参附汤加味调控TLR4/MyD88/NF-κB抑制细胞炎症和氧化损伤对LPS诱导L-02肝细胞损伤的影响

Effects of modified Shenfu Decoction on the TLR4/MyD88/NF-κB signaling pathway in inhibiting cellular inflammation and oxidative stress in LPS-induced L-02 hepatocyte injury

关键词:  参附汤加味  脓毒症  肝损伤  氧化应激

Key words:  Modified Shenfu Decoction  sepsis  liver injury  oxidative stress

摘要:

[目的] 探讨参附汤加味对脂多糖（LPS）诱导的肝细胞损伤的保护作用。[方法] 选取27只SPF级SD大鼠，进行适应性喂养，1周后，进行灌胃，分别为对照组，参附汤低剂量组（1 g/kg），参附汤中剂量组（2 g/kg），参附汤高剂量组（4 g/kg）制备含药血清。将人正常肝细胞L-02细胞随机分为正常组（正常培养）、LPS组（40 μg/mLLPS）、参附汤加味低剂量+LPS组、参附汤加味中剂量+LPS组、参附汤加味高剂量+LPS组。处理24 h后，利用蛋白质印迹法（Western Blot）检测细胞中Toll样受体4（TLR4）、髓样分化初级反应蛋白88（MyD88）、核因子κB（NF-κB）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）等关键蛋白的表达水平变化。采用实时荧光定量聚合酶链式反应（qPCR）技术检测上述蛋白对应的基因表达水平。用CCK-8实验检测细胞增殖情况，用流式细胞术检测细胞凋亡情况，用荧光探针法检测各组细胞活性氧（ROS）水平。[结果] 与正常组相比，LPS诱导的L-02细胞增殖能力显著下降（P<0.01），炎症因子TLR4、MyD88、NF-κB、IL-1β、IL-6的蛋白及mRNA表达水平显著升高（P<0.01），细胞凋亡明显增加（P<0.01），且发生明显氧化应激、ROS增多（P<0.01）。与LPS组相比，参附汤加味低、中、高剂量组可使L-02细胞增殖能力上升（P<0.01），上述炎症因子的蛋白及mRNA表达水平明显下降（P<0.01），细胞凋亡减少（P<0.01），氧化应激降低（P<0.01）、ROS减少（P<0.01），差异均有统计学意义。[结论] 参附汤加味可改善LPS诱导的肝细胞损伤，其作用机制可能与调控TLR4/MyD88/NF-κB信号通路、减轻L-02细胞的炎症反应及氧化应激有关。本研究的体外实验结果为探讨参附汤加味在脓毒症肝损伤中的潜在应用提供了初步实验依据。

Abstract:

[Objective] To investigate the protective effect of Modified Shenfu Decoction on lipopolysaccharide （LPS）-induced hepatocellular injury. [Methods] Twenty-seven SPF-grade SD rats were selected for acclimatization feeding，and after one week，gavage was performed to prepare drug-containing serums for the control group，Shenfu Decoction low-dose group（1 g/kg），medium-dose group（2 g/kg），and high-dose group（4 g/kg），respectively. Human normal hepatocyte L-02 cells were randomly divided into the Control Group（normal culture），LPS group（40 μg/mL LPS），and Modified Shenfu Decoction low-，medium-，and high-dose+LPS groups. After 24 hours of treatment，the expression levels of key proteins such as Toll-like receptor 4（TLR4），myeloid differentiation primary response protein 88（MyD88），nuclear factor kappa-B（NF-κB），interleukin-1β（IL-1β），and interleukin-6（IL-6）in cells were detected by Western blot. The mRNA expression levels of the corresponding genes were measured using quantitative real-time polymerase chain reaction（qPCR）。 Cell proliferation was assessed by the CCK- 8 assay， apoptosis was detected by flow cytometry，and reactive oxygen species（ROS） levels were measured using a fluorescent probe. [Results] Compared with the Control Group，LPS-induced L-02 cells showed a significant decrease in proliferative ability（P<0.01），a significant increase in protein and mRNA expression levels of inflammatory factors TLR4，MyD88，NF-κB，IL-1β and IL-6（P<0.01），a significant increase in apoptosis（P<0.01）， and significant oxidative stress with increased ROS（P<0.01）. Compared with the LPS group，the Modified Shenfu Decoction low-，medium-，and high-dose groups increased the proliferative ability of L-02 cells（P<0.01）， significantly decreased the protein and mRNA expression levels of the aforementioned inflammatory factors（P< 0.01），reduced apoptosis（P<0.01），and decreased oxidative stress and ROS levels（P<0.01）. All differences were statistically significant. [Conclusion] Modified Shenfu Decoction can ameliorate LPS-induced hepatocellular injury. Its mechanism of action may be related to the regulation of the TLR4/MyD88/NF-κB signaling pathway and the attenuation of inflammatory response and oxidative stress in L-02 cells. The in vitro results of this study provide a preliminary experimental basis for exploring the potential application of Modified Shenfu Decoction in septic liver injury.