小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用

邹翠云; 陈小晶; 邬颖欣; 黄锦雄; 谭小红; 胡心月; 甘松永; 吴锦辉

doi:10.12131/20210362

小柴胡汤对D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的保护作用

doi: 10.12131/20210362

1.
仲恺农业工程学院动物科技学院/广东省水环境与水产品安全工程技术研究中心/广州市水产病害与水禽养殖重点实验室，广东广州 510225
2.
广东省农业技术推广中心，广东惠州 516081

基金项目: 大学生创新创业训练计划项目 (S202011347016)；广东省现代海水种业科技示范园项目；广东省科技创新战略专项资金 (攀登计划专项资金) 项目 (pdjh2020b0292)

详细信息

作者简介:
邹翠云 (1982—)，女，副教授，博士，从事水产动物营养与免疫研究。E-mail: zoucuiyun@zhku.edu.cn

通讯作者:
吴锦辉 (1979—)，男，工程师，硕士，从事工厂化循环水养殖研究。E-mail: wjhin@sina.com

中图分类号: S 917.4
计量
- 文章访问数: 384
- HTML全文浏览量: 168
- PDF下载量: 14
- 被引次数: 0
出版历程
- 收稿日期: 2021-12-01
- 修回日期: 2022-01-31
- 录用日期: 2022-03-15
- 网络出版日期: 2022-03-30
- 刊出日期: 2022-10-05

Protective effect of Xiaochaihu Decoction on D-GalN/ LPS-induced hepatocyte injury in hybrid grouper (Epinephelus lanceolatusy♂× E. fuscoguttatus♀)

1.
College of Animal Science & Technology, Zhongkai University of Agriculture and Engineering/Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center/Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangzhou 510225, China
2.
Guangdong Provincial Agricultural Technology Extension Center, Huizhou 516081, China

摘要

摘要: 为探讨小柴胡汤对虎龙杂交石斑鱼 (Epinephelus lanceolatus♂×E. fuscoguttatus♀) 化学性肝细胞损伤的免疫功能影响，用D-氨基半乳糖 (D-GalN, 20 mmol·L^–1) 和脂多糖 (LPS, 1 μg·mL^–1) 作用肝细胞24 h构建虎龙杂交石斑鱼肝细胞损伤模型，用不同质量浓度的小柴胡汤 (100、200和400 μg·mL^–1) 对肝细胞进行前处理 [D-氨基半乳糖联合脂多糖 (D-GalN/LPS) 损伤前加药]，通过CCK-8法检测肝细胞活力，HE染色观察肝细胞形态，收集细胞上清液检测炎症因子INF-γ、COX-2和PGE2的含量，以及细胞沉淀中凋亡和免疫相关基因的表达。结果表明，小柴胡汤可以提高肝细胞的成活率，不同程度地抑制D-GalN/LPS损伤引起的肝细胞培养上清液中INF-γ活性以及COX-2和PGE2含量的升高，降低细胞内乳酸脱氢酶 (LDH)、谷草转氨酶 (AST) 和谷丙转氨酶 (ALT) 活性以及细胞凋亡率，对肝细胞结构具有明显的保护作用；同时显著抑制凋亡相关基因caspase-3、caspase-9和P53的上调，促进免疫相关基因TLR3的上调。数据统计分析显示，添加200 μg·mL^–1小柴胡汤组的抑制效果最明显。综合以上结果，小柴胡汤对虎龙杂交石斑鱼化学性肝损伤有一定的保护作用，可预防肝脏疾病的发生和发展，为鱼类保肝药物研究提供了新的见解。
- 小柴胡汤 /
- 虎龙杂交石斑鱼 /
- D-GalN/LPS /
- 肝细胞损伤
Abstract: The study aims to evaluate the protective effect of Xiaochaihu Decoction (XCHD) on chemical hepatocyte injury. We treated the hepatocytes with D-GalN (20 mmol·L^–1) and LPS (1 μg·mL^–1) for 24 h to build a hybrid grouper (Epinephelus lanceolatus♂×E. fuscoguttatus♀) hepatocyte injury model, then pretreated the liver cells with different concentrations of XCHD (100, 200 and 400 μg·mL^–1) (D-GalN/LPS damage), and measured the hepatocyte viability by the CCK-8 assay. Hepatocyte morphology was visualized by HE staining, and cell supernatants were collected to detect the content of the inflammatory factors INF-, COX-2, and PGE2, as well as the expression of apoptotic and immune-related genes in the cell cryoprecipitate. The results show that XCHD can improve the survival rate of hepatocytes, inhibit INF-γ activity and coX-2 and PGE2 contents in the supernatant of hepatocytes induced by D-GalN/LPS injury to a different extent, reduce LDH, AST and ALT contents and apoptosis rate of hepatocytes. Besides, it had a significant protective effect on the structure of hepatocytes, significantly inhibiting the up-regulation of apoptosis-related genes caspase-3, caspase-9 and P53, and enhancing the up-regulation of immune-related genes TLR3. According to the data from the statistical analysis, the inhibition effect was most obvious in the 200 μg·mL^–1 XCHD group. In conclusion, XCHD has a protective effect on the chemical hepatocyte injury of Epinephelus lanceolatus♂×E. fuscoguttatus♀, and can prevent the occurrence and development of liver disease, which provides a new insight for the study of liver protection drugs in fish.
- Xiaochaihu Decoction /
- Epinephelus lanceolatus♂×E. fuscoguttatus♀ /
- D-GalN/LPS /
- Hepatocyte injury

