黄连素对Aβ25-35诱导的HT22细胞TLR4/NF-κB信号通路的影响

Effect of Berberine on TLR4/NF-κB Pathway Induced by Aβ25-35 in HT22 Cells

目的:探讨黄连素对β-淀粉样蛋白25-35(β-amyloid protein25-35,Aβ25-35)诱导的HT22细胞的保护作用及对Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路的影响。方法:培养HT22细胞,孵育不同浓度的黄连素(0,5,10,20,40,80μmol·L-1),24 h后行甲基噻唑基四唑法确定黄连素的保护浓度。随后将HT22细胞分为空白组,Aβ组,黄连素组,TAK242组。空白组加入培养基,Aβ组加入Aβ25-35(40μmol·L-1),黄连素组和TAK 242组加入黄连素(20μmol·L-1),TAK 242(1μmol·L-1)预处理1 h后加入Aβ25-35(40μmol·L-1)。共作用24 h后行乳酸脱氢酶释放实验检测细胞生存率,并行Hoechst33342染色检测细胞凋亡率蛋白免疫印迹法(Western blot)法检测TLR4,磷酸化(p)-NF-κB和肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β)的蛋白水平。结果:与空白组比较,20μmol·L-1的黄连素促进细胞增值,40μmol·L-1和80μmol·L-1的的黄连素抑制细胞增值(P<0.01),在各药物组中,20μmol·L-1的黄连素组的细胞增殖率最高(P<0.01);与空白组比较,Aβ组的细胞生存率降低(P<0.01),与Aβ组比较,黄连素组和TAK 242组的细胞生存率升高(P<0.01);与空白组比较,Aβ组的细胞凋亡率升高(P<0.01),且染色质凝集,核萎缩,凋亡小体增多,与Aβ组比较,黄连素组和TAK 242组的细胞凋亡率降低(P<0.01),且染色质凝集,核萎缩和凋亡小体较少;与空白组比较,Aβ组的TLR4,p-NF-κB和TNF-α和IL-1β的表达增多(P<0.01),与Aβ组比较,黄连素组和TAK 242组的TLR4,p-NF-κB和TNF-α和IL-1β的表达减少(P<0.01)。结论:黄连素可能通过抑制HT22细胞中由Aβ25-35活化的TLR4/NF-κB通路,从而减少神经元的凋亡起到保护作用。

Objective:To investigate the protective mechanism of berberine on HT22 cells induced by β- amyloid protein 25-35(A/32s-35)and observe its effect of toll-like receptor 4(TLR4)/nuclear factor-KB (NF-KB)signaling pathway.Method:HT22cells were cultured and different concentrations of berberine (0,5,10,20, 40,80μmol·L^-1)were incubated.After 24h,methylthiazolyl tetrazolium (MTT)assay was used to determine the protective concentration of berberine.Then HT22cells were divided into blank group,A/3group,berberine group and TAK 242group.HT22cells in blank group were treated with serum-containing media alone;HT22cells in A/3sroup were treated with A/325_35(40μmol·L^-1);while HT22cells in berberine group and TAK 242group were treated with berberine (20μmol·L^-1)and TAK 242(20μmol·L^-1)respectively for 1h,followed by Aβ32-35 (40μmol·L^-1)treatment.After 24h,lactate dehydrogenase (LDH)release test was used to measure the cell survival rate.Hoeehst 33342staining was used to determine the apoptosis rate.Western blot was used to detect the protein expression levels of TLR4,phosphorylated (p)-NF-KB,tumor necrosis factor-a (TNF-a)and interleukin- l^ (IL-lβ).Result:As compared with the blank group,20μmol·L^-1 berberine promoted cell proliferation;40 and 80μmol·L^-1berberine inhibited cell proliferation (P <0.01);among them,20μmol.L-I berberine group had the highest cell proliferation rate (P <0.01).As compared with the blank group,the cell survival rate was decreased in Aft group (P <0.01)and the cell survival rates in berberine group and TAK 242group were higher than that of the Aft group (P <0.01).As compared with the blank group,the apoptosis rate was increased in Aft group (P <0.01),and chromatin condensation,nuclear atrophy,and increased apoptotic bodies were observed. As compared with the Aft group,the apoptotic rate was lower in berberine group and TAK 242group (P <0.01), with less chromatin condensation,nuclear atrophy and apoptotic bodies.As compared with the blank group,the expression levels of TLR4,p-NF-KB,TNF-α and IL-lβ were increased in the Aft group (P <0.01).As compared with the Aft group,the expression levels of TLR4,p-NF-KB,TNF-a and IL-lfi were lower in berberine group and TAK 242group (P <0.01).Conclusion:Berberine may protect against neuronal apoptosis by inhibiting the Aβ25-35-activated TLR4/NF-KB pathway in HT22cells.