| ObjectiveIn recent years, with the rapid development of competitive sports,the widelydevelops of body-building and the increase of life pressure, the number of personparticipate in exercise becomes more and more, the exercise amout increases day byday, exercise sudden death events take place continuously, the sudden cardiac deathcaused more than85%of this death, and quite a number of sudden death appearedafter the exercise. Therefore, the serious medical problems of long time strenuousexercise or excessive movement induced cardiac adverse effects and how to becomemore healthy by the movement became the focus of people day by day. This studybased on the previous studies, intends to observe the dynamic changes of myocardialultrastructure. changes of inflammatory factors such as TNF-α、NF-κB、ICAM-1inrat serum and myocardial tissue and cell apoptosis at different time points ofconvalescence after one week repeated exhausted exercises (at immediate,3h,6h, 12h,24h, and48h and96h after exercises); at the same time studied the effects of3.2g/kg (metered in crude drugs) and1.6g/kg Sonchus oleraceus L flavonoids to thepathology changes，the change rule of inflammatory factors such as TNF-α、NF-κB、ICAM-1in rat serum and myocardial tissue and cell apoptosis dynamic changes at24h and48h after repeated exhausted exercises，in order to investigate the delayedmotility myocardial injury mechanism of production，the relationship betweenTNF-α-NF-κB-ICAM-1inflammatory pathways and the intervention effect ofSonchus oleraceus L flavonoids, in order to prevent the occurrence of athletic suddendeath and to provide important experimental theoretical basis for motility myocardialinjury rehabilitation therapy.Methods1．Establish the motility delayed myocardial damage modelThe experimental motility delayed myocardial damage animal models wereadopted by the classic Thomas method through repeated exhausted swimming. Therats were forced to swim to exhaustion every day, for1week (7days), with2%ofbody weight attached to the tail. The exhaustion standard adopted “animals had notcome back to the water surface in10seconds”(Thomas DP，Marshall KI．1988),2．Animal groups and drug deliveryWe divided262healthy male SD rats randomly into a sedentary control group(SC,35), a exhausted exercise model group (M,155) and a Sonchus oleraceus Lextract treatment group (D.72). Sedentary control group rats were taken materialswithout any exercises, exhausted exercise model group rats were forced to takerepeated exhausted exercises. According to different recovery times after exercise,exhausted exercise model group was divided into immediately (st),3h,6h,12h,24h,48h and96h after exercise group (each group had respectively20,17,17,17,17,35,35,17rats) again, then taken materials after the different time points.3.2g/kgand1.6g/kg Sonchus oleraceus L extract treatment groups were divided into drugtreatment24h and48h groups(18) respectively in each group. Sonchus oleraceus L extract treatment24h group was deliveried drug immediately and at23h afterexercises, then taken materials after1h of the last dose; drug treatment48h groupwas deliveried drug immediately，at23h and47h after exercises，then takenmaterials after1h of the last dose.3．Materials taking and observation indexes①Rats which from each group of different time points after exercises，wereobservated general condition and weighed（Body weight, BW），calculated thepercentage of weight change.②Rats from each groups were taken2ml blood from carotid sinus respectively，then preparated serum，stored at-20℃until analysis，inorder to detected the contentof TNF-α、NF-κB and ICAM-1in rats’ serum by ELISA method (Enzyme linkedimmunosorbent assay）, detected the content of myocardial enzymes such as CK andCK-MB by fully automatic biochemical analyser.③Rats were killed, quickly taken out the heart and weighed, calculating heartcoefficient.④T he hearts were washed with4℃cold saline solution, cuted out100mgleft ventricular muscle tissue, preparated10%myocardial tissue homogenate,centrifuged and the supernatant was got, then stored at-20℃until analysis，detectedthe TNF-α、NF-κB and ICAM-1content in rats’ myocardial tissue by ELISA method.⑤Two rats’myocardial tissue of sedentary control group and each exhaustedexercise model group observed by electron microscopy, to observe the myocardialtissue ultrastructure changes.