HTML全文

图 1 小柴胡汤改善D-GalN/LPS诱导的虎龙杂交石斑鱼肝细胞损伤的成活率

注：不同小写字母表示差异显著 (P<0.05)；图 3、图 4、图 5-f、图 6 同此。

Figure 1. Hepatocyte viability of D-GalN/LPS-induced hepatocyte injury in hybrid grouper by Xiaochaihu Decoction

Note: Different small letters indicate significant difference (P<0.05); the same case in Fig. 3, Fig. 4, Fig. 5-f and Fig. 6.

下载: 全尺寸图片幻灯片

图 2 小柴胡汤对D-GalN/LPS诱导的肝细胞形态的影响

注：a. 对照组；b. 模型组；c. 100-XD；d. 200-XD；e. 400-XD；图5-a—5-e同此。

Figure 2. Effect of Xiaochaihu Decoction on morphology of D-GalN/LPS-treated hepatocytes

Note: a. Control; b. Model; c. 100-XD; d. 200-XD; e. 400-XD; the same case in Fig. 5-a−5-e.

下载: 全尺寸图片幻灯片

图 3 小柴胡汤对D-GalN/LPS诱导的上清液中细胞因子的炎症反应

Figure 3. Effect of Xiaochaihu Decoction on D-GalN/LPS-induced inflammatory responses in hepatocytes

下载: 全尺寸图片幻灯片

图 4 小柴胡汤对D-GalN/LPS诱导的细胞内肝功能指标的影响

Figure 4. Effect of Xiaochaihu Decoction on D-GalN/LPS-induced liver function index in hepatocytes

下载: 全尺寸图片幻灯片

图 5 小柴胡汤降低D-GalN/LPS诱导的细胞凋亡

注：f. 细胞凋亡率 (UR+LR)：将肝细胞进行 Annexin V-APC/7AAD 双染后，用流式细胞仪检测细胞凋亡率。

Figure 5. D-GalN/LPS-induced apoptosis reduced by Xiaochaihu Decoction

Note: f. Apoptotic cell ratio (Percentage of cells in quadrants UR and LR): The cells were double stained with Annexin V-APC/7AAD, and the apoptosis rate was determined using fow cytometry.

下载: 全尺寸图片幻灯片

图 6 小柴胡汤对D-GalN/LPS诱导的肝细胞免疫和凋亡相关基因的影响

Figure 6. Effect of Xiaochaihu Decoction on D-GalN/LPS-induced hepatocytes mRNA expression of immune and apoptosis genes

