益气活血通络解毒方对肺缺血／再灌注损伤小鼠细胞凋亡及c—Jun氨基末端激酶通路的影响

Effects of traditional Chinese medicine Yiqi Huoxue Tongluo Jiedu prescription on pneumocyte apoptosis and c-Jun N-terminal protein kinase pathway in mice after lung ischemia/reperfusion injury

目的观察益气活血通络解毒方（YHTJF）对小鼠肺缺血／再灌注（I／R）损伤（LIRI）的影响，并探讨c-Jun氨基末端激酶（JNK）是否参与其凋亡机制。方法选择雄性C57BL／6J小鼠70只，按随机数字表法分为正常对照组（C组）、羧甲基纤维素钠（CMC-Na）＋正常对照组（CMC-Na＋C组）、CMC-Na＋假手术组（CMC-Na＋S组）、CMC-Na＋I/R模型组和CMC-Na＋YHTJF低、中、高浓度干预组（CMC-Na＋YL组、CMC-Na＋YM组、CMC-Na＋YH组）。C组不进行任何处理；CMC-Na＋S组只开胸，不夹闭肺门；其余各组均开胸夹闭肺门30min，再松开动脉夹使左肺再灌注3h。术毕处死小鼠留取肺组织，光镜和电镜下观察肺组织形态学及超微结构改变，并观察肺泡损伤定量评估指标（IQA）的变化；用原位末端缺刻标记试验（TUNEL）检测肺组织细胞凋亡指数（AI）；用蛋白质免疫印迹试验（Western Blot）和反转录-聚酶链反应（RT-PCR）检测JNK、葡萄糖调节蛋白78（GRP78）的mRNA和蛋白表达；并分析肺组织AI与JNK、GRP78的mRNA和蛋白表达及IQA的相关性。结果CMC-Na＋I／R组IQA、AI及JNK和GRP78 mRNA和蛋白表达均较CMC-Na＋S组明显升高[IQA：（74．00±7．31）％比（7．00±1．23）％，AI：（64．40±11．97）％比（5．60±1．14）％，JNKmRNA（灰度值）：1．143±0．284比0．152±0．128，GRP78 mRNA（灰度值）：0．897±0．129比0．284±0．044，JNK蛋白（A值）：0．428±0．074比0．073±0．052，GRP78蛋白（A值）：1．075±0．145比0．589±0．060j，CMC-Na＋YL组、CMC-Na＋YM组、CMC-Na＋YH组IQA、AI、JNK mRNA和蛋白、GRP78mRNA表达均较CMC-Na＋I／R组明显下降，以CMC-Na＋YM组较CMC-Na＋YL和CMC-Na＋YH组下降程度更显著[IQA：（26．20±3．35）％比（34．00±5．34）％、（41．20±9．18）％，AI：（29．40±3．05）％比（48．20±3．83）％、（39．20±6．14）％，JNK mRNA（灰度值）：0．681±0．130比0．804±0．153、0．938±0．11，GRP78 mRNA（灰度值）：0．450±0．105比0．747±0．231、0．566±0．115，JNK赁白（A值）：0．188±0．049比0．261±0．065、0．209±0．063，均P〈0．01]，CMC-Na＋YH组、CMC-Na＋YL组和CMC-Na＋YM组GRP78蛋白表达较CMC-Na＋I／R组明显升高，以CMC-Na＋YH组升高程度较CMC-Na＋YL组和CMC-Na＋YM组更显著（A值：1．429±0．226比1．130±0．169、1．128±0．177，均P〈0．01）。光镜下可见各组细胞凋亡以肺血管内皮细胞、肺泡上皮细胞为主，棕色颗粒为阳性细胞。电镜下可见：CMC-Na＋I／R组细胞核固缩并边集在核膜下，胞质浓缩，板层体减少、排空增多，细胞膜上微绒毛减少或消失，线粒体肿胀，肺泡隔及毛细血管内炎性细胞附壁增多。与CMC-Na＋I／R组比较，CMC-Na＋YL组、CMC-Na＋YM组、CMC-Na＋YH组肺组织超微结构损伤减轻，肺泡结构超微结构清晰可辨，核染色质较均匀，胞质增多，Ⅱ型肺泡上皮细胞表面微绒毛较多，板层小体数量增多，线粒体肿胀减轻，以CMC-Na＋YM组减轻最明显。肺组织AI与JNK、GRP78的mRNA和蛋白表达、IQA均呈显著正相关性（r值分别为0．907、0．928、0．880、0．712、0．911，均P〈0．01）。结论YHTJF可有效减轻小鼠LIRI肺组织细胞的凋亡，其机制可能与抑制JNK通路有关。

