他达那非对分流型肺动脉高压大鼠肺血管重构的影响及其对心肌Cx43磷酸化影响的研究
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摘要
肺动脉高压(Pulmonary arterial hypertension, PAH)是不同病因导致的、以肺动脉压力升高和肺血管阻力增加为特点的一组病理生理综合征。如果不进行干预，则病程进行性恶化，最终会导致右心衰竭甚至死亡。PAH患者进行麻醉和手术时，风险极高。手术的刺激，麻醉对心肺功能的影响以及体外循环引起的炎性因子释放均可加重肺动脉高压，严重时患者不能脱离体外循环机，导致手术失败；而且，这些因素还可加重术后右心室衰竭，缺氧，心肌缺血等导致病人死亡的危险因素。目前，国内外针对PAH的研究大多集中在如何提高PAH晚期病人的生活质量，延长其寿命；或者如何在PAH治疗术过程中，降低体外循环后期肺动脉压力，而对于如何降低PAH病人术前肺动脉压力，改善其右心室肥厚，进而争取手机机会，提高手术成功率，改善术后生活质量的研究尚未见报道。
已有研究表明肺动脉压力的调节主要受内皮素-1(Endothelin-1，ET-1)和一氧化氮的调节。PAH患者ET-1表达升高，一氧化氮合酶表达下降，导致肺动脉收缩，肺小动脉平滑肌细胞增生肥厚，非肌性动脉肌化，导致肺动脉压力升高和肺血管重构。磷酸二酯酶5（Phosphodiesterase-5，PDE-5）抑制剂可选择性舒张肺血管、降低肺动脉压。他达那非（Tadalafil，TDF）是一种选择性PDE-5抑制剂，它通过增加肺血管平滑肌和内皮细胞中cGMP浓度，舒张肺血管，降低肺血管的阻力进而降低肺动脉压。然而，TDF是否可以通过影响肺动脉ET-1及ET-1受体表达起到抗肺动脉高压的作用尚未见报道。
另有研究证实，缝隙连接蛋白43(Connexins43, Cx43)是Cx基因家族中数量最为丰富的成员，不同水平磷酸化的Cx43可调控细胞信号转导，控制着细胞内许多生理过程，与心律失常、心肌缺血、心衰和心肌肥厚等许多疾病有密切联系。有学者提出通过调控Cx43的表达有助于治疗某些心血管疾病。PDE-5抑制剂能否通过调控右心室Cx43表达起到减轻右心室肌肥厚，改善心功能的作用及其可能机制目前尚无报道。
本研究应用腹主动脉-腔静脉造瘘术建立大鼠分流型肺动脉高压模型，模拟临床左向右分流先心病所致早、中期肺动脉高压。探讨中短期应用磷酸二酯酶5抑制剂TDF对PAH大鼠肺动脉高压的早、中期阶段肺血管重塑、右心室肥厚的影响及其可能机制。为临床中度肺高压病人术前短期应用磷酸二酯酶5抑制剂，降低肺动脉压力，改善心功能提供进一步的理论依据。第一部分：ET-1和cGMP参与大鼠轻中度肺动脉高压心肺功能形态变
化
目的：本实验旨在建立大鼠腹主动脉-腔静脉分流模型，模拟先心病左向右分流肺动脉高压早中期阶段，从功能学和形态学方面探讨该模型大鼠在动静脉分流1-8w内肺血管重构以及右心室肥厚发生发展过程和可能机制。
方法：健康清洁级雄性SD大鼠72只，随机分为2组，假手术组（Sham，S）分流组（Fistula，F）组。参照Garcia的方法，大鼠麻醉后，仰卧位固定，正中切口打开腹腔，暴露腹主动脉和下腔静脉，使用18G穿刺针，穿刺点为腹腔动静脉壁联合部，相当于腹主动脉肾动脉发出点与髂总动脉分叉处连线的中下1/3处,于静脉壁侧刺入进而刺破动静脉壁联合部进入腹主动脉形成瘘口。暂时用小动脉夹在肾动脉分支处上方钳夹腹主动脉，阻断血流；拔出穿刺针，以一滴氰基丙基酸盐粘合剂封住穿刺口。30s后松开小动脉夹。若观察到下腔静脉增粗并伴有波动，则证实造瘘分流建立成功，假手术组大鼠仅单纯开腹，暴露腹主动脉和下腔静脉，腹主动脉夹闭，不作分流手术。两组动物分别于术后1，2，3，4，6，8w测量肺动脉及右心室压力。经右颈动脉取血测量血清ET-1与环磷酸鸟苷(cGMP)含量。右下肺组织行HE染色观察肺动脉形态学变化；取出大鼠心脏测定心室相对重量以及Masson特殊染色观察心肌胶原总量的改变。
结果：1各组大鼠颈动脉平均动脉压差异无统计学意义(P>0.05)。与对应的S组比较，F组大鼠肺动脉平均肺动脉压（mPAP）、肺动脉收缩压（SPAP）升高；右心室发展压（+dP/dtmax）增加(P<0.05)。2与对应的S组比较，F组大鼠右心室肥厚指数RV/(LV+S)在第2w升高，第3-8w与S组相比无差异；左心室与体重比(LV+S)/BW从第4w直到第8w升高(P<0.05)。右心室与体重比（RV/BW）与对应的S组比较，从第2w到第8w升高，(P<0.05)。3与对应的S组比较，F组大鼠心肌纤维明显增粗、排列紊乱、心肌纤维间质成分增多。F组心肌胶原容积百分比（CVF%）均显著高于对应的S组(P<0.01)，并且F组动物4w后较第4w前CVF%升高更显著(P<0.01)。4与对应的S组比较，F组大鼠肺动脉管壁明显增厚，管腔变小。内皮下层不明显，中膜平滑肌层明显增厚，平滑肌纤维直径增粗，细胞层数增多。F组直径50～100μm的肌性动脉的血管厚度百分比（WT%）和血管壁面积百分比（WA%）均显著高于对应的S组(P<0.01)。5与S组比较，F组大鼠血清ET-1、cGMP浓度均升高，差异有统计学意义(P<0.05)。
结论：1本实验结果显示通过腹主动脉-下腔静脉造瘘手术可以成功模拟先心病左向右分流所致肺动脉高压早中期病理生理状态。2分流2w后大鼠开始出现右心室肥厚，分流4-6w后大鼠心室肥厚加重，并发生全心肥厚，但其右心室肌仍处于心肌肥厚代偿期。分流8w大鼠右心室心肌处于肥厚失代偿期。3肺动脉高压早中期病理生理变化与血清ET-1和cGMP浓度升高有关。
第二部分：他达那非对左向右分流肺动脉高压大鼠肺血管重构的影响
目的：通过建立分流型肺动脉高压大鼠模型，模拟先天性心脏病所致的肺动脉高压早中期阶段的病理生理状态，探讨磷酸二酯酶5抑制剂TDF短期和中期应用对分流型肺动脉高压大鼠肺血管重构的影响。
方法：健康雄性SD大鼠48只，随机分为6组，每组8只，假手术4w组6w组（S4、S6组）、分流4w组6w组（F4、F6组）、TDF4w组6w组（T4、T6组）：除S两组外其余四组麻醉后开腹行腹主动脉-腔静脉造瘘手术，S4、S6组与F4、F6组术后第4w开始使用生理盐水灌胃；T4、T6组术后第4w开始使用同等容积TDF（10mg/kg/d）灌胃。各组均从术后第三周开始灌胃，S4、F4、T4三组灌胃1w，S6、F6、T6灌胃3w。S4、F4、T4组和S6、F6、T6组分别于4w或6w后再次麻醉开胸，按上述方法测量肺动脉及右心室压力，大鼠右下肺组织行HE染色观察肺动脉形态学变化。酶联免疫法测定血清ET-1与cGMP含量。免疫组织化学染色测定肺动脉内皮素A受体（ETRA）和内皮素B受体（ETRB）表达变化。