⑥Got residual myocardial tissue of5rats from each group and fixed for threedays in neutral formalin，taken the left ventricular myocardial tissue to conventionallydehydrated, embedded in paraffin, cutted into slices，reserved for observing, afterstained by hematoxylin-eosin we observed myocardial histopathological changes；detected the protein expression of TNF-α、NF-κB and ICAM-1in rats’ myocardial tissue and apoptosis by immunohistochemistry method.⑦Got two myocardial tissue pieces which weighed about100mg of another5rats from each group，preparated myocardial frozen specimen in liquid nitrogenquickly，stored at-70℃until use, detected the protein expression of TNF-α、NF-κBand ICAM-1in rats’ myocardial tissue by Western blotting method; detected themRNA expression of TNF-α、NF-κB and ICAM-1in rats’ myocardial tissue byqRT-PCR (Real-time fluorescent quantitative PCR) method.⑧Preparated myocardial cells by heart perfusion and enzyme digestion，thenobserved the effect of Sonchus oleraceus L extract on rat myocardial cell apoptosisafter Hoechst33325staining.Results1．General condition, body weight and heart index in rats⑴Compared with sedentary control group, the rats of exhausted swim exercisemodel group could appear tachypnea, lethargy, and some rats appeared diarrheasymptoms after swim; after5to7days swim, their physical state declined obviously,the endurance time of swim shorten, surface gloss was poor, the reaction to the rest ofthe world was slow, appeared piloerection, fur might be depilated, etc.3.2g/kg dose ofSonchus oleraceus L extract could improve these conditions obviously.⑵Compared with sedentary control group, the rats’weight of each exhaustedswim exercise model group all had a significant reduction in varying degrees（P＜0.05and P＜0.01），among all model groups the weight lose of24h and96h afterexercises groups were maximum（P＜0.01）；the rats’ weighr of24h and48h groupsin3.2g/kg and1.6g/kg dose of Sonchus oleraceus L extract treatment group all hadincreased obviously.⑶Compared with sedentary control group, the rats’ heart coefficient ofimmediately,6h and48h groups in exhausted exercise model group all had increasedobviously（P＜0.05and P＜0.01）；the heart coefficient of3.2g/kg and1.6g/kg dose of Sonchus oleraceus L extract treatment group compared with that of48h exercisegroup all had reduced obviously, and had significant differences（P＜0.05）.2．Effects of Sonchus oleraceus L extract on serum myocardialenzymes such as CK and CK–MB in ratsCompared with sedentary control group, the rats’ serum CK and CK-MBactivity of24h and48h groups in exhausted exercise model group were markedlyincreased (P<0.05and P<0.01)，the rats’ serum CK and CK-MB activity ofSonchus oleraceus L extract small dose24h group all had reduced，but did not havesignificant statistical difference（P＞0.05）；the rats’ serum CK and CK-MB activityof Sonchus oleraceus L extract3.2g/kg dose24h and48h groups along with1.6g/kgdose48h group all had reduced obviously（P<0.05and P<0.01）.3．The myocardial ultrastructure and morphological changes atdifferent time points after exhausted exercises in rats and theintervention effect of Sonchus oleraceus L extract⑴Myocardial tissue ultrastructure in rats of sedentary control group observed byelectron microscope were normal. Myocardial tissue ultrastructure in rats of differenttime points after repeated exhausted exercises all had damage with different degree，the damage of48h after exercise groups were most serious among all the modelgoups.⑵Myocardial structure in rats sedentary control group observed by opticalmicroscope were normal. Myocardial structure in rats of24h and48h after repeatedexhausted exercises groups all appeared many pathologic changes such as irregulararrange of local muscle cells，cross striations unsharpness, enlargement of core，scattered focal inflammatory necrosis, etc. many muscle cells of48h group occured lyticnecrosis. The rats’ myocardial structures of Sonchus oleraceus L extract3.2g/kg dose24h group along with3.2g/kg and1.6g/kg dose48h were all improved. 4．Dynamic changes of the content of TNF-α、NF-κB andICAM-1at different time points after exhausted exercises in ratsserum and the intervention effect of Sonchus oleraceus L extract—ELISA method⑴Compared with sedentary control group, the rats’ serum TNF-α content of24h and48h after repeated exhausted exercises groups increased obviously（P＜0.05），the other groups all had rise trend，the peak of TNF-α content apeared at24hafter exercises；the rats’ serum NF-κB content of immediately,3h and6h afterexercises groups all reduced obviously（P＜0.01and P＜0.05），while the serumNF-κB content of12h、24h、48h and96h after exercises groups rised obviously（P＜0.01），peaked at，presented a first post-high-low trend；the rats’serum ICAM-1content of12h、24h、48h and96h after exercises groups all increased obviously（P＜0.01and P＜0.05），the serum ICAM-1content of immediately～6h did not havesignificant change（P＞0.05），peaked at96h after exercises.