目的 探讨黄连(RC)对脂多糖(LPS)诱导的大鼠肝细胞(BRL)炎性损伤的保护作用及其机制。方法 体外构建LPS诱导的BRL细胞损伤模型,用0.175 mg·mL-1和0.1 mg·mL-1的RC水提液(试验药物)和地塞米松(阳性对照药物)对模型细胞进行干预处理。采用CCK-8法筛选确定LPS及RC水提液的最适试验浓度,流式细胞术检测BRL细胞凋亡率,RT-PCR检测Toll样受体4/核转录因子-κB(TLR4/NF-κB)、Toll样受体4/干扰素调节因子3(TLR4/IRF3)2条信号传导通路和相关炎性因子[肿瘤细胞坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)]mRNA相对表达量,Western blot和免疫细胞荧光染色法检测NF-κB p65蛋白的表达情况。结果 ①与空白对照组相比,0.1 mg·mL-1 LPS与BRL细胞作用24 h后,BRL细胞存活率明显降低(P<0.01),细胞凋亡率明显增加(P<0.01),细胞TLR4、NF-κB、IRF3、TNF-α、IL-1β和IL-6的mRNA表达明显升高(P<0.01),NF-κB p65蛋白表达上升;②分别用0.175和0.1 mg·mL-1的RC水提液进行干预后,与模型组相比,BRL细胞存活率显著增加(P<0.01),细胞凋亡率显著降低(P<0.01),TLR4、NF-κB、IRF3、TNF-α、IL-1β和IL-6的mRNA及NF-κB p65蛋白表达量明显降低(P<0.01)。结论 RC水提液对LPS诱导的BRL细胞炎性损伤具有较好的保护作用,其机制可能与抑制细胞凋亡、减少TNF-α、IL-1β、IL-6等炎症反应因子的释放,抑制NF-κB p65蛋白核转移,阻碍R4/NF-κB、TLR4/IRF3 2条信号通路的传导等有关。
Abstract
OBJECTIVE To investigate the protective effect and possible mechanism of Rhizoma Coptis(RC) on lipopolysaccharide(LPS)-induced inflammatory injury in rat hepatocytes(BRL). METHODS LPS-induced BRL cells injury model was established in vitro, then the damaged cells were given different interventions and treatment with 0.175, 0.1 mg· mL-1 RC aqueous extract as the test drug, and dexamethasone(Dex) as positive control drug. The optimal test doses of LPS and RC aqueous extract were selected and determined by cell counting kit-8(CCK-8), the cellular apoptosis rate was determined by flow cytometry, TLR4/NF-κB and TLR4/IRF3 signaling pathways and the mRNA level of related inflammatory mediators(TNF-α, IL-1β, IL-6) were detected by RT-PCR, the NF-κB p65 protein expression was analysed by Western blot and immunofluorescence techniques. RESULTS ①Compared with normal control group, 0.1 mg·mL-1 LPS affected on BRL cells for 24 h, the cell survival rate was decreased significantly(P<0.01), the apoptotic rate increased significantly(P<0.01), the mRNA level of TLR4, NF-κB, IRF3, TNF-α, IL-1β, IL-6 were significantly increased(P<0.01), and the NF-κB p65 protein expression was increased. ②Compared with the model group, 0.1 and 0.175 mg·mL-1 RC affected on LPS-induced BRL cells for 24 h, the survival rate of BRL cells was increased significantly(P<0.05), the apoptotic rate decreased significantly(P<0.01), the mRNA level of TLR4, NF-κB, IRF3, TNF-α, IL-1β, IL-6 and the NF-κB p65 protein expression were decreased significantly(P<0.01). CONCLUSION Rhizoma Coptis has obviously protective effect on LPS-induced inflammatory injury in rat hepatocytes(BRL), the mechanism of which may be related with inhibiting apoptosis, reducing the release of inflammatory factors such as TNF-α、IL-1β and IL-6, blocking NF-κB p65 protein nuclear translocation, interfering the R4/NF-κB and TLR4/IRF3 signaling pathway.
关键词
黄连 /
脂多糖 /
大鼠肝细胞 /
炎性损伤 /
保护机制
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Key words
Rhizoma Coptis /
lipopolysaccharide /
rat hepatocyte /
inflammatory injury /
protective mechanism
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中图分类号：
R285.5
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脚注
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基金
国家自然科学基金项目资助(31172358);2015年安徽农业大学研究生创新项目资助(201520)
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