去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响

doi:10.11958/20212359

天津医药 ›› 2022, Vol. 50 ›› Issue (8): 810-816.doi: 10.11958/20212359

去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响

王勇¹(), 牛伟华², 卢成志²^,^Δ(), 许梦萍², 徐建强², 何强², 许学升¹

1商丘市第一人民医院心血管内科（邮编476100）
2天津市第一中心医院心血管内科

收稿日期:2021-10-18 修回日期:2022-04-25 出版日期:2022-08-15 发布日期:2022-08-12
通讯作者: 卢成志 E-mail:610587321@qq.com;lucz8@126.com
作者简介:王勇（1985）,男,主治医师,主要从事冠心病的基础和临床方面研究。E-mail： 610587321@qq.com
基金资助:
国家自然科学基金资助项目(81970303)

Effects of renal denervation on the vascular endothelial cell autophagy and NLRP3 activation in type 2 diabetic rats

WANG Yong¹(), NIU Weihua², LU Chengzhi²^,^Δ(), XU Mengping², XU Jianqiang², HE Qiang², XU Xuesheng¹

1 Department of Cardiology, the First People’s Hospital of Shangqiu, Shangqiu 476100, China
2 Department of Cardiology, Tianjin First Central Hospital

Received:2021-10-18 Revised:2022-04-25 Published:2022-08-15 Online:2022-08-12
Contact: LU Chengzhi E-mail:610587321@qq.com;lucz8@126.com

王勇, 牛伟华, 卢成志, 许梦萍, 徐建强, 何强, 许学升. 去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响[J]. 天津医药, 2022, 50(8): 810-816.

WANG Yong, NIU Weihua, LU Chengzhi, XU Mengping, XU Jianqiang, HE Qiang, XU Xuesheng. Effects of renal denervation on the vascular endothelial cell autophagy and NLRP3 activation in type 2 diabetic rats[J]. Tianjin Medical Journal, 2022, 50(8): 810-816.

摘要/Abstract

摘要：

目的探讨去肾交感神经（RDN）对2型糖尿病（T2DM）大鼠血管内皮细胞自噬及核苷酸结合寡聚化结构域受体蛋白3（NLRP3）炎症小体活化的影响。方法采用高脂饮食联合小剂量链脲佐菌素（STZ）腹腔注射制备T2DM大鼠模型。采用随机数字表法分为对照组（CON组）、糖尿病对照组（T2DM组）、双侧假手术组（Sham组）、双侧手术组（RDN组）,每组6只。分别于基线状态、RDN术前及术后4周测量各项指标的变化。采用离体血管环张力实验评价血管舒张功能,Western blot检测主动脉内皮细胞自噬蛋白Beclin1、微管相关蛋白1轻链3（LC3）、p62、炎症小体NLRP3、胱天蛋白酶1（Caspase-1）,白细胞介素（IL）-1β及内皮型一氧化氮合酶（eNOS）蛋白表达水平。结果与CON组比较,T2DM组大鼠主动脉内皮舒张功能明显降低,自噬蛋白Beclin1、LC3表达降低,p62表达水平升高,且NLRP3、Caspase-1、IL-1β蛋白表达水平升高,eNOS蛋白表达水平降低（P<0.05）。与T2DM组和Sham组相比,RDN组血管内皮舒张功能改善（P<0.05）;自噬蛋白Beclin1、LC3表达升高,p62表达水平降低,且NLRP3、Caspase-1、IL-1β蛋白的表达降低,eNOS蛋白表达水平升高（P<0.05）。结论 T2DM大鼠血管内皮功能异常可能与自噬水平降低及NLRP3炎症小体的过度活化有关。RDN可以增强血管内皮细胞自噬水平,抑制NLRP3炎症小体的活化,改善血管内皮功能。

关键词: 糖尿病,2型, 血管舒张, 内皮细胞, NLR家族,热蛋白结构域包含蛋白3, 自噬, 去肾交感神经

Abstract:

