【作者】 俞向梅；

【导师】 张文光；

【作者基本信息】 福建中医药大学 ， 中西医结合基础， 2011， 硕士

【摘要】 研究目的：通过观察力竭运动大鼠血清和心肌组织SOD及MDA、凋亡相关因子Bax、Bcl-2的变化,并分析心肌组织的超微结构变化,探讨丹参酮及西药磷酸肌酸钠对力竭运动大鼠心肌的保护作用机制。研究方法：采用体重250g±10g的雄性SD大鼠,按随机数字法分为正常对照组、力竭运动模型组、力竭运动+丹参酮低剂量组、力竭运动+丹参酮中剂量组、力竭运动+丹参酮高剂量组和力竭运动+磷酸肌酸钠组(西药组)各8只。正常对照组：不做任何训练,常规饲养,自由饮食；力竭运动模型组：采用游泳耐力训练；其余各组：在游泳耐力训练后腹腔注射药物。不同剂量丹参酮组在力竭游泳运动后分别给予腹腔注射低剂量3mg/kg/d、中剂量6mg/kg/d、高剂量12mg/kg/d丹参酮；磷酸肌酸钠组在力竭游泳运动后给予腹腔注射72mg/kg/d磷酸肌酸钠。经过6周力竭游泳运动实验后,各组分别取血清、心肌组织保存,采用免疫组织化学法检测心肌组织Bax、Bcl-2的蛋白表达,应用分光光度法检测血清和心肌组织SOD及MDA的含量,HE染色光镜及透射电镜观察心肌形态结构变化,并拍摄保存分析。研究结果：1.血清及心肌组织SOD检测及结果比较1.1正常对照组血清和心肌组织SOD分别为128.49±9.72、66.86±4.97；与正常对照组比较,力竭运动模型组血清及心肌组织SOD活性明显降低,分别为77.17±12.77、26.30±4.72(P<0.01)。1.2低、中、高剂量丹参酮组分别与力竭运动模型组比较,血清SOD活性均明显升高,分别为105.28±6.66、118.36±8.88、108.30±7.67(P<0.01)；心肌组织SOD活性均明显升高,分别为51.73±5.42、62.55±5.27、56.78±3.69(P<0.01)。1.3磷酸肌酸钠组与力竭运动模型组比较,血清及心肌组织SOD活性均明显升高,分别为121.19±6.98、62.77±5.79(P<0.01)。2.血清及心肌组织MDA检测及结果比较2.1正常对照组血清和心肌组织MDA分别为5.78±0.71、2.75±0.95；与正常对照组比较,力竭运动模型组血清及心肌MDA含量明显升高,分别为11.80±2.35、11.17±1.24(P<0.01)。2.2低、中、高剂量丹参酮组分别与力竭运动模型组比较,血清MDA含量均明显降低,分别为8.14±0.95、6.59±0.62、7.80±0.77(P<0.01)；心肌MDA含量均明显降低,分别为5.29±1.06、3.91±1.00、4.98±1.02(P<0.01)。2.3磷酸肌酸钠组与力竭运动模型组比较,血清及心肌组织MDA含量均明显降低,分别为6.20±0.88、3.64±1.11(P<0.01)。3.各药物组之间SOD及MDA结果比较3.1中药组不同剂量三组血清和心肌组织SOD及MDA比较,低剂量与高剂量组无差异(P>0.05),而中剂量组与低、高剂量组均有差异(P<0.05),对SOD及MDA影响效果优于二组。3.2西药组与中剂量组血清和心肌组织SOD及MDA分别比较,均无差异(P>0.05)；西药组与低、高剂量组比较均有差异(P<0.05),对SOD及MDA影响效果优于二组。4.形态学观察4.1 HE染色光镜观察结果：正常对照组：心肌纤维排列基本正常,核呈椭圆形,位居中。力竭运动模型组：心肌纤维排列紊乱、疏散,心肌纤维肿胀,间隙增宽,部分细胞核变性,间质出血。丹参酮低剂量、高剂量组：心肌纤维排列疏散、间质出血。丹参酮中剂量组、磷酸肌酸钠组：心肌纤维极性排列稍紊乱。4.2电镜观察结果：正常对照组：心肌纤维排列基本正常,轻度空泡变性,心肌细胞核正常,线粒体形态结构正常。力竭运动模型组：心肌纤维萎缩、部分溶解,排列紊乱,线粒体增生,分布紊乱,肌膜锯状突起,肌膜下水肿,血管增生。丹参酮低剂量组：心肌纤维排列稍紊乱,无明显萎缩,线粒体分布均匀。丹参酮中剂量组：心肌纤维结构、排列基本正常。丹参酮高剂量组：局部仍可见心肌纤维溶解,周围心肌纤维基本恢复。磷酸肌酸钠组：心肌纤维线粒体增多,局灶肌膜下心肌纤维轻度溶解。5.免疫组化检测结果：不同剂量丹参酮和磷酸肌酸钠均可抑制Bax表达,促进Bcl-2表达,而各药物组之间Bax、Bcl-2蛋白表达情况比较,均无显著差异。研究结论：1、本实验采用Thomas法复制大鼠力竭游泳运动心肌损伤模型,模型组心肌组织形态出现明显破坏,该方法制作的动物模型稳定,可操作性强,重复性好。2、不同剂量丹参酮和磷酸肌酸钠都能在一定程度上提高力竭游泳运动大鼠血清及心肌组织SOD活性,降低MDA含量,从而对力竭游泳运动大鼠的心肌损伤具有一定保护作用。3、不同剂量丹参酮和磷酸肌酸钠都能调整凋亡相关因子Bax、Bcl-2的变化,从而改善心肌损伤状态。4、从形态学观察,不同剂量丹参酮和磷酸肌酸钠都能减轻心肌结构的破坏,缓解心肌损伤情况。5、从形态学观察,结合氧自由基和凋亡指标变化分析,中剂量丹参酮较其他剂量有更明显的心肌保护作用。6、综上所述,丹参酮和磷酸肌酸钠在一定程度上都可减轻力竭运动心肌损伤。更多还原

【Abstract】 Objective:To Explore the effect of tanshinone and western medicine of phosphocreatine sodium to myocardium of exhaustive exercise rats by observing exhaustive exercise rat SOD and MDA of blood-serum and myocardium tissues and the change of Bax and Bcl-2 of Apoptosis correlation factor and analyzing the ultramicrostructure change of myocardium.Methods:250g±10g body weight of male SD rats were randomly divided into six groups:normal control group,exhaustive exercise model group,exhaustive exercise+low-dose tanshinone group,exhaustive exercise+middle-dose tanshinone group,exhaustive exercise +hypso-dose tanshinone group and exhaustive exercise+western medicine of phosphocreatine sodium group of 8.Normal control group:untake any exercise,common breed;exhaustive exercise model group:take swimming exercise for endurance;the rest groups:intraperitoneal inject drug after swimming exercise for endurance. Different dose tanshinone groups were given tanshinone of intraperitoneal injection low-dose3mg/kg/d,middle-dose6mg/kg/d,hypso-dosel2mg/kg/d after swimming exercise for