加味桃仁承气汤对机械通气相关性肺损伤大鼠细胞自噬的影响

doi:10.11958/20230374

天津医药 ›› 2023, Vol. 51 ›› Issue (12): 1349-1355.doi: 10.11958/20230374

加味桃仁承气汤对机械通气相关性肺损伤大鼠细胞自噬的影响

孙治霞¹(), 王丽辉¹, 索红亮²^,^△(), 陈乾¹

1.河南省中医院重症医学科（邮编450002）
2.河南省中医院心病科（邮编450002）

收稿日期:2023-03-15 修回日期:2023-07-11 出版日期:2023-12-15 发布日期:2023-12-22
通讯作者: ^△ E-mail：671818111@qq.com
作者简介:孙治霞（1980），女，副主任医师，主要从事重症呼吸及心血管病方面研究。E-mail：sunzhixia12@163.com
基金资助:
河南省卫生健康委国家中医临床研究基地科研专项(2021JDZX2033)

Effect of Jiawei Taoren Chengqi Decoction on autophagy in rats with mechanical ventilation-induced lung injury

SUN Zhixia¹(), WANG Lihui¹, SUO Hongliang²^,^△(), CHEN Qian¹

1. Department of Critical Care Medicine, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou 450002, China
2. Department of Cardiology, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou 450002, China

Received:2023-03-15 Revised:2023-07-11 Published:2023-12-15 Online:2023-12-22
Contact: ^△ E-mail：671818111@qq.com

孙治霞, 王丽辉, 索红亮, 陈乾. 加味桃仁承气汤对机械通气相关性肺损伤大鼠细胞自噬的影响[J]. 天津医药, 2023, 51(12): 1349-1355.

SUN Zhixia, WANG Lihui, SUO Hongliang, CHEN Qian. Effect of Jiawei Taoren Chengqi Decoction on autophagy in rats with mechanical ventilation-induced lung injury[J]. Tianjin Medical Journal, 2023, 51(12): 1349-1355.

摘要/Abstract

摘要：

目的探究加味桃仁承气汤通过腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路对机械通气相关性肺损伤（VILI）大鼠细胞自噬的影响。方法将60只SPF级雄性SD大鼠随机分为假手术组（Sham组）、模型组（Model组）、加味桃仁承气汤低剂量组（中药-L组，2.85 g/kg）、加味桃仁承气汤高剂量组（中药-H组，8.55 g/kg）和加味桃仁承气汤高剂量+AMPK抑制剂Compound C组（中药-H+CC组，加味桃仁承气汤8.55 g/kg+Compound C 250 μg/kg），每组12只。于插管即刻和机械通气1 h、2 h与4 h时测定氧合指数（OI）。机械通气结束后收集肺泡灌洗液（BALF），酶联免疫吸附试验（ELISA）检测BALF中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-18水平；处死大鼠，取肺组织测定湿/干重（W/D）比值；HE染色观察各组大鼠肺组织病理学改变并进行肺损伤评分；透射电镜观察肺泡上皮细胞形态。RT-qPCR检测大鼠肺组织中的AMPK、mTORC1 mRNA表达水平；Western blot检测大鼠肺组织中AMPK、p-AMPK、mTORC1、p-mTORC1和自噬相关蛋白的表达情况。结果与Sham组相比，Model组大鼠肺组织病理损伤严重，肺W/D、BALF中TNF-α、IL-1β和IL-18水平、肺损伤评分、mTORC1 mRNA表达水平、p-mTORC1蛋白表达升高（P<0.05）；机械通气2 h、4 h时OI，肺组织AMPK mRNA表达水平，p-AMPK、LC3-Ⅱ/LC3-Ⅰ、Beclin-1蛋白表达降低（P<0.05）。与Model组相比，中药-H组大鼠肺组织病理损伤减轻，相关指标的变化趋势与上述相反（P<0.05），肺泡上皮细胞自噬小体增多。Compound C减弱了加味桃仁承气汤对VILI大鼠的保护作用（P<0.05）。结论加味桃仁承气汤可能通过激活AMPK/mTOR信号通路促进细胞自噬，减轻大鼠VILI。

Abstract:

Objective To explore the impact of Jiawei Taoren Chengqi Decoction on autophagy in rats with mechanical ventilation-induced lung injury (VILI) through adenylate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Methods Sixty SPF male rats were randomly grouped into the sham operation group, the model group, the low-dose Jiawei Taoren Chengqi Decoction group (TCM-L group, 2.85 g/kg), the high-dose Jiawei Taoren Chengqi Decoction group (TCM-H group, 8.55 g/kg) and the high-dose Jiawei Taoren Chengqi Decoction + AMPK inhibitor Compound C group (TCM-H+CC group, Jiawei Taoren Chengqi Decoction 8.55 g/kg+ Compound C 250 μg/kg), 12 in each group. Oxygenation index (OI) was measured immediately after intubation and at 1 h, 2 h and 4 h after mechanical ventilation. Bronchoalveolar lavage fluid (BALF) was collected after mechanical ventilation, levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-18 in BALF were detected by enzyme-linked immunosorbent assay (ELISA). Rats were sacrificed, and lung tissue was taken to measure the wet/dry weight (W/D) ratio. HE staining was used to observe pathological changes of lung tissue in each group of rats. Lung injury scores were carried out. Morphology of alveolar epithelial cells was observed by transmission electron microscopy. RT-qPCR was applied to detect mRNA expression levels of AMPK and mTORC1 in rat lung tissue. Western blot assay was applied to detect expression levels of AMPK, p-AMPK, mTORC1, p-mTORC1 and autophagy-related proteins in rat lung tissue. Results Compared with the sham group, the pathological damage of lung tissue was serious, lung W/D, levels of TNF-α, IL-1β and IL-18 in BALF, lung injury score, mTORC1 mRNA expression level, and p-mTORC1 protein expression were increased in the model group (P<0.05). OI values at 2 h and 4 h of mechanical ventilation, AMPK mRNA expression level, p-AMPK, LC3-II/LC3-I and Beclin-1 protein expression in lung tissue were decreased (P<0.05). Compared with the model group, the pathological damage of lung tissue was alleviated in the Chinese medicine-H group, and the trend of changes in related indexes was opposite to the above (P<0.05). Autophagosomes in alveolar epithelial cells were increased. Compound C attenuated the protective effect of Jiawei Taoren Chengqi Decoction on VILI rats (P<0.05). Conclusion Jiawei Taoren Chengqi Decoction may promote autophagy and reduce VILI in rats by activating AMPK/mTOR signaling pathway.