结果：1与S4和S6比较，F4与F6组大鼠肺动脉mPAP、SPAP和右心室+dP/dtmax均升高(P<0.05)。与F4和F6组相比，T4与T6组大鼠mPAP、SPAP和右心室+dP/dtmax均升高(P<0.05)。2大鼠血清cGMP浓度：与F4和F6组相比，T4与T6组大鼠cGMP升高(P<0.05)。3大鼠血清ET-1浓度：与S4和S6比较，F4与F6组大鼠血清ET-1浓度升高(P<0.05)。与F4和F6组相比，T4与T6组大鼠血清ET-1浓度升高(P<0.05)。4与F4和F6组相比，T4与T6组大鼠肺动脉WT%和WA%均降低(P<0.01)。ETRA在肺动脉平滑肌细胞上表达, ETRB大部分在肺动脉内皮细胞上表达，少量在平滑肌细胞上表达，阳性显棕黄色颗粒。F组ETRA表达量高于S组(P<0.01)，T组表达量低于F组(P<0.01)且与S组比较无统计学差异(P<0.05)。F组ETRB表达显著低于S组(P<0.01)，T组表达量高于F组(P<0.01)。
结论：1TDF通过抑制血管壁的磷酸二酯酶-5对cGMP的降解，提高肺血管平滑肌cGMP的浓度。2cGMP水平的提高，降低了血管平滑肌的张力，从而有效降低分流型早中期肺动脉高压大鼠的肺动脉压力，改善肺血管重塑。3TDF使内源性ET-1浓度升高，可能与其改变肺血管收缩/舒张平衡调节有关。
第三部分：TDF对分流型肺动脉高压大鼠心肌Cx43磷酸化影响的研究
目的：通过建立大鼠分流型肺动脉高压模型，模拟先天性心脏病所致的早中期肺动脉高压，探讨磷酸二酯酶5抑制剂TDF短期和中期应用对分流型肺动脉高压早中期大鼠心肌肥厚的影响及心肌Cx43磷酸化的影响。
方法：分组方法和造模型同前；S4、F4、T4组和S6、F6、T6组分别于4w或6w后再次麻醉开胸，取出大鼠心脏进行Masson特殊染色观察心肌胶原含量变化。免疫组织化学染色测定大鼠心肌Cx及pCx表达及分布。Western-blot法检测Cx43和pCx43的表达。
结果：1与F4、F6组相比，T4、T6两组RV/BW、(LV+S)/BW和RV/(LV+S)均降低(P<0.05)。2与F4、F6组相比，T4、T6两组心肌CVF%显著降低(P<0.01)。3免疫组化结果：与S4相比，F4组心肌细胞Cx43与pCx43均表达明显增多(P<0.05)；F6组Cx43与pCx43表达介于S4、F4两组之间，与两者相比无统计学意义(P>0.05)；与F组相比，T组表达无明显变化，但Cx43从闰盘向心肌保质转移现象明显好转，闰盘OD/总体细胞OD明显增多(P<0.05)。4Western-blot结果：与S4相比，F4组Cx43、pCx43表达升高(P<0.05)；与F4组相比，F6组Cx43、pCx43表达降低到S4和F4两组之间水平(P>0.05)；与F4组相比，T4组Cx43、pCx43表达无明显变化(P>0.05)；与F6组相比，T6组Cx43、pCx43表达减少(P<0.05)。
结论：1TDF可改善肺动脉高压早、中期右心室收缩功能。2TDF抑制右心室肌肥厚，增强右心室收缩功能可能与对闰盘处Cx43有保护作用有关。
第四部分：香豆雌酚通过cAMP/PKA通路介导一氧化氮抑制麻醉大鼠
颈动脉窦压力感受器活动
目的：大量研究证实植物雌激素对心血管系统具有保护作用。但植物雌激素香豆雌酚（coumestrol，CMT）对动脉压力感受器活动（carotidbaroreceptor activity， CBA）的影响尚不明确。本研究拟观察CMT对CBA的影响并探讨其可能作用机制。
方法：通过隔离灌流大鼠颈动脉窦，记录窦神经传入放电Westen-blot和ELISA等方法，研究CMT对CBA的影响。
结果：CMT抑制CBA，使颈动脉窦功能曲线右下移位，并浓度依赖性的抑制窦神经放电的峰值和积分。雌激素受体拮抗剂他莫昔芬（tamoxifen）预处理不影响CMT的作用；而一氧化氮（NO）合酶抑制剂（NG-硝基-L-精氨酸甲基酯，L-NAME）能完全取消CMT对CBA的抑制作用；一氧化氮供体（SIN-1）可以模拟该抑制效应。CMT可浓度依赖性的增加颈动脉分叉组织内cAMP水平和Ser1176-eNOS磷酸化水平，并且Ser1176-eNOS磷酸化可被高选择性PKA抑制剂H89阻断。
结论：CMT可以通过cAMP/PKA通路刺激局部组织释放NO抑制CBA，且该作用与雌激素受体的基因效应无关。
Part Ⅰ：ET-1and cGMP participated in the process of lung pulmonaryhypertension
Objective: Pulmonary arterial hypertension (PAH) is described as akind of pathophysiological syndrome characterized by a progressiveraised pulmonary arterial pressure and pulmonary vascular resistance,resulting in right heart failure or even death. This study was designed toestablish the abdominal aorta-inferior vena cava shunt model in rats andto investigate the morphological and molecular mechanisms ofpulmonary vascular remodeling and right ventricular hypertrophy.