endurance;exhaustive exercise+western medicine of phosphocreatine sodium group were given phosphocreatine sodium of intraperitoneal injection 72mg/kg/d after swimming exercise for endurance.After six weeks experiment of swimming exercise for endurance,all groups take blood-serum and myocardium tissues to freeze and preserve.Adopt immunohistochemical method to detect the proteinum expression of Bax and Bcl-2 of myocardium tissues and applicat spectrophotometric method detecting the content of SOD and MDA of blood-serum and myocardium tissues and observe the change of morphous structure of myocardium tissues by HE dyeing light microscope and transmission electron microscope and preserve the shot for analyzing.Results:1.The detection of SOD of blood-serum and myocardium tissues and the comparing result.1.1 The SOD of blood-serum and myocardium tissues of normal control group are discerned 128.49±9.72 and 66.86±4.97;comparing to normal control group,the activity of SOD of blood-serum and myocardium tissues of exhaustive exercise model group are discerned 77.17±12.77 and 26.30±4.72 (P<0.01),cutting down obviously.1.2 Comparing to exhaustive exercise model group,the activity of SOD of blood-serum and myocardium tissues of low-dose, middle-dose and hypso-dose tanshinone all heightenig obviously. The activity of SOD of blood-serum are discerned 105.28±6.66,118.36±8.88 and 108.30±7.67(P< 0.01);The activity of SOD of myocardium tissues are discerned 51.73±5.42,62.55±5.27 and 56.78±3.69(P<0.01).1.3 Comparing to exhaustive exercise model group,the activity of SOD of blood-serum and myocardium tissues of phosphocreatine sodium group are discerned 121.19±6.98 and 62.77±5.79(P<0.01),all heightening obviously.2. The detection of MDA of blood-serum and myocardium tissues and the comparing result.2.1 The MDA of blood-serum and myocardium tissues of normal control group are discerned 5.78±0.71 and 2.75±0.95;comparing to normal control group,the content of MDA of blood-serum and myocardium tissues of exhaustive exercise model group are discerned 11.80±2.35 and 11.17±1.24(P<0.01),heightenig obviously.2.2 Comparing to exhaustive exercise model group,the content of MDA of blood-serum and myocardium tissues of low-dose,middle-dose and hypso-dose tanshinone all cutting down obviously. The content of MDA of blood-serum are discerned 8.14±0.95,6.59±0.62 and 7.80±0.77(P<0.01);The content of MDA of myocardium tissues are discerned 5.29±1.06,3.91±1.00 and 4.98±1.02(P<0.01).2.3 Comparing to exhaustive exercise model group,the content of MDA of blood-serum and myocardium tissues of phosphocreatine sodium group are discerned 121.19±6.98 and 62.77±5.79(P<0.01),all cutting down obviously.3.Compare the results of SOD and MDA of all drug groups.Comparing the SOD and MDA of blood-serum and myocardium tissues among the groups of different doses of Chinese crude drug,there is no difference between low-dose and middle-dose(P>0.05),but middle-dose have difference and its effect to SOD and MDA surpass to other two groups,comparing to low-dose and middle-dose(P<0.05).Comparing the SOD and MDA of blood-serum and myocardium tissues between western medicine of phosphocreatine sodium group and middle-dose tanshinone group,there is no difference(P> 0.05);Comparing western medicine of