栝楼桂枝汤调控TLR3通路对小胶质细胞活化诱导的神经细胞损伤的保护作用研究
The study of Gualou Guizhi Decoction inhibits nerve cell damage induced by microglial activation via TLR3 signaling
胡海霞 1钟兴华 2林心君 3朱晓勤2
作者信息
- 1. 福建中医药大学科技创新与转化中心,福建福州 350122
- 2. 福建中医药大学中西医结合研究院,福建福州 350122
- 3. 福建中医药大学中西医结合学院,福建福州 350122
- 折叠
摘要
目的:探讨栝楼桂枝汤通过TLR3(toll-like receptor 3)信号通路抑制小胶质细胞活化介导的炎症反应发挥神经细胞保护作用的分子机制.方法:栝楼桂枝汤预处理后,分别使用脂多糖刺激BV2细胞建立炎症模型和BV2细胞的条件性培养基(microglia-conditioned media,MCM)干预HT22细胞制备神经细胞损伤模型.CCK-8(cell counting kit-8)法测定细胞活力;ELISA法检测细胞培养上清中的细胞因子;通过免疫荧光实验检测TLR3及TRIF(toll/interleukin receptor domain-containing adaptor-inducing IFNβ)蛋白的表达情况;采用 DNA-binding ELISA 法检测转录因子 IRF3(interferon regulatory factor 3)的活化水平;细胞TLR3 siRNA(small interfering RNA)转染实验验证栝楼桂枝汤的作用靶点;采用Western blot法检测凋亡蛋白Bcl-2、Bax的表达水平;采用Caspase-9活性测定法测定其活性.结果:栝楼桂枝汤可显著减轻MCM诱导的HT22细胞损伤,并上调脂多糖刺激的BV2细胞上清中IFN-β的含量,下调IL-8的含量.同时,栝楼桂枝汤显著增加TLR3通路相关蛋白的表达水平并促进IRF3的活化水平,且敲低TLR3表达后,栝楼桂枝汤对抗炎因子IL10无促进作用.此外,栝楼桂枝汤可使经MCM干预后HT22细胞的Bcl-2表达增加,Bax表达下降,Caspase-9活性降低.结论:栝楼桂枝汤可能通过激活TLR3信号通路调节小胶质细胞活化后的炎症反应,抑制神经细胞凋亡,进而缓解神经细胞损伤.
Abstract
OBJECTIVE To explore the neuroprotective molecular mechanism of Gualou Guizhi Decoction(GLGZD)through inhibiting the microglia-mediated inflammatory response via TLR3 signaling pathway.METHODS After GLGZD pre-treatment,BV2 cells were stimulated using LPS to establish inflammatory model,HT22 cells were exposed to microglia-conditioned media of BV2 cells.Cell viability was determined by CCK-8 assay.Cytokines in the cell culture supernatants were detected by ELISA.Expression of TLR3 and TRIF protein was measured by immunofluorescence.The activation of the transcrip-tion factor IRF3 was evaluated by DNA-binding ELISA assay.TLR3 siRNA cell transfection assay was used to verify the target of GLGZD.The levels of the apoptotic proteins Bcl-2 and Bax protein were determined by Western blot.The activation of Caspase-9 was measured by Caspase-9 activity assay.RESULTS GLGZD significantly attenuated the MCM-induced HT22 cell damage,and promoted IFN-β expression and downregulated IL-8 expression in the supernatant of LPS-stimulated BV2 cells.Meanwhile,GLGZD significantly increased the expression levels of the related proteins of TLR3 pathway and IRF3 activation.After knocking down the expression of TLR3,GLGZD could not promote the anti-inflammatory factor IL-10 secretion in BV2.Furthermore,GLGZD significantly increased the expression of Bcl-2 and decreased the expression of Bax,and reduced Caspase-9 activity in HT22 cells exposed to MCM.CONCLUSION GLGZD may ameliorate brain damage through anti-inflammation via activation of theTLR3 signaling pathway following microglial activation,inhibit the neuron apoptosis,subsequently reduce brain injury.
关键词
栝楼桂枝汤/小胶质细胞/TLR3信号通路/炎症反应/神经细胞/凋亡Key words
GLGZD/microglia/TLR3 signaling pathway/inflammatory response/neuron/apoptosis引用本文复制引用
基金项目
福建省自然科学基金(2021J01937)
出版年
2024
NETL
NSTL
万方数据