Method:72male SD rats were randomly divided into two groups: thesham operation (Sham, S) group and the fistula (Fistula，F) group. Accordingto Garcia’s method, a midline abdominal incision was made and thedescending aorta above the renal bifurcation was cleared of adjacent tissuesand a snugger was placed. The infra-renal portion of the aorta and inferiorvena cava were exposed at a site where the two vessels share a common fascia.The supra-renal portion of the abdominal aorta was then occluded with asnugger to control bleeding. The shunt was then created with an18Gangiocatheter inserted between the sutures on the anterior surface of the aorta.Following release of the snugger in thirty seconds, we observed mixing ofarterial and venous blood in the IVC, with distention of and pulsations in theIVC. Sham animals were treated the same way except for IVC puncture. Ratswere anesthetized again to measure the pressure of pulmonary artery and rightventricular (RV) four or six weeks after the corresponding treatment via amicro-catheter in the right ventricular outflow tract placed by thoracotomy.The pulmonary morphological changes were observed by HE staining of the right lower lobe tissue of the lung. The relative weight of ventricular wasdetermined and the changes of myocardial collagen were identified by Massonspecial staining.
Results:1. There is no significantly difference of pulmonary arterypressure between two groups (P>0.05). Compared with S group, the meanpulmonary artery pressure (mPAP), the right ventricular systolic pressure(RVSP) and the maximal positive velcity of right ventricular pressure(+RVdP/dtmax) were increased in F group.2. Compared with S group, thepercentage of wall thickness (WT%) and the wall-to-total area ratio (WA%)were increased duing the process of the model (P<0.05).3. The two kindsmuscular artery which extremely diameter limited to50~100μm respectivelywere highly increased in F4and F6rats.4The RV/body weight ratio (RV/BW)and the percentage of (left ventricle+interventricular septum)/body weight(LV+S)/BW were markedly increased in F groups compared with that of S rats.5Compared with S4and S6, the myocardial collagen volume fraction (CVF)increased in F4and F6groups. Moreover, the CVF of F6rats was much higherthan that in F4.
Conclusions:1Arterio-venous shunt induced pulmonary hypertensionand right ventricular hypertrophy model can be successfully established in ratsby the abdominal aorta and inferior vena cava fistula surgery.2The rightventricular hypertrophy was detected at2w after aorto-caval shunt. After8wof the aorto-caval shunt, discompensation of cardiac hypertrophy was detectedin model rats.3The concentration of ET-1and cGMP was increased with theprocess of the pulmonary hypertension.
Part Ⅱ：The effect of Tadalafil on pulmonary vascular remodeling inpulmonary hypertension model caused by left to right shunt
Objective: To investigate the effects of Tadalafil, a Phosphodiesterase5inhibitor, on the pulmonary vascular remodeling in shunt-induced pulmonaryhypertension via establishing shunt pulmonary hypertension model in rats.
Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after sham operation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). The rats in T4and T6group were givenTadalafil (10mg/kg/d) by gavage four weeks after the aorta-caval fistulasurgery. The remaining rats were received the same volume of saline.Therefore, the S4, F4and T4groups received gavage for one week, while theS6, F6and T6groups for three weeks. The rats were anesthetized to measurethe mapped RVSP according to the above mentioned method after thecorresponding treatment. The pulmonary morphological changes wereobserved by HE staining of the right lower lobe tissue of the lung. ELISA isused to determinate serum content of ET-1and cGMP. The expression ofETAR and ETBR was detected with Immunohistochemistry staining.
Results:1Compared with S4and S6, the mPAP, sPAP and+PAdP/dtmaxof F4and F6group increased significantly (P<0.05). The mPAP, sPAP and+PAdP/dtmax in T4and T6rats were higher even than that of F4and F6group(P<0.05).2The serum concentration of cGMP increased in T4and T6ratscompared with that of F4and F6groups (P<0.05).3The concentration of ET-1in serum of F4and F6groups is higher than that of S4and S6rats (P<0.05).Compared with the F4and F6group, ET-1concentrations in serum wereincreased obviously in T4and T6rats (P<0.05).4Compared with the F4andF6groups, the WT%and WA%of pulmonary arterial were much lower in T4and T6rats (P<0.01). The expression of ETAR was mainly observed in theSMC of the pulmonary arteries. However, the expression of ETBR wasobserved in endothelial cells and partially in SMC of the pulmonary arteries.The amount of ETAR expression was increased in F group than that in controlgroup (P<0.01). However, in T group the expression of ETRA was obviouslydecreased compared with the control (P<0.01). Furthermore, the amount ofETRB expression was reduced significantly in F group than that in controlgroup (P<0.01). On the contrary, the expression of ETRB was obviouslyhigher in T group than F group (P<0.01), but was still lower than controlgroup (P<0.05).
Conclusions:1Tadalafil can effectively increase cGMP concentrations in pulmonary vascular smooth muscle by inhibiting widespread pulmonaryvascular phosphodiesterase-5ring guanosine monophosphate (cGMP)degradation.2The decrease of cGMP can effectively reduce the shunt withpulmonary hypertensionrat pulmonary artery pressure through the nitric oxide(NO) pathway.3Tadalafil can increase ET-1levels by adjusting pulmonaryvasoconstriction/diastolic balance.
Part Ⅲ：The role of Tadalafil on the Cx43phosphorylation in left-rightshunt pulmonary hypertension
Objective: To investigate the effects of phosphodiesterase-5inhibitorTadalafil on cardiac hypertrophy and the phosphorylation of Cx43in shunt-induced pulmonary hypertension via establishing shunt pulmonaryhypertension model in rats.
Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after shamoperation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). Masson special staining was carried out toobserve the changes of myocardial collagen. The expression of Cx43and thephosphorylation Cx43in heart were determined with immunohistochemicalstaining and Western blot.
Results:1Compared with the F4and F6groups, the RV/BW,(LV+S)/BW and RV/(LV+S) decreased in T4and T6rats (P<0.05).2Compared with F4and F6groups, the CVF in T4and T6groups wassignificantly decreased (P<0.01).3Compared with S4rats, the mean opticaldensity (OD) of the brown positive staining particles of Cx43or pCx43inmyocardium was increased significantly in F4group (P<0.05). There was nosignificant difference between F6and S4or between F6and F4(P>0.05).Compared with the F groups, the OD value of T groups remained unchanged,however, the ratio of the intercalated disc OD to total OD increasedsignificantly (P<0.05),4Western-blot: Compared with the S4group, theexpression of Cx43and pCx43was upregulated in F4group (P<0.05). Therewas no significant difference between F6and S4or between F6and F4(P> 0.05).Compared with F4group, Cx43and pCx43expression in T4group hasno significant changes (P>0.05). Compared with the F6group, the expressionof Cx43and pCx43decreased (P<0.05).
Conclusions:1Tadalafil, the Phosphodiesterase5inhibitors, can notonly reduce pulmonary artery pressure load by expensing the pulmonaryartery smooth muscle, but also attenuate the stimulating effects of beta-adrenergic and postpone the cardiac hypertrophy and remodeling viaincreasing cGMP levels.2Tadalafil can prompt the translocation of Cx43andpCx43from the intercalated disc to the cytoplasm, i.e. preventing loss ordestruction.
Part Ⅳ： Coumestrol inhibits carotid sinus baroreceptor activity bycAMP/PKA depended nitric oxide release in anesthetized male rats
Objective: Although evidences suggest that phytoestrogens offermultiple beneficial effects on the cardiovascular system, but the effects ofcoumestrol (CMT), one of the well-known phytoestrogens, on arterialbaroreceptors are still unknown.
Method: In this study we examine the effects of CMT on arterialbaroreceptors activity (CBA) for the first time by isolated carotid sinusperfusion, sinus nerve afferent discharge recording and western blot.
Results: The results showed that CMT inhibited the CBA, which shiftedthe functional curve of the carotid baroreceptor to the right and downward,with a marked decrease in the peak slope and the peak integral value of carotidsinus nerve discharge in a concentration dependent manner. Also, the possiblemechanisms underlying these effects were examined. We found thatpretreatment with an estrogen-receptor antagonist, tamoxifen, did not affectthe inhibition of CMT on CBA, while pretreatment with NG-nitro-L-argininemethyl ester, an inhibitor of nitric oxide (NO) synthase, could completelyabolish the effects of CMT, moreover, a NO donor, SIN-1, could potentiatethese inhibitory effects. In addition, we also found that the intracellular cAMPlevels and phosphorylation of Ser1176-eNOS protein expression in carotidbifurcation tissue could be increased dose-dependently by CMT, and the phosphorylation of Ser1176-eNOS was blocked by a highly selective PKAinhibitor H89.