phosphocreatine sodium group to low-dose and middle-dose tanshinone groups,it has difference and its effect to SOD and MDA surpass to other two groups.4. Observe morphology4.1 The result by observing light microscope.Normal control group:Heart muscle fiber arrange normally and cell nuclear is ellipse,locating center.Exhaustive exercise model group:Heart muscle fiber swell and arrange disorderly and distantly and its diastema widen;part of cell nucleus degenerate and cell interstitial substance bleed.Low-dose and hypso-dose tanshinone groups:Heart muscle fiber arrange distantly and cell interstitial substance bleed.Middle-dose tanshinone and western medicine of phosphocreatine sodium groups: Heart muscle fiber polarity arrange disorderly slightly.4.2 The result by observing electron microscope.Normal control group:Heart muscle fiber arrange normally,vacuolar degeneration lightly and myocardial cell nuclear and shape structure of chondriosome are normal.Exhaustive exercise model group:Heart muscle fiber atrophy,part dissolve and arrange disorderly;chondriosome hyperplasy,distribut disorderly;tunica muscularis ecphyma sawly and dropsy;Vascular proliferation.Low-dose tanshinone group:Heart muscle fiber arrange disorderly slightly and atrophy unobviously;chondriosome distribute uniformity.Middle-dose tanshinone group:The structure and arrangement of heart muscle fiber are normal basically.Hypso-dose tanshinone group:Part of heart muscle fiber fibronolysis and surrounding heart muscle fiber revive fundamentally.Western medicine of phosphocreatine sodium group:The chondriosome of heart muscle fiber mult and heart muscle fiber under tunica muscularis dissolve lightly.5. The result by detecting immunohistochemical method.Different doses of tanshinone and phosphocreatine sodium all restraint the expression of Bax and promote the expression of Bcl-2,but compering the proteinum expression of Bax and Bcl-2 among all grug groups,there is no significant deviation.Conclusion:l.The experiment adopt Thomas method copying rats’ exhaustive swimming exercise and myocardial damagement pattern;The shape of myocardium tissues of exhaustive swimming exercise model group destroy obviously.Animal model taken by this method is stable,manipulateion strongly and reproducibility easily.2. Different doses of tanshinone and phosphocreatine sodium could in some degree elevate the activity of SOD and cut down the content of MDA of blood-serum and myocardium tissues of exhaustive swimming exercise rats,preserve myocardial damage of exhaustive swimming exercise rats in some degree accordingly.3. Different doses of tanshinone and phosphocreatine sodium could regulate the change of Bax and Bcl-2 of Apoptosis correlation factor and improve the condition of myocardial damage accordingly.4. Observing by morphology,different doses of tanshinone and phosphocreatine sodium lighten destruction of myocardium structure and relieve myocardial damage.5. Observing by morphology and connetting oxygen free radical and the change of Apoptosis index,Middle-dose tanshinone take protection of myocardial preservation further evident than other doses groups.6. Sum up above statement,tanshinone and phosphocreatine sodium lighten myocardial damage of exhaustive swimming exercise in some degree.更多还原