Mertk介导NF-κb通路影响雪旺细胞炎症反应的作用

付怡丹; 陈文婷; 苏晓杨; 赵燕; 兰丹凤; 杨秋萍

doi:10.12259/j.issn.2095-610X.S20231203

Mertk介导NF-κb通路影响雪旺细胞炎症反应的作用

doi: 10.12259/j.issn.2095-610X.S20231203

1.
昆明医科大学第一附属医院干疗科
2.
重症医学科
3.
糖尿病科，云南昆明　650032
4.
云南省第一人民医院消化内科，云南昆明　650032
5.
昆明医科大学第一附属医院内分泌一科，云南昆明　650032

基金项目: 云南省科技厅科技计划基金资助项目（202001AY070001-159）

详细信息

作者简介:
付怡丹（1998～），女，河北秦皇岛人，在读硕士研究生，主要从事糖尿病及其慢性并发症的研究工作

通讯作者:
杨秋萍，E-mail：yqp22@aliyun.com

中图分类号: R587.1
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出版历程
- 收稿日期: 2023-09-28
- 网络出版日期: 2023-12-20
- 刊出日期: 2023-12-25

Role of Mertk-mediated NF-κb Pathway in Inflammatory Response of Schwann Cells

1.
Dept. of Geriatrics
2.
Dept. of Critical Care Medicine
3.
Dept. of Diabetes，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032
4.
Dept. of Gastroenterology，The 1st People's Hospital of Yunnan Province，Kunming Yunnan 650032
5.
Dept. of Endocrinology，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

摘要

摘要: 目的探究Mertk表达水平对大鼠雪旺细胞NF-κb通路的调控作用及其可能的机制。方法体外培养大鼠雪旺细胞，免疫印迹法检测Mertk在高糖环境雪旺细胞中表达情况。免疫共沉淀法检测内源性Mertk与Ikbkb相互作用。免疫印迹法检测沉默Mertk后雪旺细胞内Ikbkb、P65、肿瘤坏死因子-α表达水平。免疫荧光法检测沉默Mertk后Mertk、Ikbkb、P65蛋白表达。结果 Mertk在雪旺细胞中存在表达，且表达量随葡萄糖浓度增加而提高；免疫共沉淀实验表明Mertk与Ikbkb在大鼠雪旺细胞内相互作用；免疫印迹实验显示，将雪旺细胞内Mertk敲减表达后，与对照组相比，Mertk表达水平降低（P < 0.05），Ikbkb、P65、TNF-α明显升高（P < 0.05）；免疫荧光实验显示，沉默后的雪旺细胞内Mertk荧光减弱，Ikbkb、P65荧光增强。结论 Mertk表达降低后，可以通过与Ikbkb进行相互作用，介导调控雪旺细胞NF-κb通路，上调P65和炎症因子TNF-α的表达。
- Mertk /
- Ikbkb /
- 核因子κB /
- 雪旺氏细胞 /
- 糖尿病周围神经病变
Abstract: Objective To explore the regulatory effect of Mertk expression level on NF-κb pathway in rat Schwann cells and its possible mechanism. Methods Rat Schwann cells were cultured in vitro, and the expression of Mertk in Schwann cells exposed to high glucose was detected by Western blot. Co-immunoprecipitation was used to detect the interaction between endogenous Mertk and Ikbkb. Western blot was used to detect the expression levels of Ikbkb, P65 and tumor necrosis factor-α in Schwann cells after Mertk silencing. The protein expressions of Mertk, Ikbkb and P65 after silencing Mertk were detected by immunofluorescence. Results Mertk was expressed in Schwann cells, and the expression level increased with the increase of glucose concentration. Co-immunoprecipitation assay showed that Mertk interacted with Ikbkb in rat Schwann cells. Compared with the control group, the expression level of Mertk was significantly decreased（P < 0.05）, while Ikbkb, P65 and TNF-α were significantly increased（P < 0.05） after knock down expression of Mertk in Schwann cells. Immunofluorescence experiments showed that the fluorescence of Mertk was decreased, and the fluorescence of Ikbkb and P65 was increased in the silenced Schwann cells. Conclusion After the expression of Mertk is decreased, it can mediate the regulation of NF-κb pathway in Schwann cells through interaction with Ikbkb, and up-regulate the expression of P65 and inflammatory factor TNF-α.
- Mertk /
- Ikbkb /
- Nuclear factor kappa-b /
- Schwann cells /
- Diabetes peripheral neuropathy