Conclusions: These findings indicate that CMT could inhibit CBA viathe local release of NO, which were mediated by the cAMP/PKA pathway andwere unrelated to the estrogenic effect.
已有研究表明肺动脉压力的调节主要受内皮素-1(Endothelin-1，ET-1)和一氧化氮的调节。PAH患者ET-1表达升高，一氧化氮合酶表达下降，导致肺动脉收缩，肺小动脉平滑肌细胞增生肥厚，非肌性动脉肌化，导致肺动脉压力升高和肺血管重构。磷酸二酯酶5（Phosphodiesterase-5，PDE-5）抑制剂可选择性舒张肺血管、降低肺动脉压。他达那非（Tadalafil，TDF）是一种选择性PDE-5抑制剂，它通过增加肺血管平滑肌和内皮细胞中cGMP浓度，舒张肺血管，降低肺血管的阻力进而降低肺动脉压。然而，TDF是否可以通过影响肺动脉ET-1及ET-1受体表达起到抗肺动脉高压的作用尚未见报道。
另有研究证实，缝隙连接蛋白43(Connexins43, Cx43)是Cx基因家族中数量最为丰富的成员，不同水平磷酸化的Cx43可调控细胞信号转导，控制着细胞内许多生理过程，与心律失常、心肌缺血、心衰和心肌肥厚等许多疾病有密切联系。有学者提出通过调控Cx43的表达有助于治疗某些心血管疾病。PDE-5抑制剂能否通过调控右心室Cx43表达起到减轻右心室肌肥厚，改善心功能的作用及其可能机制目前尚无报道。
本研究应用腹主动脉-腔静脉造瘘术建立大鼠分流型肺动脉高压模型，模拟临床左向右分流先心病所致早、中期肺动脉高压。探讨中短期应用磷酸二酯酶5抑制剂TDF对PAH大鼠肺动脉高压的早、中期阶段肺血管重塑、右心室肥厚的影响及其可能机制。为临床中度肺高压病人术前短期应用磷酸二酯酶5抑制剂，降低肺动脉压力，改善心功能提供进一步的理论依据。第一部分：ET-1和cGMP参与大鼠轻中度肺动脉高压心肺功能形态变
化
目的：本实验旨在建立大鼠腹主动脉-腔静脉分流模型，模拟先心病左向右分流肺动脉高压早中期阶段，从功能学和形态学方面探讨该模型大鼠在动静脉分流1-8w内肺血管重构以及右心室肥厚发生发展过程和可能机制。
方法：健康清洁级雄性SD大鼠72只，随机分为2组，假手术组（Sham，S）分流组（Fistula，F）组。参照Garcia的方法，大鼠麻醉后，仰卧位固定，正中切口打开腹腔，暴露腹主动脉和下腔静脉，使用18G穿刺针，穿刺点为腹腔动静脉壁联合部，相当于腹主动脉肾动脉发出点与髂总动脉分叉处连线的中下1/3处,于静脉壁侧刺入进而刺破动静脉壁联合部进入腹主动脉形成瘘口。暂时用小动脉夹在肾动脉分支处上方钳夹腹主动脉，阻断血流；拔出穿刺针，以一滴氰基丙基酸盐粘合剂封住穿刺口。30s后松开小动脉夹。若观察到下腔静脉增粗并伴有波动，则证实造瘘分流建立成功，假手术组大鼠仅单纯开腹，暴露腹主动脉和下腔静脉，腹主动脉夹闭，不作分流手术。两组动物分别于术后1，2，3，4，6，8w测量肺动脉及右心室压力。经右颈动脉取血测量血清ET-1与环磷酸鸟苷(cGMP)含量。右下肺组织行HE染色观察肺动脉形态学变化；取出大鼠心脏测定心室相对重量以及Masson特殊染色观察心肌胶原总量的改变。
结果：1各组大鼠颈动脉平均动脉压差异无统计学意义(P>0.05)。与对应的S组比较，F组大鼠肺动脉平均肺动脉压（mPAP）、肺动脉收缩压（SPAP）升高；右心室发展压（+dP/dtmax）增加(P<0.05)。2与对应的S组比较，F组大鼠右心室肥厚指数RV/(LV+S)在第2w升高，第3-8w与S组相比无差异；左心室与体重比(LV+S)/BW从第4w直到第8w升高(P<0.05)。右心室与体重比（RV/BW）与对应的S组比较，从第2w到第8w升高，(P<0.05)。3与对应的S组比较，F组大鼠心肌纤维明显增粗、排列紊乱、心肌纤维间质成分增多。F组心肌胶原容积百分比（CVF%）均显著高于对应的S组(P<0.01)，并且F组动物4w后较第4w前CVF%升高更显著(P<0.01)。4与对应的S组比较，F组大鼠肺动脉管壁明显增厚，管腔变小。内皮下层不明显，中膜平滑肌层明显增厚，平滑肌纤维直径增粗，细胞层数增多。F组直径50～100μm的肌性动脉的血管厚度百分比（WT%）和血管壁面积百分比（WA%）均显著高于对应的S组(P<0.01)。5与S组比较，F组大鼠血清ET-1、cGMP浓度均升高，差异有统计学意义(P<0.05)。
结论：1本实验结果显示通过腹主动脉-下腔静脉造瘘手术可以成功模拟先心病左向右分流所致肺动脉高压早中期病理生理状态。2分流2w后大鼠开始出现右心室肥厚，分流4-6w后大鼠心室肥厚加重，并发生全心肥厚，但其右心室肌仍处于心肌肥厚代偿期。分流8w大鼠右心室心肌处于肥厚失代偿期。3肺动脉高压早中期病理生理变化与血清ET-1和cGMP浓度升高有关。
第二部分：他达那非对左向右分流肺动脉高压大鼠肺血管重构的影响
目的：通过建立分流型肺动脉高压大鼠模型，模拟先天性心脏病所致的肺动脉高压早中期阶段的病理生理状态，探讨磷酸二酯酶5抑制剂TDF短期和中期应用对分流型肺动脉高压大鼠肺血管重构的影响。
方法：健康雄性SD大鼠48只，随机分为6组，每组8只，假手术4w组6w组（S4、S6组）、分流4w组6w组（F4、F6组）、TDF4w组6w组（T4、T6组）：除S两组外其余四组麻醉后开腹行腹主动脉-腔静脉造瘘手术，S4、S6组与F4、F6组术后第4w开始使用生理盐水灌胃；T4、T6组术后第4w开始使用同等容积TDF（10mg/kg/d）灌胃。各组均从术后第三周开始灌胃，S4、F4、T4三组灌胃1w，S6、F6、T6灌胃3w。S4、F4、T4组和S6、F6、T6组分别于4w或6w后再次麻醉开胸，按上述方法测量肺动脉及右心室压力，大鼠右下肺组织行HE染色观察肺动脉形态学变化。酶联免疫法测定血清ET-1与cGMP含量。免疫组织化学染色测定肺动脉内皮素A受体（ETRA）和内皮素B受体（ETRB）表达变化。
结果：1与S4和S6比较，F4与F6组大鼠肺动脉mPAP、SPAP和右心室+dP/dtmax均升高(P<0.05)。与F4和F6组相比，T4与T6组大鼠mPAP、SPAP和右心室+dP/dtmax均升高(P<0.05)。2大鼠血清cGMP浓度：与F4和F6组相比，T4与T6组大鼠cGMP升高(P<0.05)。3大鼠血清ET-1浓度：与S4和S6比较，F4与F6组大鼠血清ET-1浓度升高(P<0.05)。与F4和F6组相比，T4与T6组大鼠血清ET-1浓度升高(P<0.05)。4与F4和F6组相比，T4与T6组大鼠肺动脉WT%和WA%均降低(P<0.01)。ETRA在肺动脉平滑肌细胞上表达, ETRB大部分在肺动脉内皮细胞上表达，少量在平滑肌细胞上表达，阳性显棕黄色颗粒。F组ETRA表达量高于S组(P<0.01)，T组表达量低于F组(P<0.01)且与S组比较无统计学差异(P<0.05)。F组ETRB表达显著低于S组(P<0.01)，T组表达量高于F组(P<0.01)。
