半夏厚朴汤对脂多糖诱导神经炎症损伤的保护机制

Protective Mechanism of Banxia Houputang on Lipopolysaccharide-induced Neuroinflammatory Injury

目的:研究半夏厚朴汤(BHT)对脂多糖(LPS)诱导小胶质细胞(BV2细胞)炎症的抑制作用以及对人神经母细胞瘤细胞(SH-SY5Y细胞)的神经保护作用。方法:经LPS诱导BV2细胞构建神经炎症模型后,分别给予模型组(LPS 100μg·L^(-1))、给药组(LPS+1 g·L^(-1)BHT、LPS+2 g·L^(-1)BHT、LPS+5 g·L^(-1)BHT、LPS+10 g·L^(-1)BHT),空白组同体积DEME培养基给药;同时建立LPS诱导BV2细胞炎症培养基与SH-SY5Y细胞共培养(LPS-DMEM)系统构建小胶质细胞炎症反应诱导的神经元凋亡模型按上分组分别给药。通过细胞增殖与活性检测(CCK-8)法检测细胞活性,Griess法测定一氧化氮(NO)的含量,酶联免疫吸附测定法(ELISA)测定肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL^(-1)β)、白细胞介素-6(IL-6)的含量,实时荧光定量聚合酶链式反应(Real-time PCR)测定TNF-α、IL^(-1)β、IL-4、一氧化氮合酶(iNOS)、IL^(-1)0 mRNA的水平,蛋白免疫印迹法(Western blot)测定细胞内信号传导及转录激活蛋白3(STAT3)、Janus激酶2(JAK2)、核转录因子-κB p65(NF-κB p65)、蛋白激酶B(Akt)、NF-κB抑制蛋白激酶-α(IκBα)、B细胞淋巴瘤-2(Bcl-2)及Bcl-2相关X蛋白(Bax)表达水平。结果:与空白组比较,模型组可增加BV2细胞NO释放,增加TNF-α、IL^(-1)β、IL-6、iNOS水平,减少IL-4、IL^(-1)0的含量,增加Akt、NF-κB p65、IκBα、JAK2和STAT3蛋白的表达,并引起共培养的SH-SY5Y细胞的凋亡(P<0.01)。与模型组比较,而BHT能显著降低NO、TNF-α、IL^(-1)β、iNOS含量(P<0.01),显著升高IL-4、IL^(-1)0的含量(P<0.01),显著降低Akt、NF-κB p65、IκBα、JAK2和STAT3蛋白的表达(P<0.01)。同时,BHT能抑制LPS-DMEM引起的SH-SY5Y细胞的凋亡(P<0.01)。结论:实验揭示BHT通过调控Akt/NF-κB/JAK2/STAT3信号通路抑制LPS诱导的BV2细胞炎症反应,并在SH-SY5Y细胞中表现出神经保护作用。

Objective:To study the inhibitory effect of Banxia Houputang(BHT)on lipopolysaccharide(LPS)-induced inflammation of microglia(BV2)cells and the neuroprotective effect on human neuroblastoma(SH-SY5Y)cells.Method:After the neuroinflammatory model was constructed by LPS inducing BV2 cells,model group(LPS 100μg·L^(-1)),administration groups(LPS+1 g·L^(-1)BHT,LPS+2 g·L^(-1)BHT,LPS+5 g·L^(-1)BHT,LPS+10 g·L^(-1)BHT),and blank group were given DEME medium at the same volume.In addition,neuronal apoptosis model was established by co-culture of LPS-induced BV2 cell inflammation medium and SH-SY5Y cells(LPS-DMEM)and was administrated according to the above grouping.Cell viability was detected by Cell Counting Kit-8(CCK-8)assay.The content of nitric oxide(NO)and that of tumor necrosis factor-α(TNF-α),interleukin-1β(IL^(-1)β),and interleukin-6(IL-6)were determined by Griess aasay and enzyme-linked immunosorbent assay(ELISA),respectively.The mRNA levels of TNF-α,IL^(-1)β,interleukin-4(IL-4),nitric oxide synthase(iNOS),and interleukin-10(IL^(-1)0)were measured by realtime polymerase chain reaction(Real-rime PCR).Western blot was used to detect the expression levels of signal transducer and activator of transcription 3(STAT3),Janus kinase 2(JAK2)and nuclear factor kappa-B(NF-κB p65),protein kinase B(Akt),inhibitor of nuclear factorκBα(IκBα),B-cell lymphoma-2(Bcl-2),and Bcl-2associated X protein(Bax).Result:Compared with blank group,LPS increased the NO release,levels of TNF-α,IL^(-1)β,IL-6,and iNOS and protein expression of Akt,NF-κB p65,IκBα,JAK2 and STAT3,decreased the content of IL-4 and IL^(-1)0 in BV2 cells,and induced apoptosis of co-cultured SH-SY5Y cells(P<0.01).Compared with model group,BHT reduced the content of NO,TNF-α,IL^(-1)β,and iNOS(P<0.01)and protein expression of Akt,NF-κB p65,IκBα,JAK2 and STAT3(P<0.01),elevated the content of IL-4 and IL^(-1)0(P<0.01),and inhibited the apoptosis of SH-SY5Y cells induced by LPS-DMEM(P<0.01).Conclusion:This experiment reveals that BHT inhibited LPS-induced inflammation in BV2 cells by regulating Akt/NF-κB/JAK2/STAT3 signaling pathway and showed neuroprotective effects on SH-SY5Y cells.