Objective To explore the effects of renal denervation (RDN) on the autophagy of vascular endothelial cells and NLRP3 inflammatory complexes in type 2 diabetic mellitus (T2DM) rats. Methods T2DM rat model was established by high-fat diet (HFD) combined with low-dose streptozotocin (STZ) intraperitoneal injection. Rats were randomly divided into four groups: the control group (CON, n=6), the diabetic group (T2DM, n=6), the bilateral sham surgery group (Sham, n=6) and the bilateral RDN group (RDN, n=6). Changes in all indicators were recorded at baseline, before and after RDN and 4 weeks after RDN, respectively. In vitro vascular ring tension test was used to evaluate vascular relaxation function. Western blot assay was used to detect the aorta endothelial cell autophagy of Beclin1, LC3 and p62, and the expression of NLRP3 protein and cysteine aspartate protease 1 (Caspase-1) and endothelial nitric oxide synthase (eNOS) protein. Results Compared with the control group, the endothelium diastolic function of the thoracic aorta was significantly decreased in the T2DM group, the expression of autophagy-related proteins Beclin1 and LC3 were decreased, the expression of p62 was increased. The expression levels of NLRP3, Caspase-1 and IL-1β protein were higher in the T2DM group than those of the control group (P<0.05), the level of eNOS protein was decreased (P<0.05). Compared with the T2DM group and the Sham group, the endothelium diastolic function was significantly improved in the RDN group, and the expression levels of Beclin1 and LC3 were increased, the expression of p62 was decreased. The protein expression levels of NLRP3, Caspase-1 and IL-1β were decreased, the level of eNOS increased (P<0.05). Conclusion The vascular endothelial dysfunction in T2DM rats may be associated with the decreased autophagy level and overexpression of NLRP3 inflammatory complex protein. RDN can enhance the autophagy in vascular endothelial cells, decrease the expression of NLRP3 inflammatory complex protein and improve vascular endothelial dysfunction.

Key words: diabetes mellitus, type 2, vasodilation, endothelial cells, NLR family, pyrin domain-containing 3 protein, autophagy, renal denervation

中图分类号:

R543

图/表 13

参考文献 18

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基因名称	引物序列（5'→3'）	产物大小（bp）
NLRP3	上游：AGTCTTCGAGATTTCCTGGTAG	114
NLRP3	下游：CTTGTGCTCTGCTTGTGAGG	114
GAPDH	上游：CCTTCCGTGTTCCTACCC	130
GAPDH	下游：CCCAAGATGCCCTTCAGT	130

基因名称	引物序列（5'→3'）	产物大小（bp）
NLRP3	上游：AGTCTTCGAGATTTCCTGGTAG	114
NLRP3	下游：CTTGTGCTCTGCTTGTGAGG	114
GAPDH	上游：CCTTCCGTGTTCCTACCC	130
GAPDH	下游：CCCAAGATGCCCTTCAGT	130

组别	SBP（mmHg）	HR（次/min）	FPG（mmol/L）	FINS（IU/L)	NE（ng/L）	vWF（μg/L）	NO（μmol/L）
CON组	100.33±6.97	319.17±5.19	4.17±0.44	16.16±1.95	66.87±2.01	273.55±14.38	71.60±7.29
T2DM组	98.14±5.24	316.29±6.18	4.01±0.62	15.91±1.42	67.48±2.04	284.39±12.30	74.15±6.69
Sham组	102.28±4.86	314.43±4.43	4.31±0.37	17.21±1.79	66.50±2.27	281.41±14.61	72.96±5.79
RDN组	102.70±6.02	317.20±8.51	3.94±0.43	16.24±2.18	66.29±1.92	280.44±14.49	73.94±6.99
F	0.991	0.583	0.983	0.575	0.384	0.645	0.181

组别	SBP（mmHg）	HR（次/min）	FPG（mmol/L）	FINS（IU/L)	NE（ng/L）	vWF（μg/L）	NO（μmol/L）
CON组	100.33±6.97	319.17±5.19	4.17±0.44	16.16±1.95	66.87±2.01	273.55±14.38	71.60±7.29
T2DM组	98.14±5.24	316.29±6.18	4.01±0.62	15.91±1.42	67.48±2.04	284.39±12.30	74.15±6.69
Sham组	102.28±4.86	314.43±4.43	4.31±0.37	17.21±1.79	66.50±2.27	281.41±14.61	72.96±5.79
RDN组	102.70±6.02	317.20±8.51	3.94±0.43	16.24±2.18	66.29±1.92	280.44±14.49	73.94±6.99
F	0.991	0.583	0.983	0.575	0.384	0.645	0.181

组别	SBP（mmHg）	HR（次/min）	FPG（mmol/L）	FINS（IU/L)	NE（ng/L）	vWF（μg/L）	NO（μmol/L）
CON组	105.50±5.39	319.50±5.61	4.02±0.46	17.02±1.86	67.69±3.22	275.41±16.38	71.73±6.16
T2DM组	136.33±8.78^a	346.17±5.74^a	18.87±2.73^a	35.44±3.87^a	103.10±4.09^a	478.74±20.21^a	38.92±4.90^a
Sham组	135.50±5.99^a	348.83±7.31^a	19.45±2.60^a	33.61±3.33^a	105.01±3.73^a	491.75±18.51^a	37.56±4.18^a
RDN组	136.67±6.41^a	347.50±8.09^a	19.50±2.49^a	35.17±3.38^a	102.03±4.05^a	488.07±21.63^a	36.76±4.99^a
F	29.751^＊＊	25.813^＊＊	67.700^＊＊	46.338^＊＊	133.809^＊＊	179.718^＊＊	66.513^＊＊