结论：1TDF通过抑制血管壁的磷酸二酯酶-5对cGMP的降解，提高肺血管平滑肌cGMP的浓度。2cGMP水平的提高，降低了血管平滑肌的张力，从而有效降低分流型早中期肺动脉高压大鼠的肺动脉压力，改善肺血管重塑。3TDF使内源性ET-1浓度升高，可能与其改变肺血管收缩/舒张平衡调节有关。
第三部分：TDF对分流型肺动脉高压大鼠心肌Cx43磷酸化影响的研究
目的：通过建立大鼠分流型肺动脉高压模型，模拟先天性心脏病所致的早中期肺动脉高压，探讨磷酸二酯酶5抑制剂TDF短期和中期应用对分流型肺动脉高压早中期大鼠心肌肥厚的影响及心肌Cx43磷酸化的影响。
方法：分组方法和造模型同前；S4、F4、T4组和S6、F6、T6组分别于4w或6w后再次麻醉开胸，取出大鼠心脏进行Masson特殊染色观察心肌胶原含量变化。免疫组织化学染色测定大鼠心肌Cx及pCx表达及分布。Western-blot法检测Cx43和pCx43的表达。
结果：1与F4、F6组相比，T4、T6两组RV/BW、(LV+S)/BW和RV/(LV+S)均降低(P<0.05)。2与F4、F6组相比，T4、T6两组心肌CVF%显著降低(P<0.01)。3免疫组化结果：与S4相比，F4组心肌细胞Cx43与pCx43均表达明显增多(P<0.05)；F6组Cx43与pCx43表达介于S4、F4两组之间，与两者相比无统计学意义(P>0.05)；与F组相比，T组表达无明显变化，但Cx43从闰盘向心肌保质转移现象明显好转，闰盘OD/总体细胞OD明显增多(P<0.05)。4Western-blot结果：与S4相比，F4组Cx43、pCx43表达升高(P<0.05)；与F4组相比，F6组Cx43、pCx43表达降低到S4和F4两组之间水平(P>0.05)；与F4组相比，T4组Cx43、pCx43表达无明显变化(P>0.05)；与F6组相比，T6组Cx43、pCx43表达减少(P<0.05)。
结论：1TDF可改善肺动脉高压早、中期右心室收缩功能。2TDF抑制右心室肌肥厚，增强右心室收缩功能可能与对闰盘处Cx43有保护作用有关。
第四部分：香豆雌酚通过cAMP/PKA通路介导一氧化氮抑制麻醉大鼠
颈动脉窦压力感受器活动
目的：大量研究证实植物雌激素对心血管系统具有保护作用。但植物雌激素香豆雌酚（coumestrol，CMT）对动脉压力感受器活动（carotidbaroreceptor activity， CBA）的影响尚不明确。本研究拟观察CMT对CBA的影响并探讨其可能作用机制。
方法：通过隔离灌流大鼠颈动脉窦，记录窦神经传入放电Westen-blot和ELISA等方法，研究CMT对CBA的影响。
结果：CMT抑制CBA，使颈动脉窦功能曲线右下移位，并浓度依赖性的抑制窦神经放电的峰值和积分。雌激素受体拮抗剂他莫昔芬（tamoxifen）预处理不影响CMT的作用；而一氧化氮（NO）合酶抑制剂（NG-硝基-L-精氨酸甲基酯，L-NAME）能完全取消CMT对CBA的抑制作用；一氧化氮供体（SIN-1）可以模拟该抑制效应。CMT可浓度依赖性的增加颈动脉分叉组织内cAMP水平和Ser1176-eNOS磷酸化水平，并且Ser1176-eNOS磷酸化可被高选择性PKA抑制剂H89阻断。
结论：CMT可以通过cAMP/PKA通路刺激局部组织释放NO抑制CBA，且该作用与雌激素受体的基因效应无关。
Part Ⅰ：ET-1and cGMP participated in the process of lung pulmonaryhypertension
Objective: Pulmonary arterial hypertension (PAH) is described as akind of pathophysiological syndrome characterized by a progressiveraised pulmonary arterial pressure and pulmonary vascular resistance,resulting in right heart failure or even death. This study was designed toestablish the abdominal aorta-inferior vena cava shunt model in rats andto investigate the morphological and molecular mechanisms ofpulmonary vascular remodeling and right ventricular hypertrophy.
Method:72male SD rats were randomly divided into two groups: thesham operation (Sham, S) group and the fistula (Fistula，F) group. Accordingto Garcia’s method, a midline abdominal incision was made and thedescending aorta above the renal bifurcation was cleared of adjacent tissuesand a snugger was placed. The infra-renal portion of the aorta and inferiorvena cava were exposed at a site where the two vessels share a common fascia.The supra-renal portion of the abdominal aorta was then occluded with asnugger to control bleeding. The shunt was then created with an18Gangiocatheter inserted between the sutures on the anterior surface of the aorta.Following release of the snugger in thirty seconds, we observed mixing ofarterial and venous blood in the IVC, with distention of and pulsations in theIVC. Sham animals were treated the same way except for IVC puncture. Ratswere anesthetized again to measure the pressure of pulmonary artery and rightventricular (RV) four or six weeks after the corresponding treatment via amicro-catheter in the right ventricular outflow tract placed by thoracotomy.The pulmonary morphological changes were observed by HE staining of the right lower lobe tissue of the lung. The relative weight of ventricular wasdetermined and the changes of myocardial collagen were identified by Massonspecial staining.