Key words: ventilator-induced lung injury, Tao He Cheng Qi Tang, autophagy, AMP-activated protein kinases, TOR serine-threonine kinases

中图分类号:

R563

图/表 8

参考文献 18

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基因名称	引物序列（5′→3′）	产物大小/bp
AMPK	上游：GCTGTGGATCGCCAAATTAT	228
AMPK	下游：CACGTGCTCATCATCGAAAG	228
mTORC1	上游：AATACGCCATGAAACACTTCG	214
mTORC1	下游：GGTCTTCCTTGTTTGTGTCCA	214
GAPDH	上游：TGTGTCCGTCGTGGATCTGA	195
GAPDH	下游：TTGCTGTTGAAGTCGCAGGAG	195

基因名称	引物序列（5′→3′）	产物大小/bp
AMPK	上游：GCTGTGGATCGCCAAATTAT	228
AMPK	下游：CACGTGCTCATCATCGAAAG	228
mTORC1	上游：AATACGCCATGAAACACTTCG	214
mTORC1	下游：GGTCTTCCTTGTTTGTGTCCA	214
GAPDH	上游：TGTGTCCGTCGTGGATCTGA	195
GAPDH	下游：TTGCTGTTGAAGTCGCAGGAG	195

组别	插管即刻	通气1 h	通气2 h	通气4 h
Sham组	566.23±34.35	557.68±26.92	548.44±28.48	546.35±25.87
Model组	572.36±32.62	549.73±29.66	265.52±20.31^a	208.94±15.47^a
中药-L组	568.40±33.14	553.17±30.83	286.50±21.22	223.64±16.42
中药-H组	563.83±30.15	555.24±28.35	521.86±27.96^bc	515.30±23.65^bc
中药-H+CC组	568.36±31.07	551.29±29.64	432.64±20.33^d	410.68±23.52^d
F	0.114	0.141	357.164^＊＊	658.911^＊＊

组别	插管即刻	通气1 h	通气2 h	通气4 h
Sham组	566.23±34.35	557.68±26.92	548.44±28.48	546.35±25.87
Model组	572.36±32.62	549.73±29.66	265.52±20.31^a	208.94±15.47^a
中药-L组	568.40±33.14	553.17±30.83	286.50±21.22	223.64±16.42
中药-H组	563.83±30.15	555.24±28.35	521.86±27.96^bc	515.30±23.65^bc
中药-H+CC组	568.36±31.07	551.29±29.64	432.64±20.33^d	410.68±23.52^d
F	0.114	0.141	357.164^＊＊	658.911^＊＊

组别	TNF-α	IL-1β	IL-18
Sham组	16.59±2.48	19.25±2.03	22.34±2.78
Model组	128.45±12.06^a	102.34±11.65^a	139.05±12.59^a
中药-L组	117.63±13.55	96.41±9.50	128.55±11.31
中药-H组	80.27±8.22^bc	68.42±6.22^bc	85.65±8.24^bc
中药-H+CC组	104.20±11.39^d	83.69±8.24^d	116.52±12.35^d
F	223.256^＊＊	197.278^＊＊	257.616^＊＊

加味桃仁承气汤对机械通气相关性肺损伤大鼠细胞自噬的影响

Effect of Jiawei Taoren Chengqi Decoction on autophagy in rats with mechanical ventilation-induced lung injury

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组别	AMPK	mTORC1
Sham组	1.00±0.00	1.00±0.00
Model组	0.57±0.05^a	1.42±0.12^a
中药-L组	0.63±0.06	1.35±0.13
中药-H组	0.85±0.09^bc	1.14±0.11^bc
中药-H+CC组	0.68±0.07^d	1.33±0.12^d
F	48.738^＊＊	15.555^＊＊

组别	p-AMPK/ AMPK	p-mTORC1/ mTORC1	LC3-Ⅱ/ LC3-Ⅰ	Beclin-1
Sham组	0.65±0.07	0.41±0.04	1.78±0.20	0.82±0.08
Model组	0.34±0.03^a	0.86±0.09^a	0.88±0.09^a	0.25±0.03^a
中药-L组	0.39±0.04	0.78±0.09	0.92±0.15	0.32±0.05
中药-H组	0.58±0.06^bc	0.47±0.05^bc	1.57±0.18^bc	0.75±0.08^bc
中药-H+CC组	0.42±0.04^d	0.72±0.07^d	1.16±0.12^d	0.44±0.04^d
F	41.738^＊＊	46.393^＊＊	40.661^＊＊	110.444^＊＊

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