Effect of astaxanthin on inflammatory response of RAW264.7 cells induced by lipopolysaccharide and its mechanism
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摘要:目的
研究虾青素(AST)对脂多糖(LPS)诱导的RAW264.7细胞炎症反应的影响及作用机制，为将虾青素应用于炎症治疗奠定理论基础。
方法采用不同浓度梯度的脂多糖及虾青素对RAW264.7细胞进行不同时间段的处理，通过MTT法确定最佳处理浓度和时间，对细胞进行最佳处理后，用ELISA法、荧光定量PCR技术和Western blot法分别检测细胞炎症因子的分泌量、mRNA相对表达量和蛋白相对表达量。
结果100 μmol/L虾青素和2 μg/mL脂多糖处理3 h的RAW264.7细胞活力处于峰值。与对照组相比，脂多糖组RAW264.7细胞中TNF-α、IL-6和Caspase-1的分泌量分别降低了12.83%、9.66%和20.80%(P<0.05)，脂多糖对于TLR4/MyD88/NF-кB通路相关蛋白表达有促进作用，其中，TLR4和NF-кB p65蛋白相对表达量分别提高了195.40%和226.95%(P<0.05)；与LPS组相比，AST+LPS组中虾青素对炎性因子的分泌及mRNA表达有抑制作用，TLR4、MyD88和NF-кB p65蛋白相对表达量降低了54.99%、45.70%和28.20%(P<0.05)。
结论虾青素预保护能抑制TLR4/MyD88/NF-кB通路相关蛋白的表达，进而缓解脂多糖刺激RAW264.7细胞产生的炎症反应。
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关键词:
- 炎症反应 /
- 虾青素 /
- 脂多糖 /
- TLR4/MyD88/NF-кB信号通路
Abstract:ObjectiveTo study the effect of astaxanthin (AST) on the inflammatory response of RAW264.7 cells induced by lipopolysaccharide (LPS) and the mechanism, and provide a theoretical basis for using AST in inflammation therapy.
MethodDifferent concentrations of LPS and AST were used to treat RAW264.7 cells for different time. The optimal treatment concentration and time were determined by MTT method. After applying the optimal treatment, the secretion, mRNA relative expression and protein relative expression of inflammatory factors were detected by ELISA, fluorescence quantitative PCR and Western blot method respectively.
ResultWhen treated with 100 μmol/L AST and 2 μg/mL LPS for 3 h, the viability of RAW264.7 cells was at the peak. Compared with the control group, the secretion of TNF-α, IL-6 and Caspase-1 in RAW264.7 cells of LPS group reduced by 12.83%, 9.66% and 20.80% respectively(P<0.05). LPS promoted the expression of TLR4/MyD88/NF-κB pathway-related proteins with relative expression of TLR4 and NF-кB p65 proteins enhanced by 195.40% and 226.95% respectively(P<0.05). Compared with LPS group, AST had inhibitory effects on the secretion and mRNA expression of inflammatory factors in AST+LPS group, and the relative expression of TLR4, MyD88 and NF-кB p65 proteins reduced by 54.99%, 45.70% and 28.20% respectively (P<0.05).
ConclusionAST pre-protection can inhibit the expression of TLR4/MyD88/NF-κB pathway-related proteins, thereby alleviate the inflammatory response in RAW264.7 cells induced by LPS.
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图 1 不同浓度虾青素对RAW264.7细胞活力的影响
相同处理时间柱子上的不同小写字母表示不同处理组间差异显著(P<0.05，Duncan’s法)
Figure 1. Effect of astaxanthin on the viability of RAW264.7 cells at different concentration
Different lowercase letters on bars of the same treatment time indicate significant difference among treatment groups (P<0.05, Duncan’s method)
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图 2 不同质量浓度脂多糖对RAW264.7细胞活力的影响
相同处理时间柱子上的不同小写字母表示不同处理组间差异显著(P<0.05，Duncan’s法)
Figure 2. Effect of different content of lipopolysaccharide on the viability of RAW264.7 cells
Different lowercase letters on bars of the same treatment time indicate significant difference among treatment groups (P<0.05, Duncan’s method)
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图 3 虾青素预保护对脂多糖刺激RAW264.7细胞炎症因子分泌量的影响
虾青素(AST)浓度为100 μmol/L，脂多糖(LPS)质量浓度为2 μg/mL；各图中，柱子上方的不同小写字母表示差异显著(P<0.05，Duncan’s法)
Figure 3. Effects of astaxanthin pre-protection on inflammatory factor secretion from RAW264.7 cells stimulated by lipopolysaccharide
The astaxanthin(AST) concentration is 100 μmol/L, the lipopolysaccharide(LPS) content is 2 μg/mL; In each figure, different lowercase letters on bars indicate significant difference (P<0.05, Duncan’s method)
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图 4 虾青素预保护对脂多糖刺激的RAW264.7细胞中炎症因子mRNA相对表达量的影响
虾青素(AST)浓度为100 μmol/L，脂多糖(LPS)质量浓度为2 μg/mL；各图中，柱子上方的不同小写字母表示差异显著(P<0.05，Duncan’s法)
Figure 4. Effects of astaxanthin pre-protection on mRNA expression of inflammatory factor in RAW264.7 cells stimulated by lipopolysaccharide
The astaxanthin(AST) concentration is 100 μmol/L, the lipopolysaccharide (LPS) content is 2 μg/mL；In each figure, different lowercase letters on bars indicate significant difference (P<0.05, Duncan’s method)
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图 5 虾青素预保护下脂多糖刺激的RAW264.7细胞中炎症因子蛋白的电泳图
Figure 5. Electrophoresis of inflammatory factor in RAW264.7 cells stimulated by lipopolysaccharide under astaxanthin pre-protection
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图 6 虾青素预保护对脂多糖刺激的RAW264.7细胞中炎症因子蛋白相对表达量的影响
各图中，柱子上方的不同小写字母表示差异显著(P<0.05，Duncan’s法)
Figure 6. Effects of astaxanthin pre-protection on relative protein expression of inflammatory factor in RAW264.7 cells stimulated by lipopolysaccharide
In each figure, different lowercase letters on bars indicate significant difference (P<0.05, Duncan’s method)
表 1 qPCR的引物序列
Table 1 qPCR primer sequence
基因 Gene 正向引物(5′→3′) Forward primer 反向引物(5′→3′) Reverse primer 18s RNA CTCAACACGGGAAACCTCAC CGCTCCACCAACTAAGAACG IL-1β GGCTACTGCCTTCCCTACC CCTGATTGAACCCAGATTGG TNF-α ACCTGCCTGTGCTGAGTT ATGAAGTGCTGGGACACC IL-18 GGCCGACTTCACTGTACAACCG GGTCACAGCCAGTCCTCTTACTTC 
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