灯盏乙素对OX-LDL损伤的RAW264.7细胞中PKC和TNF-α表达的影响

张莉; 余丹; 王雨婷; 苟志梅; 杨仁华; 何波; 沈志强; 柴文英; 陈鹏

doi:10.12259/j.issn.2095-610X.S20211040

灯盏乙素对OX-LDL损伤的RAW264.7细胞中PKC和TNF-α表达的影响

doi: 10.12259/j.issn.2095-610X.S20211040

张莉¹,
余丹¹,
王雨婷¹,
苟志梅¹,
杨仁华¹,
何波¹,
沈志强^1,,
柴文英^2, ,,
陈鹏^1, ,

1.
昆明医科大学药学院暨云南省天然药物药理重点实验室，云南昆明　650500
2.
昆明医科大学第一附属医院乳腺外科，云南昆明　6500031

基金项目: 国家自然科学基金资助项目（81860641）；云南省基础研究计划重点项目（202001AS070035）；昆明医科大学博士研究生创新基金资助项目（2021D05）

详细信息

作者简介:
张莉（1984～），女，河南南阳人，在读博士研究生，主要从事老年病药理研究工作

通讯作者:
柴文英，E-mail：wenyingchai74@126.com

陈鹏，E-mail：ynkmcp@gmail.com

中图分类号: R966
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出版历程
- 收稿日期: 2021-07-12
- 网络出版日期: 2021-10-29
- 刊出日期: 2021-10-30

Effects of Scutellarin on Expressions of PKC and TNF-α in OX-LDL Injured RAW264.7 Cells in vitro

1.
School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products，Kunming Medical University，Kunming Yunnan 650500
2.
Dept. of Breast Surgery，First Affiliated Hospital of Kunming Medical University， Kunming Yunnan 650031，China

摘要

摘要: 目的研究灯盏乙素（scutellarin）对OX-LDL诱导损伤的巨噬细胞RAW264.7脂质吞噬能力及对PKC和TNF-α的蛋白和mRNA表达调控的影响。方法体外培养小鼠单核巨噬细胞RAW264.7，复制OX-LDL诱导损伤模型，通过油红O染色检测脂质吞噬及试剂盒检测细胞损伤模型胆固醇酯的含量。Western blot和RT-PCR方法检测RAW264.7细胞中PKC和TNF-α表达变化。 结果（ 1）油红O结果显示：与正常对照组比较，模型组细胞红染程度明显高于正常对照组；与模型组比较，辛伐他汀组、灯盏乙素50和100 μmol /L组细胞红染程度均低于模型组;（ 2）胆固醇酯含量检测显示:与正常对照组比较，各时间点模型组细胞CE/TC值均明显升高；与模型组比较，各时间点用药组细胞CE/TC值均降低。（ 3） WB和RT-PCR结果显示：与正常对照组比较，模型组PKC和TNF-α的蛋白和mRNA表达升高；与模型组比较，辛伐他汀组(simvastatin) PKC和TNF-α的蛋白和mRNA表达降低，且灯盏乙素组呈浓度依赖性下调PKC和TNF-α的蛋白和mRNA表达。结论（1）灯盏乙素能明显抑制OX-LDL诱导损伤的RAW264.7细胞脂质吞噬能力。（ 2）灯盏乙素能够明显下调OX-LDL诱导损伤的RAW264.7中PKC和TNF-α的蛋白和mRNA表达水平。
- 灯盏乙素 /
- 巨噬细胞RAW264.7 /
- 氧化型低密度脂蛋白 /
- 蛋白激酶C /
- 肿瘤坏死因子-α
Abstract: Objectives To investigate the effects of scutellarin on lipid phagocytosis and the regulation of PKC, and TNF-α expressions in low density lipoprotein （OX-LDL） injured RAW264.7 cells. Methods Murine RAW264.7 macrophages was cultured in vitro. Then, the OX-LDL-induced injury model was replicated. Combined with oil red O staining, cellular cholesteryl ester content was also detected in OX-LDL-induced injury model. Furthermore, Western blot and RT-PCR methods were used to detect changes in PKC and TNF-α protein as well as mRNA expression in RAW264.7 cells. Results （1） The results of oil red O showed that the degree of cell red staining in model group was significantly higher than that in normal control group; compared with model group, the degree of cell red staining in simvastatin group, scutellarin 50 and 100 μmol/L group were lower than that in the model group; （2） The cholesteryl ester content assay showed that: compared with the normal control group, the CE/TC values of the model group cells were significantly increased at each time point; compared with model group, and cellular CE/TC values were significantly decreased in the dosing group at each time point. （3） WB and RT-PCR results showed that compared with normal control group, protein and mRNA expressions of PKC and TNF-α were increased in the model group; compared with the model group, protein and mRNA expression of PKC and TNF-α were decreased in simvastatin group, and scutellarin group showed a concentration-dependent downregulation of PKC and TNF-α expression. Conclusions （1） Scutellarin can significantly inhibit the levels of cholesterol in OX-LDL injuried RAW264.7 cells. （2） Scutellarin can down-regulated the protein and mRNA expressions of PKC, TNF-α in OX-LDL induced RAW264.7 cells.
- Scutellarin /
- RAW264.7 macrophages /
- oxidized low-density lipoprotein （OX-LDL） /
- PKC /
- TNF-α

