Effects of Scutellarin on Expressions of PKC and TNF-α in OX-LDL Injured RAW264.7 Cells in vitro
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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。 -
关键词:
- 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. -
Key words:
- Scutellarin /
- RAW264.7 macrophages /
- oxidized low-density lipoprotein （OX-LDL） /
- PKC /
- TNF-α
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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×）
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]
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]
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]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′ 
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