Department of Emergency, Xiangya Hospital Central South University, Changsha, 410008, Hunan Province, People's Republic of China.
Department of Infectious Diseases/Hunan Provincial Key Laboratory of Viral Hepatitis, Xiangya Hospital Central South University, Changsha, 410008, Hunan Province, People's Republic of China.
Inflamm Res. 2022 Nov;71(10-11):1213-1227. doi: 10.1007/s00011-022-01603-0. Epub 2022 Jul 8.
Acute liver injury is liver cell injury that occurs rapidly in a short period of time. Caffeine has been shown to maintain hepatoprotective effect with an unclear mechanism. Endoplasmic reticulum stress (ERS) has significant effects in acute liver injury. Induction of GRP78 is a hallmark of ERS. Whether or not caffeine's function is related to GRP78 remains to be explored.
Acute liver injury model was established by LPS-treated L02 cells and in vivo administration of LPS/D-Gal in mice. Caffeine was pre-treated in L02 cells or mice. Gene levels was determined by real-time PCR and western blot. Cell viability was tested by CCK-8 assay and cell apoptosis was tested by flow cytometry. The interaction of GRP78 and NEDD4L was determined by Pull-down and co-immunoprecipitation (Co-IP) assay. The ubiquitination by NEDD4L on GRP78 was validated by in vitro ubiquitination assay.
Caffeine protected liver cells against acute injury induced cell apoptosis and ERS both in vitro and in vivo. Suppression of GRP78 could block the LPS-induced cell apoptosis and ERS. NEDD4L was found to interact with GRP78 and ubiquitinate its lysine of 324 site directly. Caffeine treatment induced the expression of NEDD4L, resulting in the ubiquitination and inhibition of GRP78.
Caffeine mitigated the acute liver injury by stimulating NEDD4L expression, which inhibited GRP78 expression via ubiquitination at its K324 site. Low dose of caffeine could be a promising therapeutic treatment for acute liver injury.
急性肝损伤是指在短时间内迅速发生的肝细胞损伤。咖啡因已被证明具有肝保护作用，但作用机制尚不清楚。内质网应激（ERS）在急性肝损伤中具有重要作用。诱导 GRP78 的表达是 ERS 的一个标志。咖啡因的作用是否与 GRP78 有关仍有待探讨。
通过 LPS 处理的 L02 细胞和 LPS/D-Gal 在小鼠体内给药建立急性肝损伤模型。用咖啡因预处理 L02 细胞或小鼠。通过实时 PCR 和 Western blot 测定基因水平。用 CCK-8 测定细胞活力，用流式细胞术测定细胞凋亡。通过下拉和免疫共沉淀（Co-IP）实验测定 GRP78 与 NEDD4L 的相互作用。通过体外泛素化实验验证 NEDD4L 对 GRP78 的泛素化作用。
咖啡因在体外和体内均能保护肝细胞免受急性损伤诱导的细胞凋亡和 ERS。抑制 GRP78 可阻断 LPS 诱导的细胞凋亡和 ERS。发现 NEDD4L 与 GRP78 相互作用，并直接泛素化其赖氨酸 324 位。咖啡因处理诱导 NEDD4L 的表达，导致 GRP78 的泛素化和抑制。
咖啡因通过刺激 NEDD4L 的表达减轻急性肝损伤，通过泛素化 GRP78 的 K324 位点抑制其表达。低剂量的咖啡因可能是治疗急性肝损伤的一种有前途的治疗方法。
