实验性肺动脉高压大鼠心室间糖酵解代谢相关基因筛选和差异

Glycoltic metabolism gene changes in left and right ventricles in experimental rat pulmonary arterial hypertension model

目的 探讨野百合碱（MCT）所致肺动脉高压（PAH）大鼠左、右心室能量代谢转移及可能的差异位点.方法 成年Sprague-Dawley大鼠,颈背部皮下一次性注射MCT（50 mg/kg）建立PAH右心衰竭模型,同时设立对照组,模型组按MCT作用时间又分为3组,即MCT-2周组、MCT-3周组和MCT-4周组（MCT-2w组、MCT-3w组和MCT-4w组）.采用右心导管法检测各组大鼠血液动力学指标,右心室/（左心室＋室间隔）重量计算右心室肥厚指数,免疫组织化学法检测心室壁细胞增殖细胞核抗原（PCNA）以及原位末端转移酶标记（TUNEL）的变化,实时定量PCR方法从糖酵解途径候选位点中筛选表达差异基因.结果 （1）血液动力学指标及右心室肥厚指数：MCT-3w组大鼠平均肺动脉压（mPAP）和右心室收缩末压（RVSP）均高于对照组（P均＜0.01）,提示PAH模型建立成功.MCT-4w组大鼠mPAP和RVSP亦均高于对照组（P均＜0.01）,但与MCT-3w组比较差异无统计学意义.对照组、MCT-2w组、MCT-3w组和MCT-4w组大鼠右心室肥厚指数呈递增趋势,MCT-3w组和MCT-4w组与对照组比较差异均有统计学意义,P分别＜0.05和0.01.（2）左、右心室心肌组织形态学：HE结果显示对照组大鼠左、有心室组织心肌细胞排列整齐,肌纤维走行一致,心肌细胞胞核清楚.MCT-3w组大鼠心肌细胞则排列紊乱,肌纤维走行不清晰,心肌细胞肌质溶解呈网状,肌间质增大,模型组各亚组间左、右心室组织形态学差异不明显.（3）左、右心室心室壁细胞中PCNA和TUNEL表达：对照组、MCT-2w组、MCT-3w组和MCT-4w组大鼠左、右心室室壁细胞数呈递增趋势,PCNA阳性细胞比例递增;而TUNEL阳性细胞比例递减,且MCT-3w组和MCT-4w组大鼠右心室室壁细胞中阳性细胞数多于左心室室壁细胞（P均＜0.05）.（4）左、右心室心肌组织中糖酵解途径候选基因的筛选：MCT-4w组大鼠左心室心肌组织中HK1、HK2、PDHα1和LDHA基因表达水平均高于对照组（P均＜0.05）.MCT-4w组大鼠右心室心肌组织LDHA基因表达水平高于对照组（P＜0.05）,而MCT-3w组和MCT-4w组HK1基因表达水平则均高于对照组（P均＜0.05）,且MCT-4w组高于MCT-3w组（尸＜0.05）.（5）左、右心室心肌组织中HK1阳性细胞表达及其蛋白表达：免疫组织化学结果表明,对照组、MCT-2w组、MCT-3w组和MCT-4w组大鼠左、右心室心肌组织中HK1阳性细胞表达递增,且MCT-3w组和MCT-4w组大鼠右心室的阳性数均多于左心室（P均＜0.05）.MCT-3w组和MCT-4w组大鼠左、右心室心肌组织中HK1蛋白表达水平均高于对照组（P均＜0.05）.结论 PAH时左、右心室均发生能量代谢转移,右心室HK1表达早于左心室,提示干预右心室糖酵解途径可能成为延缓PAH右心衰竭的策略之一.

Objective To explore potential divergent glycolytic metabolism gene changes between left and right ventricle in the monocmtaline （MCT） induced pulmonary arterial hypertension （PAH） rat model.Methods PAH was induced by a single subcutaneous injection of MCT （50 mg/kg） in rats.Control rats were injected with normal saline.MCT-PAH rats were randomly divided into MCT-2week,MCT-3week and MCT-4week groups （MCT-2w,3w,4w）.At the end of study,the hemodynamics and right ventricular hypertrophy were compared among groups.The expression levels of proliferating cell nuclear antigen （PCNA） and TdT-mediated dUTP nick end labeling （TUNEL） in left and right ventricular cells were compared.The glycolytic key candidate genes expression was screened between two ventricles.Results After three to four weeks MCT injection,mean pulmonary arterial pressure,right ventricular systolic pressure and right ventricular hypertrophy index were all significantly increased compared to control group （all P ＜ 0.05）.Both left and right ventricular morphology and structure changes were observed in all PAH rats and were similar between left and right ventricular cells.Left and right ventricular cells increased while apoptotic cells decreased in proportion to the duration post MCT injection and the PCNA positive cells in the right ventricle were higher than in the left ventricle in rats post 3 and 4 weeks MCT injection （P ＜0.05）.The HK1,HK2,PDHα1 and LDHA mRNA expression in the left ventricle and LDHA mRNA expression were significantly upregulated after 4 weeks MCT injection compared to control rats （all P ＜ 0.05）.Moreover,HK1 mRNA expression in the left ventricle was significantly higher in the MCT-PAH-4w group than in MCT-PAH-3w group （P ＜0.05）.Immunohistochemistry analysis evidenced increasing HK1 positive cells in both left and right ventricle in proportion to MCT injection time and positive HK1 cells were significantly higher in the right ventricle than in left ventricle of MCT-PAH-3w and MCT-PAH-4w rats.Furthermore,the HK1 protein expression in left ventricular tissue form MCT-PAH-4w group and in right ventricular tissue from MCT-PAH-3w and MCT-PAH-4w groups were also significantly upregulated compared to control group （P ＜ 0.05）.Conclusions Energy metabolic shift occurs both in the left and right ventricles in this PAH model.Upregulated HK1 expression appeares earlier in right ventricle compared to left ventricle.Interference on right ventricular glycolysis may be a potential novel therapy target of PAH.