下载: 全尺寸图片幻灯片

表 1 本研究所用相关的引物序列

Table 1. Primer sequence of related genes in this study

基因 Gene	引物序列 (5'—3') Primer sequence (5'–3')
半胱氨酸天冬氨酸蛋白酶-3 Caspase-3	F: CGCAAAGAGTAGCGACGGA
半胱氨酸天冬氨酸蛋白酶-3 Caspase-3	R: CGATGCTGGGGAAATTCAGAC
53抑癌基因 P53	F: GGCACCAAACAAACCAAAAAAC
53抑癌基因 P53	R: GTCAAGCAACTCCAGACCATCA
半胱氨酸天冬氨酸蛋白酶-9 Caspase-9	F: TTTTCCTGGTTATGTTTCGTGG
半胱氨酸天冬氨酸蛋白酶-9 Caspase-9	R: TTGCTTGTAGAGCCCTTTTGC
抑制蛋白κBα IKKα	F: ACACCGACACAACGGCTCAT
抑制蛋白κBα IKKα	R: CCAGACGGCACAGTTTCACAG
谷胱甘肽过氧化物酶 GPx	F: TACCCTACCAAGTCCTCCAACC
谷胱甘肽过氧化物酶 GPx	R: AACAAACACCCGACACCCA
Toll样受体3 TLR3	F: TCTCCATTCCGTCACCTTCC
Toll样受体3 TLR3	R: TCATCCAGCCCGTTACTATCC
内参 β-Actin	F: TACGAGCTGCCTGACGGACA
内参 β-Actin	R: GGCTGTGATCTCCTTCTGC

下载: 导出CSV

参考文献(30)

