Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Eur Rev Med Pharmacol Sci. 2018 Nov;22(21):7543-7550. doi: 10.26355/eurrev_201811_16296.
To investigate whether CD31 could regulate paracetamol-induced liver injury, thereby providing a new direction for the prevention and treatment of drug-induced hepatitis.
Wild-type (WT) mice were treated with acetaminophen (APAP) (250 mg/kg) or isodose of phosphate-buffered saline (PBS). 1, 3, 6 and 12 h after the treatment, the messenger RNA (mRNA) and protein expression level of CD31 in the liver of mice were determined by Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Once CD31 was confirmed to be involved in APAP-induced liver injury, the acute liver injury model in WT mice and CD31 gene deficient (CD31-/-) mice induced by APAP was established. Serum samples were collected at 8 and 24 h after APAP injection (250 mg/kg), and the activity of serum alanine aminotransferase (ALT) was measured. The liver tissues of mice were isolated and analyzed by hematoxylin and eosin (HE) staining. Meanwhile, mononuclear cells (MNCs) were isolated from the liver tissues of mice. The number of infiltrating macrophages and neutrophils was detected by flow cytometry, and the activation level of these cells was analyzed. The expression levels of proinflammatory cytokines in liver tissues, such as TNF-α, IL-1β, keratinocyte chemoattractant (KC), MCP-1 and IL-6, were determined by RT-PCR. The expression levels of cytokines in serum were detected by enzyme-linked immunosorbent assay (ELISA). Moreover, the protein expression levels of JNK, Caspase-3, and cytochrome P450 2E1 (CYP2E1) in liver tissues were detected by Western blotting.
After APAP treatment, we found that WT mice were more sensitive to APAP-induced liver injury. The level of ALT in WT mice was significantly higher than that of CD31-/- mice, meanwhile, more necrotic or apoptotic cells were found in WT mice. Results also indicated that the expression levels of inflammatory cytokines, including KC, IL-1β, MCP-1 and IL-6, were significantly higher in WT mice. Meanwhile, the number of infiltrating macrophages and neutrophils in the liver tissues of WT mice were much more than that of CD31-/- mice.
APAP-treated CD31-/- mice exhibited less liver injury when compared with WT mice. We also confirmed that CD31 was greatly involved in APAP-induced inflammatory response by promoting hepatic inflammatory and cell apoptosis, which might provide a new strategy for the prevention and treatment of drug-induced hepatitis.
研究 CD31 是否可以调控对乙酰氨基酚（APAP）诱导的肝损伤，从而为防治药物性肝炎提供新的方向。
采用野生型（WT）小鼠给予对乙酰氨基酚（APAP）（250mg/kg）或等剂量磷酸盐缓冲液（PBS）处理。在处理后 1、3、6 和 12 小时，通过实时逆转录聚合酶链反应（RT-PCR）和 Western blot 分别检测小鼠肝脏中 CD31 的信使 RNA（mRNA）和蛋白表达水平。一旦证实 CD31 参与 APAP 诱导的肝损伤，就会在 WT 小鼠和缺乏 CD31 基因（CD31-/-）的小鼠中建立 APAP 诱导的急性肝损伤模型。在注射 APAP（250mg/kg）后 8 和 24 小时收集血清样本，并测定血清丙氨酸氨基转移酶（ALT）的活性。分离小鼠肝脏组织，进行苏木精和伊红（HE）染色分析。同时，从小鼠肝脏组织中分离单核细胞（MNC）。通过流式细胞术检测浸润巨噬细胞和中性粒细胞的数量，并分析这些细胞的激活水平。通过 RT-PCR 检测肝组织中促炎细胞因子，如 TNF-α、IL-1β、角质细胞化学引诱物（KC）、单核细胞趋化蛋白-1（MCP-1）和 IL-6 的表达水平。通过酶联免疫吸附测定（ELISA）检测血清中细胞因子的表达水平。此外，通过 Western blot 检测肝组织中 JNK、Caspase-3 和细胞色素 P450 2E1（CYP2E1）的蛋白表达水平。
APAP 处理后，我们发现 WT 小鼠对 APAP 诱导的肝损伤更为敏感。WT 小鼠的 ALT 水平明显高于 CD31-/-小鼠，同时，WT 小鼠中发现更多的坏死或凋亡细胞。结果还表明，WT 小鼠中 KC、IL-1β、MCP-1 和 IL-6 等炎症细胞因子的表达水平显著升高。同时，WT 小鼠肝脏组织中浸润的巨噬细胞和中性粒细胞数量也明显多于 CD31-/-小鼠。
与 WT 小鼠相比，APAP 处理的 CD31-/- 小鼠肝损伤程度较轻。我们还通过促进肝内炎症和细胞凋亡证实了 CD31 可显著参与 APAP 诱导的炎症反应，这可能为防治药物性肝炎提供新的策略。