Results:1. There is no significantly difference of pulmonary arterypressure between two groups (P>0.05). Compared with S group, the meanpulmonary artery pressure (mPAP), the right ventricular systolic pressure(RVSP) and the maximal positive velcity of right ventricular pressure(+RVdP/dtmax) were increased in F group.2. Compared with S group, thepercentage of wall thickness (WT%) and the wall-to-total area ratio (WA%)were increased duing the process of the model (P<0.05).3. The two kindsmuscular artery which extremely diameter limited to50~100μm respectivelywere highly increased in F4and F6rats.4The RV/body weight ratio (RV/BW)and the percentage of (left ventricle+interventricular septum)/body weight(LV+S)/BW were markedly increased in F groups compared with that of S rats.5Compared with S4and S6, the myocardial collagen volume fraction (CVF)increased in F4and F6groups. Moreover, the CVF of F6rats was much higherthan that in F4.
Conclusions:1Arterio-venous shunt induced pulmonary hypertensionand right ventricular hypertrophy model can be successfully established in ratsby the abdominal aorta and inferior vena cava fistula surgery.2The rightventricular hypertrophy was detected at2w after aorto-caval shunt. After8wof the aorto-caval shunt, discompensation of cardiac hypertrophy was detectedin model rats.3The concentration of ET-1and cGMP was increased with theprocess of the pulmonary hypertension.
Part Ⅱ：The effect of Tadalafil on pulmonary vascular remodeling inpulmonary hypertension model caused by left to right shunt
Objective: To investigate the effects of Tadalafil, a Phosphodiesterase5inhibitor, on the pulmonary vascular remodeling in shunt-induced pulmonaryhypertension via establishing shunt pulmonary hypertension model in rats.
Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after sham operation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). The rats in T4and T6group were givenTadalafil (10mg/kg/d) by gavage four weeks after the aorta-caval fistulasurgery. The remaining rats were received the same volume of saline.Therefore, the S4, F4and T4groups received gavage for one week, while theS6, F6and T6groups for three weeks. The rats were anesthetized to measurethe mapped RVSP according to the above mentioned method after thecorresponding treatment. The pulmonary morphological changes wereobserved by HE staining of the right lower lobe tissue of the lung. ELISA isused to determinate serum content of ET-1and cGMP. The expression ofETAR and ETBR was detected with Immunohistochemistry staining.
