile granulocytes (less than control groups) , occasionally found acidophil and vacuolar degeneration.4. CardiohemodynamicsPd - la (0. 5, 1.0 and 2. 0 mg/kg) dose - dependency decreased MAP from (15.5 ±5.0) Kpa to ( 14. 2 ±3. 7) , ( 13. 0 ±5. 6) and (12.3 ±2.4) Kpa, respectively ( P < 0. 05, vs Sol). Pd -la induced HR slightly, but non significant in statistics ( P >0. 05).The hypotensive effects of Nif was seemed to that of Pd - la, but the severe inhibition of HR was observed (P <0.01, vs MIRI) .Conclusions1. Acute ischemic reperfusion may induce cardiac myocyte injury and abatement of desmin and vimentin.2. The myocardial protection of Pd - la were found through inhibiting the decrease of desmin and vimentin induced by myocardial ischemic reperfusion injury of rats.3. The myocardial protection of Nifedipine were found through attenuating myocardial ischemic reperfusion injury of rats and inhibiting the drop of desmin induced by MIRI, but there was no influence on vimentin.
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