去肾交感神经对2型糖尿病大鼠血管内皮细胞自噬及NLRP3活化的影响

Effects of renal denervation on the vascular endothelial cell autophagy and NLRP3 activation in type 2 diabetic rats

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参考文献 18

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组别	SBP（mmHg）	HR（次/min）	FPG（mmol/L）	FINS（IU/L)	NE（ng/L）	vWF（μg/L）	NO（μmol/L）	肾脏NE（ng/L）
CON组	102.16±7.83	320.16±9.82	3.92±0.36	16.41±1.99	68.39±3.13	273.78±16.84	71.07±6.15	91.52±3.23
T2DM组	138.67±4.46^a	349.67±6.12^a	18.98±2.07^a	35.05±3.96^a	101.59±4.01^a	477.64±21.96^a	38.23±3.68^a	149.07±4.73^a
Sham组	137.33±5.92^a	347.00±9.59^a	19.98±2.39^a	34.76±4.53^a	103.92±3.46^a	484.48±16.49^a	37.79±2.97^a	151.91±5.13^a
RDN组	126.33±6.77^abc	328.67±7.06^abc	14.03±1.67^abc	25.40±3.07^abc	89.91±3.26^abc	380.00±21.83^abc	56.15±4.86^abc	126.88±1.38^abc
F	42.276^＊＊	17.736^＊＊	102.640^＊＊	38.093^＊＊	341.806^＊＊	155.961^＊＊	73.097^＊＊	215.628^＊＊

组别	lg（SNP）
组别	-10	-9	-8	-7	-6	-5
CON组	2.25±0.82	8.97±1.19	35.26±6.97	61.54±7.93	81.54±2.20	91.68±2.48
T2DM组	1.63±0.23^a	4.85±1.05^a	10.46±1.75^a	33.64±7.27^a	60.42±8.09^a	82.78±2.99^a
Sham组	1.66±0.36^a	4.64±0.81^a	11.03±1.81^a	37.42±7.02^a	63.82±5.74^a	84.74±2.32^a
RDN组	2.33±0.60^abc	8.09±1.72^abc	33.73±5.89^abc	55.60±5.72^abc	77.61±4.08^abc	88.46±2.65^abc
F	2.311	15.952^＊＊	42.037^＊＊	18.705^＊＊	17.678^＊＊	12.033^＊＊

组别	lg（Ach）
组别	-9	-8	-7	-6	-5	-4
CON组	1.72±0.94	8.87±1.19	30.14±5.05	47.54±7.06	61.54±5.79	75.57±4.98
T2DM组	1.69±0.53^a	2.09±0.48^a	10.61±2.21^a	27.64±4.33^a	36.22±5.67^a	45.98±6.39^a
Sham组	1.46±0.49^a	1.40±0.55^a	14.09±2.15^a	31.42±7.12^a	43.82±6.75^a	48.34±5.92^a
RDN组	2.33±0.97^abc	6.50±1.35^abc	26.94±4.88^abc	43.82±13.81^abc	59.61±7.97^abc	72.46±5.19^abc
F	1.137	68.918^＊＊	30.948^＊＊	7.191^＊＊	17.264^＊＊	38.033^＊＊

组别	Beclin1	LC3	p62	cleaved Caspase-3	eNOS
CON组	1.07±0.20	1.45±0.37	0.27±0.12	0.30±0.05	1.01±0.13
T2DM组	0.60±0.09^a	0.68±0.07^a	0.82±0.12^a	1.57±0.44^a	0.59±0.15^a
Sham组	0.54±0.06^a	0.55±0.06^a	0.91±0.16^a	1.09±0.21^a	0.60±0.05^a
RDN组	0.85±0.11^abc	0.95±0.17^abc	0.56±0.11^abc	0.72±0.22^abc	0.85±0.09^abc
F	7.366^＊＊	10.776^＊＊	12.212^＊＊	11.874^＊＊	9.000^＊＊

组别	NLRP3 mRNA	NLRP3	Caspase-1	IL-1β
CON组	1.03±0.05	0.46±0.04	0.49±0.10	0.48±0.11
T2DM组	6.05±0.31^a	1.10±0.12^a	1.06±0.14^a	1.04±0.16^a
Sham组	6.01±0.28^a	1.19±0.06^a	1.05±0.14^a	1.02±0.17^a
RDN组	3.05±0.29^abc	0.69±0.12^abc	0.83±0.19^abc	0.68±0.09^abc
F	91.472^＊＊	84.385^＊＊	18.828^＊＊	23.319^＊＊

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