Department of Gastroenterology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China.
World J Gastroenterol. 2013 Jun 28;19(24):3781-91. doi: 10.3748/wjg.v19.i24.3781.
To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apoptosis in rats via a p53-dependent mitochondrial pathway.
Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis.
After 8 wk of treatment, GA significantly reduced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P < 0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P < 0.05), attenuated the changes in liver histopathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P < 0.05) in CCl4-treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7.87% ± 0.66% to 3.68% ± 0.32%, P < 0.05) compared with the CCl4 group. GA also decreased the expression level of cleaved caspase-3 compared to the CCl4 group. TUNEL assay indicated that GA significantly diminished the number of TUNEL-positive cells compared with the CCl4 group (P < 0.05). GA treatment clearly decreased the level of p53 (P < 0.05) detected by immunohistochemistry and Western blotting analysis. Compared with the CCl4 group, we also found that GA reduced the Bax/Bcl-2 ratio (P < 0.05), the expression of cleaved caspase-3 (P < 0.05), cleaved caspase-9 (P < 0.05), and inhibited cytochrome C and second mitochondria-derived activator of caspases (Smac) release from mitochondria to cytoplasm, i.e., GA reduced the expression level of Smac, which inhibited c-IAP1 activity (P < 0.05), ultimately inhibiting the activity of caspase-3, according to Western blotting analysis. As a result, GA suppressed activation of the caspase cascades and prevented hepatocyte apoptosis.
GA can inhibit CCl4-induced hepatocyte apoptosis via a p53-dependent mitochondrial pathway to retard the progress of liver fibrosis in rats.
研究甘草酸（GA）通过 p53 依赖性线粒体途径对四氯化碳（CCl4）诱导的大鼠肝细胞凋亡的影响。
将 45 只雄性 Sprague-Dawley 大鼠随机均分为 3 组，对照组、CCl4 组和 GA 治疗组。为了在该模型中诱导肝纤维化，大鼠每周接受两次皮下注射 40%的 CCl4 在橄榄油中的溶液，剂量为 0.3 mL/100 g 体重，共 8 周，而对照组则通过皮下注射等容量的橄榄油，初始剂量为两倍。在 GA 组中，大鼠还按照先前发表的方法，每周三次通过腹腔注射 3 mL 的 40%的 CCl4 加双蒸水的 0.2%GA 溶液进行治疗，有一些修改。对照组给予等容量的双蒸水。通过测定丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）等肝功能参数来确定肝损伤情况。通过苏木精和伊红染色检测肝组织病理学变化。通过天狼星红染色评估胶原纤维。使用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸末端标记（TUNEL）检测和裂解的 caspase-3 免疫组化检测来研究肝细胞凋亡。通过免疫组化或 Western 印迹分析评估 p53 和凋亡相关蛋白的表达水平。
经过 8 周的治疗，GA 显著降低了血清 ALT 活性（从 526.7 ± 57.2 降至 342 ± 44.8，P < 0.05）和 AST 活性（从 640 ± 33.7 降至 462.8 ± 30.6，P < 0.05），减轻了 CCl4 处理大鼠肝组织病理学变化，并降低了分期评分（从 3.53 ± 0.74 降至 3.00 ± 0.76，P < 0.05）。GA 显著降低了 Sirius 红的阳性面积和肝纤维化区域的比例（从 7.87% ± 0.66%降至 3.68% ± 0.32%，P < 0.05）与 CCl4 组相比。GA 还降低了与 CCl4 组相比，裂解的 caspase-3 的表达水平。TUNEL 检测表明，GA 与 CCl4 组相比，显著减少了 TUNEL 阳性细胞的数量（P < 0.05）。GA 处理明显降低了免疫组化和 Western blot 分析检测到的 p53 水平（P < 0.05）。与 CCl4 组相比，我们还发现 GA 降低了 Bax/Bcl-2 比值（P < 0.05）、裂解的 caspase-3（P < 0.05）、裂解的 caspase-9（P < 0.05）的表达，并抑制了细胞色素 C 和第二线粒体衍生的凋亡激活因子（Smac）从线粒体释放到细胞质，即 GA 降低了 Smac 的表达水平，抑制了 c-IAP1 的活性（P < 0.05），最终抑制了 caspase-3 的活性，根据 Western blot 分析。结果表明，GA 通过 p53 依赖性线粒体途径抑制 CCl4 诱导的肝细胞凋亡，从而延缓大鼠肝纤维化的进展。