HTML全文

图 1 灯盏乙素的化学结构式

Figure 1. Chemical structural of Scutellarin

下载: 全尺寸图片幻灯片

图 2 OX-LDL诱导损伤RAW264.7细胞的油红O染色（400×）

Figure 2. Oil red O staining of OX-LDL injured RAW264.7 cells （400×）

下载: 全尺寸图片幻灯片

图 3 灯盏乙素对OX-LDL诱导损伤的RAW264.7内脂质的影响[（$\bar x \pm s$），n = 6]

Figure 3. Effects of Scutellarin on OX-LDL injured RAW264.7 cells lipid levels [（$\bar x \pm s$），n = 6]

下载: 全尺寸图片幻灯片

图 4 灯盏乙素对OX-LDL损伤的RAW264.7中PKC和TNF-α蛋白表达的影响[（$ \bar x \pm s $），n = 6]

注：a：PKC和TNF-α的蛋白表达电泳图；b,c：细胞损伤模型中PKC和TNF-α蛋白表达变化。用ImageJ 1.4.3.67对蛋白条带进行统计分析，βactin是内参。与空白对照组相比较，^#P < 0.05；与模型组比较，^*P < 0.05，^**P < 0.01。

Figure 4. Effects of scutellarin on OX-LDL induced RAW264.7 cells PKC and TNF-α protein expression [（$ \bar x \pm s $），n = 6]

下载: 全尺寸图片幻灯片

图 5 灯盏乙素对OX-LDL诱导的RAW264.7中PKC和TNF-α的mRNA表达的影响[（$ \bar x \pm s $），n = 6]

注：a，b：细胞损伤模型中PKC和TNF-α的 mRNA表达变化。与空白对照组相比较，^#P < 0.05；与模型组比较，^*P < 0.05，^**P < 0.01。

Figure 5. Effects of Scutellarin on OX-LDL induced RAW264.7 cells PKC and TNF-α mRNA expression [（$ \bar x \pm s $），n = 6]

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因名称	双向引物序列
β-actin	Forward：5′ TGAGAGGGAAATCGTGCGTGAC 3′
	Reverse：5′ GCTCGTTGCCAATAGTGATGACC 3′
PKC	Forward：5′ TCACCAAGGAATCCAAGGAC3′
	Reverse：5′ CCAGGAGGGACCAGTTGATA3′
TNF-α	Forward：5′ ACGGCATAAATCTCAAAGAC 3′
	Reverse：5′ GTGGGTGAGGAGCACGTAGT 3′

下载: 导出CSV

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