⑵Compared with24h after repeated exhausted exercises group，the rats’serumTNF-α, NF-κB and ICAM-1content of Sonchus oleraceus L extract3.2g/kg dose24h group all reduced obviously（P＜0.05），1.6g/kg dose of Sonchus oleraceus Lextract could caused reduction of the rats’ serum NF-κB content obviously（P＜0.05）；Compared with48h after exercises group，Sonchus oleraceus L extract3.2g/kg dose48h all induced reduction of the rats’ serum TNF-α, NF-κB andICAM-1content prominently（P＜0.05and P＜0.01），1.6g/kg dose of Sonchusoleraceus L extract all caused the reduction of the TNF-α and NF-κB serum contentprominently（P＜0.05）.5．Dynamic changes of the content of TNF-α、NF-κB andICAM-1at different time points after exhausted exercises in ratsmyocardial tissue and the intervention effect of Sonchus oleraceus L extract—ELISA method⑴C ompared withsedentary control group，the rats’ TNF-α content in themyocardial tissue of3h、24h and48h after repeated exhausted exercises groups allincreased obviously（P＜0.05），the TNF-α content of immediately,6h,12h and96h after exercise had a rise trend，peaked at3h and48h respectively，presented twinpeaks changes；the rats’ NF-κB content in the myocardial tissue of3h,6h,12h,24h,48h and96h after repeated exhausted exercises groups all increased obviously（P＜0.05and P＜0.01），peaked at3h and48h respectively，presented twin peakschanges；the rats’ ICAM-1content in the myocardial tissue of immediately,3h,6h,12h and24h after repeated exhausted exercises groups all increased obviously（P＜0.05），peaked at3h，then reduced gradually.⑵Compared with24h after repeated exhausted exercises group，the rats’TNF-α, NF-κB and ICAM-1content in the myocardial tissue of Sonchus oleraceus Lextract3.2g/kg dose24h group all reduced obviously（P＜0.05），1.6g/kg dose ofSonchus oleraceus L extract could caused reduction of the rats’ NF-κB content in themyocardial tissue obviously（P＜0.05）；Compared with48h after exercises group，Sonchus oleraceus L extract3.2g/kg dose48h all induced reduction of the rats’TNF-α and NF-κB content in the myocardial tissue prominently（P＜0.05），1.6g/kgdose of Sonchus oleraceus L extract all caused the reduction of the TNF-α andNF-κB content in the myocardial tissue prominently（P＜0.05）.6．Dynamic changes of the expression of TNF-α、NF-κB andICAM-1proteins at different time points after exhausted exercisesin rats myocardial tissue and the intervention effect of Sonchusoleraceus L extract—immunohistochemical method⑴Compared with sedentary control group，the rats’ TNF-α, NF-κB andICAM-1protein expression in the myocardial tissue of different time points after repeated exhausted exercises groups all had somewhat increase，among the modelgroups the TNF-α protein expression of48h after exercises was the most.，the NF-κBprotein expression of3h after exercises was the most., the ICAM-1proteinexpression of immediately after exercises was the most.⑵Compared with24h after repeated exhausted exercises group，the rats’TNF-αprotein expression in the myocardial tissue of Sonchus oleraceus L extract3.2g/kgdose24h group all had somewhat reduce，the rats’ TNF-α protein expression in themyocardial tissue reduced obviously；Compared with48h after exercises group，therats’ TNF-α, NF-κB and ICAM-1protein expression in the myocardial tissue ofSonchus oleraceus L extract3.2g/kg dose48h all reduced prominently,1.6g/kg doseof Sonchus oleraceus L extract all caused the reduction of the TNF-α and NF-κB andICAM-1protein expression in the myocardial tissue prominently.7．Dynamic changes of the expression of TNF-α、NF-κB andICAM-1proteins at different time points after exhausted exercisesin rats myocardial tissue and the intervention effect of Sonchusoleraceus L extract—Western-blotting method⑴The results of western-blotting show that，compared with sedentary controlgroup，the rats’ TNF-α, NF-κB and ICAM-1protein expression in the myocardialtissue of different time points after repeated exhausted exercises groups all increasedobviously，among the model groups the TNF-α protein expression of48h afterexercises was the most.，the NF-κB protein expression peaked at immediately，12hand48h after exercises respectively，the ICAM-1protein expression peaked at24hafter exercises.