目的 探讨葛黄颗粒含药血清对乙醇诱导的L-02肝细胞损伤的影响及其可能机制。方法 SD雄性大鼠45只，随机分为三组（15只/组）：空白对照组、葛黄颗粒组及美他多辛组。空白对照组予以蒸馏水灌胃、葛黄颗粒组（3.1 g·100 g^-1·d^-1）、美他多辛组（0.1 g·100 g^-1·d^-1）灌胃，连续干预5天，腹主动脉取血制备含药血清。将L-02肝细胞分为六组并按以下方式干预：正常组（加入10%正常大鼠血清）、模型组（在正常组基础上加入3%乙醇）、葛黄颗粒高、中、低剂量组（分别加入不同浓度葛黄颗粒含药血清及正常大鼠血清及3%乙醇）、美他多辛组（加入3%乙醇、美他多辛及正常大鼠血清各5%），将L-02肝细胞培养24 h后进行之后实验部分。ELISA法检测NF-κB、TNF-α、IL-1β、IL-18水平；RT-PCR法检测NLRP3、ASC、Caspase-1 mRNA的表达；Western blot法检测TLR4、NLRP3、ASC、Caspase-1的蛋白表达。结果 与正常组比较：模型组NF-κB、TNF-α、IL-1β、IL-18明显升高，NLRP3、ASC、Caspase-1 mRNA表达明显升高，TLR4、NLRP3、ASC、Caspase-1的蛋白表达均亦明显升高（P<0.05）；与模型组相比较：以上指标在葛黄颗粒高、中剂量组及美他多辛组表达水平均有所降低（P<0.05）；与美他多辛组比较：葛黄颗粒高剂量组中TNF-α、IL-18有统计学意义（P<0.05），而IL-1β、NF-KB差异则不显著（P>0.05）；葛黄颗粒低剂量组中NLRP3、ASC、Caspase-1 mRNA差异显著（P<0.05），而在高、中剂量组差异则均无统计学意义（P>0.05）；TLR4、NLRP3、ASC、Caspase-1的蛋白表达水平在葛黄颗粒各剂量组均有统计学意义（P<0.05）。结论 葛黄颗粒含药血清可能通过TLR4/NLRP3信号通路改善乙醇诱导的肝细胞炎症损伤。

Objective To investigate the effect of serum containing Gehuang granules on ethanol-induced L-02 hepatocytes injury and its possible mechanism.Methods 45 SD male rats were randomly divided into three groups (15 rats/group): blank control group, Gehuang granule group and Metadoxine group. The blank control group was intragastrically administered with distilled water, Gehuang Granule group (3.1 g·100 g^-1·d^-1) and Metadoxine group (0.1 g·100 g^-1·d^-1) for 5 days. The blood was collected from abdominal aorta to prepare drug-containing serum. The L-02 hepatocytes were divided into six groups and intervened according to the following methods: normal group (adding 10% normal rat serum), model group (adding 3% ethanol on the basis of normal group), Gehuang granules high, medium and low dose groups (adding different concentrations of Gehuang granules medicated serum and normal rat serum and 3% ethanol respectively), and Metadoxine group (adding 3% ethanol, Metadoxine and normal rat serum 5% each). The L-02 hepatocytes were cultured for 24 h and then the experimental part was carried out. The levels of NF-κB, TNF-α, IL-1β and IL-18 were detected by ELISA. RT-PCR was used to detect the expression of NLRP3, ASC and Caspase-1. The protein expressions of TLR4, NLRP3, ASC and Caspase-1 were detected by Western blot.Results Compared with the normal group, the expressions of NF-κB, TNF-α, IL-1β, IL-18, and the expression of NLRP3, ASC, Caspase-1 mRNA and the levels of TLR4, NLRP3, ASC and caspase-1 protein in the model group were significantly increased (P<0.05). Compared with the model group, the expression levels of the above indexes in Gehuang granules high, medium dose group and Metadoxine group were decreased (P<0.05). Compared with the Metadoxine group: Gehuang granules high dose group TNF-α,IL-18 was statistically significant (P<0.05),However, there was no significant difference between high and medium dose groups (P>0.05). NLRP3, ASC, Caspase-1mRNA in Gehuang granules low dose group difference was statistically significant (P<0.05), however, IL-1β, NF-κB difference was not significant (P>0.05). The protein expression levels of TLR4, NLRP3, ASC and caspase-1 were statistically significant in each dose group of Gehuang Granules (P<0.05).Conclusion Gehuang Granule medicated serum may improve ethanol-induced inflammatory injury of hepatocytes through TLR4/NLRP3 signaling pathway.

四川省教育厅重点项目（16ZA0193）：葛黄颗粒对大鼠酒精性肝损伤的防治作用及其分子机制研究，负责人：魏嵋。