HTML全文

图 1 不同葡萄糖水平RSC96内Mertk蛋白表达

A：Mertk蛋白在不同葡萄糖水平下的表达；B：对Mertk/β-actin结果进行统计分析；ns表示差异无统计学意义，^*P < 0.05。

Figure 1. Mertk protein expression within RSC96 at different glucose levels

下载: 全尺寸图片幻灯片

图 2 Mertk与Ikbkb免疫共沉淀

A：以Mertk抗体调取Ikbkb的免疫沉淀物；B：以Ikbkb抗体调取Mertk的免疫沉淀物。　　　　　　　　　　　　

Figure 2. Co-Immunoprecipitation of Mertk with Ikbkb

下载: 全尺寸图片幻灯片

图 3 沉默Mertk后Ikbkb、P65、TNF-α蛋白表达水平

A：siRNA处理后Mertk、Ikbkb、P65、TNF-α的蛋白表达；B：对Mertk/β-actin结果进行统计分析；C：对Ikbkb/β-actin结果进行统计分析；D：对P65/β-actin结果进行统计分析；E：对TNF-α/β-actin结果进行统计分析；ns表示差异无统计学意义，^*P < 0.05。

Figure 3. Ikbkb，P65，and TNF-α protein expression levels after silencing Mertk

下载: 全尺寸图片幻灯片

图 4 免疫荧光检测siRNA3转染后Mertk、Ikbkb、P65蛋白表达（200x）

Figure 4. Immunofluorescence detection of Mertk，Ikbkb，and P65 protein expression after siRNA3 transfection（200x）

下载: 全尺寸图片幻灯片

表 1 Mertk小干扰核苷酸序列

Table 1. Mertk-RNA oligo sequences

寡核苷酸	正义序列（5′-3′）	反义序列（5′-3′）
Mertk-Rat-1	GGGUCGUACAUCUGUAAGATT	UCUUACAGAUGUACGACCCTT
Mertk-Rat-2	CACCCACUGAAGUCCAUAUTT	AUAUGGACUUCAGUGGGUGTT
Mertk-Rat-3	GAGGAAAGAUACAUCUUAATT	UUAAGAUGUAUCUUUCCUCTT

下载: 导出CSV

表 2 不同葡萄糖水平RSC96内Mertk蛋白表达

Table 2. Mertk protein expression within RSC96 at different glucose levels

灰度值比值	组别					F	P
灰度值比值	25 mmol/L	50 mmol/L	75 mmol/L	100 mmol/L	125 mmol/L	F	P
Mertk/β-actin	0.60 ± 0.20	0.78 ± 0.19	0.81 ± 0.06	0.95 ± 0.09^*	0.87 ± 0.11^*	4.452	0.0098^*
与对照组（25 mmol/L组）比较，^*P < 0.05。

下载: 导出CSV

表 3 沉默Mertk后Ikbkb、P65、TNF-α蛋白表达量

Table 3. Ikbkb，P65，and TNF-α protein expression after silencing Mertk

组别	Mertk /β-actin	Ikbkb /β-actin	P65 /β-actin	TNF-α /β-actin
CON	1.13 ± 0.22	0.38 ± 0.11	0.60 ± 0.12	0.60 ± 0.09
NC	1.28 ± 022	0.44 ± 0.22	0.57 ± 0.16	0.62 ± 0.19
siRNA1	0.68 ± 0.08^*	1.10 ± 0.18^*	1.22 ± 0.18^*	1.05 ± 0.25^*
siRNA2	0.60 ± 0.24^*	1.12 ± 0.02^*	1.02 ± 0.09^*	0.85 ± 0.04^*
siRNA3	0.60 ± 0.12^*	1.18 ± 0.28^*	0.95 ± 0.11^*	1.19 ± 0.09^*
F	8.927	13.76	12.62	8.646
P	0.0025^*	0.0004^*	0.0006^*	0.0028^*
与CON组比较，^*P < 0.05。

下载: 导出CSV

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