茵陈蒿汤调控lncRNA PVT1/miRNA-30a-5p信号通路对重症急性胰腺炎大鼠模型的保护作用

项红; 胡凤林; 陶旭锋; 齐心; 张金楠; 尚东

doi:10.3969/j.issn.1001-5256.2021.01.029

茵陈蒿汤调控lncRNA PVT1/miRNA-30a-5p信号通路对重症急性胰腺炎大鼠模型的保护作用

DOI: 10.3969/j.issn.1001-5256.2021.01.029

项红^1a,
胡凤林^{1a, 2},
陶旭锋³,
齐心^{1a, 2},
张金楠^{1a, 2},
尚东^{1a, 1b, 2, ,}

1a.
大连医科大学附属第一医院中西医结合临床重点学科实验室，辽宁大连 116011
1b.
大连医科大学附属第一医院普外三科，辽宁大连 116011
2.
大连医科大学中西医结合研究院，辽宁大连 116044
3.
大连理工大学化工学院，辽宁大连 116024

基金项目:

国家自然科学基金面上项目 (81873156);

国家重点研发计划“政府间国际科技创新合作/港澳台科技创新合作”重点专项项目 (2018YFE0195200)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：项红、胡凤林负责课题设计，资料分析，撰写论文; 陶旭锋参与收集数据，修改论文; 齐心、张金楠协助收集数据，整理参考文献; 尚东负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
项红(1988—)，女，助理研究员，博士，主要从事胰腺炎发病机制及中西医结合防治研究

胡凤林(1989—)，男，主要从事胆胰疾病的中西医结合治疗研究

通信作者:
尚东，shangdong@dmu.edu.cn

二者对本文贡献相同，同为第一作者

中图分类号: R285.5;R576
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出版历程
- 收稿日期: 2020-07-22
- 录用日期: 2020-08-14
- 出版日期: 2021-01-20

Artemisia capillaris Thunb. decoction exerts a protective effect on rats with severe acute pancreatitis by regulating the lncRNA PVT1/miRNA-30a-5p signaling pathway

Hong XIANG^1a,
Fenglin HU^{1a, 2},
Xufeng TAO³,
Xin QI^{1a, 2},
Jinnan ZHANG^{1a, 2},
Dong SHANG^{1a, 1b, 2
, ,}

1a.
Clinical Laboratory of Integrative Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China
1b.
Third Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China
2.
Institute (College) of Integrative Medicine, Dalian Medical University, Dalian, Liaoning 116044, China
3.
School of Chemical Engineering, Dalian University of Technology, Dalian, Liaoning 116024, China

摘要

摘要: 目的探讨清热利胆的经典中药方剂——茵陈蒿汤(YCHT)对牛磺胆酸钠诱导的大鼠重症急性胰腺炎(SAP)的保护作用及其机制。方法 30只SD大鼠随机分成假手术(SO)组、SAP模型组和YCHT (4.0 g/kg)治疗组，每组10只。造模成功24 h后，留取大鼠胰腺组织和血浆待检测。HE染色观察胰腺病理损伤情况; ELISA法检测血浆淀粉酶、TNFα和IL-1β水平; 免疫荧光染色检测LC-3蛋白的荧光强度，TUNEL检测细胞凋亡情况。Western Blot检测胰腺组织LC-3、Beclin-1、X连锁凋亡抑制蛋白(XIAP)、Caspase-3和NF-κB蛋白表达。实时荧光定量PCR检测lncRNA PVT1和miRNA-30a-5p的表达水平。采用单因素方差分析和Tukey’s检验用于分析多个独立样本之间的差异。结果 YCHT能明显减轻SAP大鼠胰腺组织水肿、坏死、出血及炎性细胞浸润等病理学损伤。与SO组相比，SAP模型组大鼠血浆淀粉酶、炎性因子TNFα和IL-1β水平显著升高，YCHT治疗后血浆淀粉酶及TNFα和IL-1β水平显著降低(P值均＜0.05)。与SO组相比，SAP模型组LC-3Ⅱ/LC-3Ⅰ比值及Beclin-1、XIAP、Caspase-3和NF-κB蛋白表达明显上调，YCHT治疗组LC-3Ⅱ/LC-3Ⅰ、Beclin-1、XIAP和NF-κB的表达水平较SAP模型组显著降低，Caspase-3水平显著升高(P值均＜0.05)。与SO组相比，SAP模型组大鼠胰腺lncRNA PVT1表达显著升高，miRNA-30a-5p表达显著降低(P值均＜0.05);与SAP模型组相比，YCHT显著降低了lncRNA PVT1的表达，增加了miRNA-30a-5p的表达(P值均＜0.05)。结论 lncRNA PVT1/miRNA-30a-5p介导的细胞自噬和凋亡可能是YCHT治疗SAP的一个药物靶点，这为进一步开发中药方剂YCHT治疗SAP提供了实验基础和理论依据。
- 胰腺炎 /
- 大鼠，Sprague-Dawley /
- 茵陈蒿汤 /
- RNA，长链非编码 /
- 微RNAs
Abstract: Objective To investigate the protective effect and mechanism of Artemisia capillaris Thunb. decoction (YCHT), a classic heat-clearing and cholagogic traditional Chinese medicine (TCM) prescription, on rats with severe acute pancreatitis (SAP) induced by sodium taurocholate. Methods A total of 30 Sprague-Dawley rats were randomly divided into sham-operation (SO) group, SAP model group, and YCHT (4.0 g/kg) treatment group, with 10 rats in each group. At 24 hours after successful modeling, pancreatic tissue and plasma samples were collected for analysis. HE staining was used to observe pathological injury of the pancreas; ELISA was used to measure the plasma levels of amylase, tumor necrosis factor-α (TNFα), and interleukin-1β (IL-1β); immunofluorescent staining was used to measure the fluorescence intensity of LC-3 protein, and TUNEL was used to measure cell apoptosis. Western blot was used to measure the protein expression of LC-3, Beclin-1, X-linked inhibitor of apoptosis protein (XIAP), caspase-3, and nuclear factor-kappa B (NF-κB) in the pancreas, and quantitative real-time PCR was used to measure the expression levels of lncRNA PVT1 and miRNA-30a-5p. A one-way analysis of variance and the Tukey's test were used to analyze the differences between multiple independent samples. Results YCHT significantly alleviated the pathological injury of the pancreas of SAP rats, such as edema, necrosis, hemorrhage, and inflammatory cell infiltration. Compared with the SO group, the SAP group had significant increases in the plasma levels of amylase and the inflammatory factors TNFα and IL-1β, and there were significant reductions in the plasma levels of amylase, TNFα, and IL-1β after YCHT treatment (all P < 0.05). Compared with the SO group, the SAP group had significant increases in LC-3II/LC-3I ratio and the protein expression of Beclin-1, XIAP, caspase-3, and NF-κB, and compared with the SAP group, the YCHT group had significant reductions in LC-3II/LC-3I ratio and the protein expression of Beclin-1, XIAP, and NF-κB (all P < 0.05). Compared with the SO group, the SAP group had a significant increase in the expression of lncRNA PVT1 and a significant reduction in the expression of miRNA-30a-5p in the pancreas (both P < 0.05), and compared with the SAP group, the YCHT group had a significant reduction in the expression of lncRNA PVT1 and a significant increase in the expression of miRNA-30a-5p (both P < 0.05). Conclusion Cell autophagy and apoptosis mediated by lncRNA PVT1/miRNA-30a-5p may be a drug target for YCHT treatment of SAP, which provides experimental and theoretical bases for further development of the TCM prescription YCHT for the treatment of SAP.
- Pancreatitis /
- Rats, Sprague-Dawley /
- Artemisia Capillaris Thunb. Decoction /
- RNA, Long Noncoding /
- MicroRNAs