[1]	张林宝, 陈海刚, 田斐, 等. 三唑磷对翡翠贻贝神经毒性与氧化损伤效应的性别差异研究[J]. 南方水产科学, 2020, 16(6): 75-80.
[2]	JUNIOR G B, de SOUZA C F, da SILVA H N P, et al. Combined effect of florfenicol with linalool via bath in combating Aeromonas hydrophila infection in silver catfish (Rhamdia quelen)[J]. Aquaculture, 2021, 545: 737247. doi: 10.1016/j.aquaculture.2021.737247
[3]	ARNOLD H, PLUTA H J, BRAUNBECK T. Simultaneous exposure of fish to endosulfan and disulfoton in vivo: ultrastructural, stereological and biochemical reactions in hepatocytes of male rainbow trout (Oncorhynchus mykiss)[J]. Aquat Toxicol, 1995, 33(1): 17-43. doi: 10.1016/0166-445X(95)00006-P
[4]	ZHENG T, JIA R, CAO L, et al. Alleviative effects of Ginkgo biloba extract on oxidative stress, inflammatory response and immune suppression induced by long-term glyphosate exposure in tilapia (Oreochromis niloticus)[J]. Aquaculture, 2022, 546: 737325. doi: 10.1016/j.aquaculture.2021.737325
[5]	ZHENG T, JIA R, CAO L, et al. Effects of chronic glyphosate exposure on antioxdative status, metabolism and immune response in tilapia (GIFT, Oreochromis niloticus)[J]. Comp Biochem Physiol C, 2020, 239: 108878.
[6]	SINGLEMAN C, HOLTZMAN N G. PCB and TCDD derived embryonic cardiac defects result from a novel AhR pathway[J]. Aquat Toxicol, 2021, 233: 105794. doi: 10.1016/j.aquatox.2021.105794
[7]	FU D, HU Y, CHU P, et al. Histopathological and calreticulin changes in the liver and gill of Takifugu fasciatus demonstrate the effects of copper nanoparticle and copper sulphate exposure[J]. Aquac Rep, 2021, 20(126): 100662.
[8]	AUTHMAN M M N, ABBAS W T, ABBAS H H, et al. Ameliorative effect of the dietary Egyptian leek (Allium ampeloprasum L. var. Kurrat) on zinc toxicity of the African catfish Clarias gariepinus (Burchell, 1822)[J]. Aquac Res, 2021, 52(11): 5656-5672. doi: 10.1111/are.15440
[9]	AZEVEDO A C B, BOZZA D A, DORIA H B, et al. Low levels of inorganic copper impair reproduction parameters in Oreochromis niloticus after chronic exposure[J]. Aquaculture, 2021, 545: 737186. doi: 10.1016/j.aquaculture.2021.737186
[10]	翟亚南, 王晶晶, 李梦, 等. D-氨基半乳糖联合脂多糖诱导小鼠慢性肝损伤模型的建立[J]. 中国比较医学杂志, 2014, 24(5): 62-65. doi: 10.3969/j.issn.1671-7856.2014.05.013
[11]	LIU B, LU Y, CHEN X, et al. Protective role of Shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice[J]. Nutrients, 2020, 12(2): 477. doi: 10.3390/nu12020477
[12]	徐安乐, 黎中宝, 上官静波, 等. 复方中草药对珍珠龙胆石斑鱼生长、非特异性免疫及消化酶活性的影响[J]. 海洋学报, 2018, 40(12): 49-57.
[13]	黄景军, 何琪, 赵会宏, 等. 饲料中添加复方中草药对鞍带石斑鱼生长与消化酶活性的影响[J]. 饲料工业, 2021, 42(4): 53-58.
[14]	GAO S, WANG T, HUANG X K, et al. Exploring the protective effect of modified Xiaochaihu Decoction against hepatic steatosis and inflammation by network pharmacology and validation in ageing rats[J]. Chin Med, 2020, 15(1): 1-19. doi: 10.1186/s13020-019-0281-6
[15]	ZHU W, LUO H, XIONG M, et al. Xiaochaihu Decoction for nonalcoholic fatty liver disease: a protocol for a systematic review and meta-analysis[J]. Medicine, 2021, 100(12): e25267. doi: 10.1097/MD.0000000000025267
[16]	詹理辉, 黄古叶, 韦翠婷, 等. 经方治疗肝纤维化的研究进展[J]. 中医药临床杂志, 2021, 33(7): 1406-1409.
[17]	张志雄, 刘春芳, 刘明洋, 等. 小柴胡汤的药理作用及临床应用研究进展[J]. 中医药临床杂志, 2021, 33(3): 580-584.
[18]	ZOU C, XU M, CHEN L, et al. Xiaochaihu Decoction reduces hepatic steatosis and improves D-GalN/LPS-induced liver injury in hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)[J]. Fish Shellfish Immunol, 2019, 91: 293-305. doi: 10.1016/j.fsi.2019.05.025
[19]	TAN X, SUN Z, LIU Q, et al. Effects of dietary ginkgo biloba leaf extract on growth performance, plasma biochemical parameters, fish composition, immune responses, liver histology, and immune and apoptosis-related genes expression of hybrid grouper (Epinephelus lanceolatus♂× Epinephelus fuscoguttatus♀) fed high lipid diets[J]. Fish Shellfish Immunol, 2018, 72: 399-409. doi: 10.1016/j.fsi.2017.10.022
[20]	郭楠, 郑怀东, 刘学光. 孔雀石绿在不同品种鱼类中残留量的研究与分析[J]. 河南水产, 2014(2): 29-30.
[21]	OGUNWOLE G A, ABIYA S E, AMAEZE N H, et al. Antioxidant markers in gills, liver and muscle tissue of the African sharptooth catfish (Clarias gariepinus) exposed to subchronic levels of Ibuprofen and Dibutyl phthalate[J]. Sci Afr, 2021, 12(12): e00816.
[22]	FU Q, SCHEIDEGGER A, LACZKO E, et al. Metabolomic profiling and toxicokinetics modeling to assess the effects of the pharmaceutical diclofenac in the aquatic invertebrate Hyalella azteca[J]. Environ Sci Technol, 2021, 55: 7920-7929. doi: 10.1021/acs.est.0c07887
[23]	翟雪珍. 小柴胡汤加减联合恩替卡韦治疗慢性乙型病毒性肝炎肝郁脾虚型疗效观察[J]. 实用中医药杂志, 2020, 36(1): 39-40.
[24]	王丽敏, 张丽艳, 谷松. 解析小柴胡汤“和法”调节自身免疫性疾病机制[J]. 中华中医药学刊, 2018, 36(1): 218-221.
[25]	AMADI C N, ORISAKWE O E. Herb-induced liver injuries in developing nations: an update[J]. Toxics, 2018, 6(2): 24. doi: 10.3390/toxics6020024
[26]	蔡乐. 基于肝器官芯片的中草药成分肝毒性评价[D]. 大连: 大连理工大学, 2019: 13-14.
[27]	商海涛, 庞欣欣, 张晴晴, 等. 中草药致肾损伤及其相关成分毒性机制研究进展[J]. 医药导报, 2021, 40(9): 1210-1215.
[28]	焦豪妍, 许梦怡, 师大智, 等. 蓝莓叶总黄酮对LPS/D-GalN诱导小鼠急性肝损伤的保护作用[J]. 食品研究与开发, 2020, 41(17): 1-6.
[29]	ZHANG T, CHEN Y, ZHAN Z, et al. Oridonin alleviates d-Galn/lps-induced acute liver injury by inhibiting NLRP3 inflammasome[J]. Drug Dev Res, 2021, 82(4): 575-580. doi: 10.1002/ddr.21776
[30]	TSUKAYAMA I, MEGA T, HOJO N, et al. Diosgenin suppresses COX-2 and mPGES-1 via GR and improves LPS-induced liver injury in mouse[J]. Prostag Oth Lipid M, 2021, 156: 106580.