Results:1Compared with S4and S6, the mPAP, sPAP and+PAdP/dtmaxof F4and F6group increased significantly (P<0.05). The mPAP, sPAP and+PAdP/dtmax in T4and T6rats were higher even than that of F4and F6group(P<0.05).2The serum concentration of cGMP increased in T4and T6ratscompared with that of F4and F6groups (P<0.05).3The concentration of ET-1in serum of F4and F6groups is higher than that of S4and S6rats (P<0.05).Compared with the F4and F6group, ET-1concentrations in serum wereincreased obviously in T4and T6rats (P<0.05).4Compared with the F4andF6groups, the WT%and WA%of pulmonary arterial were much lower in T4and T6rats (P<0.01). The expression of ETAR was mainly observed in theSMC of the pulmonary arteries. However, the expression of ETBR wasobserved in endothelial cells and partially in SMC of the pulmonary arteries.The amount of ETAR expression was increased in F group than that in controlgroup (P<0.01). However, in T group the expression of ETRA was obviouslydecreased compared with the control (P<0.01). Furthermore, the amount ofETRB expression was reduced significantly in F group than that in controlgroup (P<0.01). On the contrary, the expression of ETRB was obviouslyhigher in T group than F group (P<0.01), but was still lower than controlgroup (P<0.05).
Conclusions:1Tadalafil can effectively increase cGMP concentrations in pulmonary vascular smooth muscle by inhibiting widespread pulmonaryvascular phosphodiesterase-5ring guanosine monophosphate (cGMP)degradation.2The decrease of cGMP can effectively reduce the shunt withpulmonary hypertensionrat pulmonary artery pressure through the nitric oxide(NO) pathway.3Tadalafil can increase ET-1levels by adjusting pulmonaryvasoconstriction/diastolic balance.
Part Ⅲ：The role of Tadalafil on the Cx43phosphorylation in left-rightshunt pulmonary hypertension
Objective: To investigate the effects of phosphodiesterase-5inhibitorTadalafil on cardiac hypertrophy and the phosphorylation of Cx43in shunt-induced pulmonary hypertension via establishing shunt pulmonaryhypertension model in rats.
Method: Male Spague-Dawley rats (250-290g) were randomly dividedinto the following six groups (n=8): S4, S6(four or six weeks after shamoperation), F4, F6(four or six weeks after shunt), T4and T6group (treatmentwith four or six weeks after shunt). Masson special staining was carried out toobserve the changes of myocardial collagen. The expression of Cx43and thephosphorylation Cx43in heart were determined with immunohistochemicalstaining and Western blot.
Results:1Compared with the F4and F6groups, the RV/BW,(LV+S)/BW and RV/(LV+S) decreased in T4and T6rats (P<0.05).2Compared with F4and F6groups, the CVF in T4and T6groups wassignificantly decreased (P<0.01).3Compared with S4rats, the mean opticaldensity (OD) of the brown positive staining particles of Cx43or pCx43inmyocardium was increased significantly in F4group (P<0.05). There was nosignificant difference between F6and S4or between F6and F4(P>0.05).Compared with the F groups, the OD value of T groups remained unchanged,however, the ratio of the intercalated disc OD to total OD increasedsignificantly (P<0.05),4Western-blot: Compared with the S4group, theexpression of Cx43and pCx43was upregulated in F4group (P<0.05). Therewas no significant difference between F6and S4or between F6and F4(P> 0.05).Compared with F4group, Cx43and pCx43expression in T4group hasno significant changes (P>0.05). Compared with the F6group, the expressionof Cx43and pCx43decreased (P<0.05).
Conclusions:1Tadalafil, the Phosphodiesterase5inhibitors, can notonly reduce pulmonary artery pressure load by expensing the pulmonaryartery smooth muscle, but also attenuate the stimulating effects of beta-adrenergic and postpone the cardiac hypertrophy and remodeling viaincreasing cGMP levels.2Tadalafil can prompt the translocation of Cx43andpCx43from the intercalated disc to the cytoplasm, i.e. preventing loss ordestruction.
Part Ⅳ： Coumestrol inhibits carotid sinus baroreceptor activity bycAMP/PKA depended nitric oxide release in anesthetized male rats
Objective: Although evidences suggest that phytoestrogens offermultiple beneficial effects on the cardiovascular system, but the effects ofcoumestrol (CMT), one of the well-known phytoestrogens, on arterialbaroreceptors are still unknown.
Method: In this study we examine the effects of CMT on arterialbaroreceptors activity (CBA) for the first time by isolated carotid sinusperfusion, sinus nerve afferent discharge recording and western blot.
Results: The results showed that CMT inhibited the CBA, which shiftedthe functional curve of the carotid baroreceptor to the right and downward,with a marked decrease in the peak slope and the peak integral value of carotidsinus nerve discharge in a concentration dependent manner. Also, the possiblemechanisms underlying these effects were examined. We found thatpretreatment with an estrogen-receptor antagonist, tamoxifen, did not affectthe inhibition of CMT on CBA, while pretreatment with NG-nitro-L-argininemethyl ester, an inhibitor of nitric oxide (NO) synthase, could completelyabolish the effects of CMT, moreover, a NO donor, SIN-1, could potentiatethese inhibitory effects. In addition, we also found that the intracellular cAMPlevels and phosphorylation of Ser1176-eNOS protein expression in carotidbifurcation tissue could be increased dose-dependently by CMT, and the phosphorylation of Ser1176-eNOS was blocked by a highly selective PKAinhibitor H89.
Conclusions: These findings indicate that CMT could inhibit CBA viathe local release of NO, which were mediated by the cAMP/PKA pathway andwere unrelated to the estrogenic effect.
引文
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