HTML全文

图 1 YCHT对SAP大鼠胰腺病理的影响(HE染色，×200)

下载: 全尺寸图片幻灯片

图 2 SO组、SAP模型组和YCHT治疗组胰腺组织TUNEL染色(红色)典型图像(×200)

下载: 全尺寸图片幻灯片

图 3 SO组、SAP模型组和YCHT治疗组胰腺组织LC-3蛋白表达(红色)典型图像(×200)

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图 4 YCHT对自噬和凋亡标记蛋白表达的影响

注：1.SO组; 2.SAP模型组; 3.YCHT治疗组

下载: 全尺寸图片幻灯片

表 1 引物序列

基因	引物序列
lncRNA PVT1	R：5′-ACCAGGATATAAACCTAGTGATAAA-3′
	F：5′-AAGCACCCAATGCAGAATAG-3′
GAPDH	R：5′-ATGTTCCAGTATGACTCTA-3′
	F：5′-CACCCCATTTGATGTTAG-3′
U6	R：5′-GGAACGATACAGAGAAGATTAGC-3′
	F：5′-TGGAACGCTTCACGAATTTGCG-3′

下载: 导出CSV

表 2 各组大鼠胰腺组织病理学评分以及血浆淀粉酶、TNFα、IL-1β水平的比较

组别	大鼠数(只)	病理学评分	淀粉酶(μmol/L)	TNFα(ng/L)	IL-1β(ng/L)
SO组	10	1.667±0.577	2.259±0.935	4.444±1.684	2.129±0.931
SAP模型组	10	13.000±1.000¹⁾	16.156±3.916¹⁾	42.791±10.502¹⁾	11.054±0.850¹⁾
YCHT治疗组	10	5.000±1.000²⁾	4.400±1.089²⁾	14.250±2.821²⁾	4.739±0.727²⁾
F值		130.90	95.61	98.13	298.50
P值		＜0.001	＜0.001	＜0.001	＜0.001
注：与SO组相比，1)P＜0.05;与SAP模型组相比，2)P＜0.05。

下载: 导出CSV

表 3 各组自噬和凋亡标记蛋白相对表达量的比较

组别	大鼠数(只)	LC-3Ⅱ/LC-3Ⅰ	Beclin-1	XIAP	Caspase-3	NF-κB
SO组	10	0.360±0.049	0.767±0.017	0.227±0.002	0.264±0.035	0.701±0.007
SAP模型组	10	3.945±0.093¹⁾	1.289±0.013¹⁾	0.254±0.004¹⁾	0.618±0.035¹⁾	0.754±0.000¹⁾
YCHT治疗组	10	1.667±0.062²⁾	0.310±0.021²⁾	0.102±0.016²⁾	1.278±0.061²⁾	0.682±0.014²⁾
F值		1498.0	2482.0	204.4	272.4	48.9
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与SO组相比，1)P＜0.05;与SAP模型组相比，2)P＜0.05。

下载: 导出CSV

表 4 YCHT对胰腺组织中lncRNA-PVT1和miRNA-30a-5p相对表达量的影响

组别	大鼠数(只)	lncRNA-PVT1	miRNA-30a-5p
SO组	10	1.003±0.019	1.003±0.089
SAP模型组	10	1.626±0.060¹⁾	0.021±0.003¹⁾
YCHT治疗组	10	0.253±0.178²⁾	3.404±0.129²⁾
F值		119.7	1111.0
P值		＜0.001	＜0.001
注：与SO组相比，1)P＜0.05;与SAP模型组相比，2)P＜0.05。

下载: 导出CSV

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