益气温阳活血化痰方对大鼠低氧高二氧化碳性肺动脉高压的干预作用

Effect of Yiqi Wenyang Huoxue Huatan Formula on Pulmonary Hypertension Induced by Hypoxia and Hypercapnia in Rats

目的研究益气温阳活血化痰方(Yiqi Wenyang Huoxue Huatan Formula,YWHHF)对大鼠低氧高二氧化碳性肺动脉高压的干预作用及机制研究。方法将50只SD大鼠随机分为5组:常氧对照(N)组、低氧高二氧化碳(HH)组、YWHHF高剂量(YH)组、YWHHF中剂量(YM)组、YWHHF低剂量(YL)组,每组10只。除N组置于仓外,其余各组均置于常压低氧高二氧化碳氧仓中,每天8h,每周6天,持续4周。YH、YM及YL组在进仓前0.5h分别予益气温阳活血化痰方0.6、0.3、0.15g/kg灌胃,其余各组大鼠分别用等量生理盐水灌胃,每天1次,连续灌胃4周。采用右心导管法测量大鼠的肺动脉平均压(mPAP)、颈动脉平均压(mCAP)及右心室肥大指数(RVHI);光镜、电镜观察大鼠肺动脉形态学变化,测定肺动脉管壁面积/管总面积(WA/TA)和管腔面积/管总面积(LA/TA)的比值;采用RT-PCR、Western blot分别检测大鼠内质网应激相关蛋白及mRNA(GRP78、CHOP、JNK、Caspase-12)的表达;原位末端标记法(TUNEL)检测各组肺动脉平滑肌细胞的凋亡指数(AI)。结果与N组比较,HH组mPAP、RVHI和WA/TA值增大,LA/TA减小(均P<0.01),ERS相关蛋白及mRNA(GRP78、JNK、Caspase-12、CHOP)表达量均升高(P<0.01),肺动脉平滑肌细胞AI减少(P<0.01);与HH组比较,YH、YM、YL组mPAP、RVHI及WA/TA值减小,LA/TA增大(P<0.05,P<0.01),ERS相关蛋白及mRNA表达量降低(P<0.05,P<0.01),YH组及YM组肺动脉平滑肌细胞AI增加(P<0.05,P<0.01)RVHI;与YL组比较,YH、YM组上述各项指标均有明显改善(P<0.05,P<0.01)。结论YWHHF可通过抑制JNK、Caspase-12、CHOP降低大鼠低氧高二氧化碳性肺动脉高压,综合考虑以中剂量(0.3g/kg)效果最适宜。

Objective To observe the effects and mechanism of Yiqi Wenyang Huoxue Huatan Formula（YWHHF） on hypoxia-hypercapnia pulmonary hypertension rats. Methods Totally 55 SD rats were randomly divided into five groups, i.e., the normoxia control（N） group, the hypoxia-hypercapnia （HH） group, the high-dose YWHHF（YH） group, the middle-dose YWHHF（YM） group, the low-dose YWH- HF（YL） group, 10 in each group. Rats in N group were raised outside the chamber, rats in the rest groups were raised in the oxygen chamber with low oxygen concentration and high CO2 concentration for 8 h daily, 6 days a week for 4 weeks. Rats in YH, YM and YL groups were fed with YWHHF at the daily dose of 0.6, 0.3 and 0.15 g/kg respectively by gastrogavage 0.5 h before entering chamber, equal vol- ume of normal saline were administered to rats in the N group and HH group by gastrogavage. All treat-ment lasted for 4 successive weeks. The mean pulmonary artery pressure（mPAP）, mean carotid artery pressure （mCAP） and right ventricular hypertrophy index （RVHI） were measured by right heart cathe- terization. The morphological changes of pulmonary arteries were observed with light microscope and e- lectron microscope. The ratio of pulmonary vascular wall area and total area（WA/TA）, the ratio of vascu- lar lumen area and total area（LA/TA） were determined. Reverse transcription polymerase chain reaction （RT-PCR） and Western blot were used to detect the expression of ERS related protein and mRNA （GRP78, CHOP, JNK, Caspase-12）. The apoptotic index of pulmonary artery smooth muscle cells was detected by TUNEL. Results Compared with the N group, mPAP, RVHI and WA/TA were increased in the HH group, LA/TA were decreased（P 〈0.01 ）, expression of ERS related protein and mRNA （GRP78, JNK, Caspase-12, CHOP） were increased in the HH group （P 〈0.01 ）, and pulmonary artery smooth muscle cells apoptosis rate were decreased （P 〈0.01）. Compared with the HH group, mPAP, RVHI and WA/TA in YH, YM and YL groups were decreased, LA/-rA were increased （P 〈0.05, P 〈0.01 ）, and ex- pression of ERS related protein and mRNA were decreased in the YH, YM and YL groups （P 〈0.05, P 〈 0.01 ）. The apoptotic rates of pulmonary artery smooth muscle cells in the YH and YM group were signifi- cantly higher than those of in the HH group（P 〈0.05, P 〈0.01）. Compared with the YL group, the above indexes of YH and YM groups were significantly improved （P 〈 0.05, P 〈 0.01 ）. Conclusions YWHHF can inhibit JNK, Caspase-12 and CHOP to reduce the hypoxia-hypercapnia pulmonary hypertension in rats. Comprehensive consideration, the effect of medium dose （0.3 g/kg） is the most appropriate.