黄诺玛苷通过抑制LPS诱导BV2细胞炎性活化保护HT22细胞的机制

Flavanomarein Protects HT22 Cells by Inhibiting LPS-induced Inflammatory Activation of BV2 Cells

目的:探讨黄诺玛苷通过抑制脂多糖(LPS)诱导的小鼠小胶质细胞(BV2)炎症反应,对小鼠海马神经元细胞(HT22)的保护作用以及相关机制。方法:BV2细胞分为正常组(Con),模型组(LPS 1μg/mL),黄诺玛苷(flavanomarein,FL)低、中、高剂量组(50、100、200μmol/L),TLR4抑制剂组(TAK,10μmol/L),抑制剂+黄诺玛苷组(TAK+FL,10μmol/L+200μmol/L)。检测细胞存活率、细胞上清液中NO、肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)的含量,Western blot检测BV2细胞中TLR4、Myd88、NF-кB、p-NF-кB蛋白表达。各组BV2细胞上清作为条件培养基(CM)作用于HT22细胞,检测细胞活力并进行形态学观察,Western blot检测HT22细胞中Bax、Bcl-2、cleaved-Caspase-3、Caspase-3蛋白表达。结果表明,不同浓度的FL干预BV2细胞,与Con组相比,细胞存活率无显著性差异。与LPS组相比,FL(200μmol/L)组NO释放量、TNF-α和IL-6分泌量均显著性降低(P<0.001)。Western blot结果表明,FL各组TLR4、Myd88、NF-κBp65、p-NF-κBp65的蛋白表达均降低。条件培养基(CM)处理后,与CM-Con组相比,CM-LPS组中HT22细胞存活率显著性降低(P<0.001),细胞皱缩,破裂较多;CM-FL的不同剂量组HT22细胞存活率逐渐恢复,呈现剂量依赖性,细胞形态逐渐恢复,呈现梭形。Western blot结果表明,与CM-Con组相比,CM-LPS中cleaved-Caspase3和Bax表达升高,Bcl-2表达降低,FL会逆转上述蛋白的表达情况。综上,黄诺玛苷可以通过抑制神经炎症,进而保护神经细胞。

Objective:To investigate the protective effects of flavonomaside on HT22 mouse hippocampal neuronal cells by inhibiting the inflammatory response induced by lipopolysaccharide(LPS)in mouse microglia(BV2)and elucidate the underlying mechanisms.Methods:BV2 cells were categorized into control(Con),LPS model(1μg/mL),flavonomaside low,medium,and high dose groups(50,100,200μmol/L respectively),TLR4 inhibitor group(TAK,10μmol/L),and TAK+flavonomaside group(10μmol/L+200μmol/L).Nitric oxide(NO),tumor necrosis factor-alpha(TNF-α),and interleukin-6(IL-6)levels in supernatants and cell viability were quantified.Western blot assessed the protein levels of TLR4,Myd88,NF-κB p65,and phosphorylated NF-κB p65 in BV2 cells.The BV2 cell supernatant from each group acted as conditioned medium(CM)on HT22 cells to measure cell viability and observe morphological changes.Western blot also detected the protein levels of Bax,Bcl-2,cleaved Caspase-3,and Caspase-3 in HT22 cells.Results:No significant difference in BV2 cell viability was observed with varying flavonomaside concentrations compared to the Con group.The flavonomaside(200μmol/L)group showed a significant reduction in NO,TNF-α,and IL-6 secretion compared to the LPS group(P<0.001).Western blot results indicated significant differences in TLR4,Myd88,NF-κB p65,and phosphorylated NF-κB p65 protein levels in the flavonomaside groups,respectively.Compared to the CM-Con group,HT22 cell viability significantly decreased in the CM-LPS group(P<0.001),with cells exhibiting more shrinkage and rupture.HT22 cell viability in the CM-flavonomaside groups gradually improved in a dose-dependent manner,with cell morphology progressively normalizing to a spindle shape.Western blot analysis revealed increased expression of cleaved Caspase-3 and Bax and decreased Bcl-2 expression in the CM-flavonomaside groups compared to the CM-Con group,indicating flavonomaside's reversal of protein expression changes.In conclusion,flavonomaside can protect nerve cells by inhibiting neuroinflammation.