⑵Compared with24h after repeated exhausted exercises group，the rats’TNF-α, NF-κB and ICAM-1protein expression in the myocardial tissue of Sonchusoleraceus L extract3.2g/kg and1.6g/kg dose24h goups all had somewhat reduce；Compared with48h after exercises group，the rats’ TNF-α and NF-κB protein expression in the myocardial tissue of Sonchus oleraceus L extract3.2g/kg dose48h reduces were more obvious.8．Dynamic changes of the expression of TNF-α、NF-κB andICAM-1mRNA at different time points after exhausted exercises inrats myocardial tissue and the intervention effect of Sonchusoleraceus L extract—qRT-PCR method⑴The results of qRT-PCR show that，compared with sedentary control group，the rats’ TNF-α, NF-κB and ICAM-1mRNA expression in the myocardial tissue ofdifferent time points after repeated exhausted exercises groups all increasedobviously（P＜0.05'P＜0.01），among the model groups that peaked at48h afterexercise，⑵Compared with24h after repeated exhausted exercises group，Sonchusoleraceus L extract3.2g/kg dose caused the rats’ TNF-α, NF-κB and ICAM-1mRNA expression in the myocardial tissue of24h goup reduced obviously, Sonchusoleraceus L extract1.6g/kg dose caused TNF-α mRNA expression reducedobviously，the NF-κB mRNA and ICAM-1mRNA expression level had somewhatreduce. Compared with48h after exercises group，the rats’ TNF-α, NF-κB andICAM-1protein expression in the myocardial tissue of Sonchus oleraceus L extract3.2g/kg dose48h goups all reduced obviously（P＜0.05'P＜0.01）.9．Dynamic changes of myocardial apoptosis at different timepoints after exhausted exercises in rats myocardial tissue and theintervention effect of Sonchus oleraceus L extract—TUNELmethod and Hoechst33325staining method⑴The results of TUNEL show that，the myocardial tissue of sedentary controlgroup existed a few sporadic apoptosis；a lot of apoptosis were all apeared in the myocardial tissue of different time points after repeated exhausted exercises，myocardial cell apoptosis indexes all increased obviously（P<0.01），peaked at48h，and had time dependence characteristics.⑵The results of Hoechst33325stain show that，most myocardial suspensioncell nucleus after perfusion of sedentary control group in rats were complete，thegroups of24h and48h after repeated exhausted exercises have observed a lot ofmorphology of cell apoptosis such as nuclear fragmentation, condensation,pyknosis, nucleoplasm uneven and so on; compared with24h and48h afterrepeated exhausted exercises groups，3.2g/kg and1.6g/kg Sonchus oleraceus Lextract all could improve apoptosis state obviously，the number of apoptosis cellsall reduced，the effect of3.2g/kg dose was more obvious.Conclusion1. One week repeated exhausted exercises could established delayed myocardialinjury model successfully.2. Rat myocardial enzymes and myocardial tissue structural changes areconfirmed that repeated exhausted exercise could cause rats’ myocardial tissuedamage in different degrees at different time points.3.2g/kg and1.6g/kg Sonchusoleraceus L extract all could improve myocardial ultrastructure damage andmorphological changes induced by exhausted exercise.3. ELISA, immunohistochemical and Western-blotting methods all confirmedthat repeated exhausted exercise could cause the rats’ TNF-α, NF-κB andICAM-1protein expression in the serum and myocardial tissue rised in differentdegrees at different time points,3.2g/kg and1.6g/kg Sonchus oleraceus L extract allcould reduced the TNF-α, NF-κB and ICAM-1protein expression level.4. qRT-PCR method confirmed that the TNF-αmRNA, NF-κBmRNA andICAM-1mRNA expression in the myocardial tissue at different time points after repeated exhausted exercise all rised in different degrees，3.2g/kg and1.6g/kgSonchus oleraceus L extract could reduced the the TNF-αmRNA, NF-κBmRNA andICAM-1mRNA protein expression level obviously.5. TUNEL method and Hoechst33325staining method all confirmed that therats’ myocardial tissue all appeared a lot of apoptosis cells at different time pointsafter repeated exhausted exercise,3.2g/kg and1.6g/kg Sonchus oleraceus L extractcould all reduced apoptosis myocardial cells obviously.6. Different methods further confirmed that inflammatory mediator such asNF-κB, TNF-α, ICAM-1and myocardial apoptosis played an important role in thedevelopment of delayed myocardial damage induced by exhausted exercise.7. Sonchus oleraceus L extract had protect effects on delayed myocardial damageinduced by exhausted exercise，the mechanism may be related to reduced the NF-κB、TNF-α、ICAM-1expression and apoptosis in the myocardial tissue. |
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