Objective To observe the effects of Yiqi Huoxue Tongluo Jiedu fang （YHTJF） on pneumocyte apoptosis after lung isehemia/reperfusion （I/R） injury （LIRI） in mice and to investigate whether c-Jun N-terminal protein kinase （,INK） is involved in the mechanism of apoptosis. Methods Seventy C57BL/6J male mice were randomly divided into seven groups： normal control group （C group）, carboxyl methyl cellulose-Na＋ormal control group （CMC-Na＋ group）, CMC-Na＋ham group （CMC-Na＋ group）, CMC-Na＋/R group （CMC-Na＋/R group） and CMC-Na＋HTJF-low, -middle, -high dose groups （CMC-Na＋L, CMC-Na＋M, CMC-Na＋H groups）. C group did not undergo any processing; in CMC-Na＋ group, only was chest opened without clipping the lung hilum; in the rest of the four groups, they all underwent opening of the chest and clipping the lung hilum for 30 minutes, then the clipping of artery was relieved and left lung repeffusion was carried out for 3 hours. After operation, the mice were sacrificed, the lung tissues were harvested. Under light and electron microscopes, the lung morphological and ultra-structural changes were observed, and the changes of index of quantitative evaluation for alveolar damage （IQA） were determined. The terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） was applied to evaluate the apoptosis index （AI） of the lung tissues. The protein and mRNA expressions of JNK and glucose regulating protein 78 （GRP78） in lung tissues were detected by Western Blot and reverse transcription-polymerase chain reaction （RT-PCR）; the correlations between lung AI and the expressions of mRNA and protein of JNK and GRP78, IQA were analyzed. Results Compared with CMC-Na＋ group, IQA, AI and mRNA and the protein expressions of JNK and GRP78 in CMC-Na＋/R group were obviously higher [IQA： （74.00 ± 7.31）% vs. （7.00± 1.23）%, AI： （64.40 ± 11.97）% vs. （5.60 ± 1.14）%, JNK mRNA （gray value）： 1.143 ± 0.284 vs. 0.152 ± 0.128, GRP78 mRNA （gray value）： 0.897 ± 0.129 vs. 0.284± 0.044, JNK protein （A value）： 0.428 ±0.074 vs. 0.073 20.052, GRP78 protein （A value）： 1.075 ± 0.145 vs. 0.589 ± 0.060]. Compared with CMC-Na＋/R group, the IQA, AI, protein and mRNA expressions of JNK and GRP78 in CMC-Na＋L, CMC-Na＋M, CMC-Na＋H groups were all lower, and the degree of reduction in group CMC-Na＋M was the most remarkable, greater than that in CMC-Na＋L or CMC-Na＋H group [IQA： （26.20 ± 3.35）% vs. （34.00±5.34）%, （41.20±9.18）%, AI： （29.40±3.05）% vs. （48.20±3.83）%, （39.20±6.14）%, JNK mRNA （gray value）： 0.681 ± 0.130 vs. 0.804 ± 0.153, 0.938 ± 0.11, GRP78 mRNA （gray value）： 0.450 ± 0.105 vs. 0.747 ± 0.231, 0.566± 0.115, JNK protein （A value）： 0.188± 0,049 vs. 0.261 ±0.065, 0.209 ± 0.063, all P 〈 0.01], compared with the CMC-Na＋/R group, the expression of GRP78 protein was obviously higher in CMC-Na＋H, CMC-Na＋L, CMC-Na＋M groups and the most remarkably high was in CMC-Na＋H group （A value： 1.429 ± 0.226 vs. 1.130± 0.169, 1.128 ±0.177, all P 〈 0.01）. The apoptosis of each group was mainly in the pulmonary vascular endothelial cells and alveolar epithelial cells, and brown particles were positive cells under light microscope. Under transmission electron microscope： nuclear pyknosis and margination under the nuclear membrane, cytoplasm condensed, lamellar bodies decreased and emptying increased, cell membrane microvilli decreased or disappeared, mitochondria swelling, inflammatory cells increased in alveolar septum and adhering onto the capillary walls could be seen in CMC-Na＋/R group. Compared with CMC-Na＋/R group, the lung tissue ultrastructural damage alleviated, uhrastructure of alveoli clearly seen, nuclear chromatin relatively uniform, cytoplasm increased, type Ⅱ alveolar epithelial cell surface microvilli relatively plenty, lamellar corpuscle number increased, mitochondria swelling ameliorated in CMC-Na＋H, CMC-Na＋L, CMC-Na＋M groups and the most remarkable one was CMC-Na＋M group. AI was significantly positive correlated with the mRNA and protein expressions of JNK, GRP78 and IQA （r = 0.907, 0.928, 0.880, 0.712, 0.911, all P 〈 0.0l）. Conclusions YHTJF may effectively alleviate the cell apoptosis in mice LIR1, and its mechanism may be related to the inhibition of JNK pathway.