| 金莉,潘怡宏.祛瘀消肿方对LPS刺激的巨噬细胞TLR-4信号通路调控机制的硏究[J].浙江中西医结合杂志,2022,32(12): |
| 祛瘀消肿方对LPS刺激的巨噬细胞TLR-4信号通路调控机制的硏究 |
| The study of Quyu Xiaozhong Prescription on the regulation mechanism of LPS-stimulated TLR-4 signaling pathway in macrophages |
| 投稿时间：2022-05-03 修订日期：2022-08-06 |
| DOI： |
| 中文关键词: 急性乳腺炎 巨噬细胞 TLR4 / NF-κB 祛瘀消肿方 |
| 英文关键词:Acute mastitis Macrophage TLR4 / NF-κB Quyu Xiaozhong Recipe |
| 基金项目:浙江省中医药科技计划项目(No.2020ZA086)；2019年省级名中医传承人项目 |
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| 中文摘要: |
| 目的 本研究探讨了祛瘀消肿方在LPS诱导的乳腺炎过程中对巨噬细胞的影响及祛瘀消肿方在这一过程中对炎症的抑制作用和对TLR-4信号通路的调控机制。方法用含不同剂量（低、中、高）祛瘀消肿方血清预处理RAW264.7巨噬细胞，再进行LPS刺激，MTT检测细胞活性；DAPI染核检测细胞凋亡，同时qRT-PCR及Westernblot检测凋亡蛋白（Bax，Bcl2）表达；ELISA检测炎症细胞因子（TNF-α，IL-1β和IL-6）的水平。为了探讨与祛瘀消肿方有关的信号通路，通过qRT-PCR检测TLR4相对表达水平，并用Westernblotting检测TLR4，NF-κB-p65，p-NF-κB-p65，IκB-α和p-IκB-α蛋白的变化。结果 祛瘀消肿方可以增强LPS诱导的RAW264.7巨噬细胞活性[（36.32±4.32）%、（51.69±4.08）%、（74.33±4.92）%比（21.36±4.08）%，P均<0.05]，增多活细胞数目[（41.67±6.24）、（56.00±4.32）、（63.67±5.79）比（22.67±5.25），P均<0.05]，下调Bax mRNA水平[（1.91±0.06）、（1.68±0.06）、（1.35±0.04）比（2.19±0.13），P均<0.05]与蛋白表达，上调Bcl2 mRNA水平[（0.46±0.05）、（0.62±0.04）、（0.87±0.05）比（0.22±0.07），P均<0.05]与蛋白表达，减少细胞凋亡以及抑制炎症反应，TNF-α [（1126.67±61.28）pg/ml、（873.33±55.58）pg/ml、（456.67±67.98）pg/ml比（1916.67±102.74）pg/ml，P均<0.05]、IL-1β[（67.67±5.56）pg/ml、（58.67±2.87）pg/ml、（35.33±4.49）pg/ml比（87.33±5.25）pg/ml，P均<0.05]、IL-6[（72.69±7.93）pg/ml、（55.36±3.68）pg/ml、（44.96±3.74）pg/ml比（96.33±4.18）pg/ml，P均<0.05]水平显著下调。此外，祛瘀消肿方能够剂量依赖性地抑制与TLR4 / NF-κB通路相关的TLR4，pNF-κB-p65和pIκBα的激活，抑制TLR4，p-NF-κB-p65及 p-IκB-α表达；并使NF-κB-p65和IκB-α表达增加。结论 本研究阐明了祛瘀消肿方通过抑制TLR4 / NF-κB信号通路，产生抗炎作用，为中医药治疗乳腺炎提供了理论依据。 |
| 英文摘要: |
| This study explored the effect of Quyu Xiaozhong Recipe on macrophages in the process of LPS induced mastitis, the inhibitory effect of Quyu Xiaozhong Recipe on inflammation and the regulatory mechanism of TLR-4 signaling pathway.MethodsRAW264.7 macrophages were pretreated with serum containingQuyu Xiaozhong Recipe with different doses (low, medium and high), and then stimulated by LPS.MTT was used to detect cell viability;apoptosis was detected by DAPIstaining.The expression of apoptotic protein (Baxand Bcl2) was detected by qRT-PCR and western blot.The levels of inflammatory cytokines (TNF-α, IL-1βand IL-6) were detected by ELISA.In order to explore the signal pathway related to Quyu Xiaozhong Recipe, the relative expression level of TLR4 was detected by qRT-PCR, and the changes of TLR4, NF-κB-p65, p-NF-κB-p65, IκB-αand p-IκB-α proteins were detected by western blotting.ResultsQuyu Xiaozhong Recipe can enhance the activity of LPS-treated RAW264.7 macrophages (36.32±4.32, 51.69±4.08 and 74.33±4.92 vs. 21.36±4.08 %; P<0.05), increase the number of living cells (41.67±6.24, 56.00±4.32 and 63.67±5.79 vs. 22.67±5.25; P<0.05), downregulate the mRNA level (1.91±0.06, 1.68±0.06 and 1.35±0.04 vs. 2.19±0.13; P<0.05) and protein expression of Bax, upregulate the mRNA level (0.46±0.05, 0.62±0.04 and 0.87±0.05 vs. 0.22±0.07; P<0.05) and protein expression of Bcl2,reduce apoptosis and inhibit inflammatory reaction. The levels of TNF-α (1126.67±61.28, 873.33±55.58 and 456.67±67.98 vs. 1916.67±102.74 pg/ml; P<0.05), IL-1β (67.67±5.56, 58.67±2.87 and 35.33±4.49 vs. 87.33±5.25 pg/ml; P<0.05) and IL-6 (72.69±7.93, 55.36±3.68 and 44.96±3.74 vs. 96.33±4.18 pg/ml; P<0.05) were significantly downregulated. In addition, Quyu Xiaozhong Recipe can inhibit the activation of TLR4, p-NF-κB-p65and p-IκB-α related to TLR4 / NF-κB pathway in a dose-dependent manner, downregulate the expression of TLR4, p-NF-κB-p65 and p-IκB-α, and upregulate the expression of NF-κB-p65 and IκB-α.ConclusionThis study elucidated the anti-inflammatory effect of Quyu Xiaozhong Recipe by inhibiting TLR4 / NF-κB signaling pathway, which provides theoretical basis for the treatment of mastitis with traditional Chinese medicine. |
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