收稿日期: 2023-03-14
网络出版日期: 2023-09-26
基金资助
国家自然科学基金地区科学基金项目(82260408);云南省科技厅基础研究专项面上项目(202101AT070054);昆明市卫健委卫生科研课题(2022-03-08-005)
Gentiopicroside promotes autophagy to alleviate nonalcoholic steatohepatitis in rats via up⁃regulating the expression of LC3Ⅱ
Received date: 2023-03-14
Online published: 2023-09-26
目的 明确龙胆苦甙对大鼠非酒精性脂肪性肝炎（non-alcoholic steatohepatitis，NASH）的疗效，并初步探讨LC3Ⅱ基因及自噬在其中所起的作用及机制。 方法 建立SD大鼠NASH模型，分为：正常对照组、模型对照组、龙胆苦甙组、龙胆苦甙+3?MA组、二甲双胍阳性对照组，每组8只大鼠。给予相应治疗和处理4周后，通过q-PCR及Western blot分别检测大鼠肝组织LC3Ⅱ、LC3Ⅰ的mRNA和蛋白质表达，HE染色检测肝组织病理学情况，结合大鼠血清的血脂（CHOL和TG）、谷丙转氨酶以及炎症因子（IL-1β和TNF-α）水平的变化，评价龙胆苦甙对大鼠NASH的治疗效果，并初步探讨LC3Ⅱ及自噬在这一过程中所起的作用和机制。 结果 （1）正常对照组肝小叶结构正常，未见脂肪变及炎症损伤；模型对照组肝细胞索结构紊乱，出现严重肝脂肪变及炎症损伤，并可见点、片状坏死形成；龙胆苦甙组、二甲双胍阳性对照组肝脂肪变及炎症损伤均有所减轻；龙胆苦甙+3?MA组肝脂肪变和炎症损伤较龙胆苦甙组明显加重，肝小叶结构紊乱，坏死增多。（2）模型对照组ALT、CHOL、TG、IL-1β、TNF-α水平均较正常对照组升高，龙胆苦甙组、二甲双胍阳性对照组的ALT、CHOL、TG、IL-1β、TNF-α水平一定程度上得到逆转，而龙胆苦甙+3?MA组ALT、CHOL、TG、IL-1β、TNF-α水平又较龙胆苦甙组升高，差异有统计学意义（P < 0.05）。（3）模型对照组大鼠肝组织LC3Ⅱ mRNA和蛋白质表达较正常对照组明显下调，这一趋势在龙胆苦甙组和二甲双胍阳性对照组中出现逆转，而龙胆苦甙+3?MA组的LC3Ⅱ mRNA和蛋白质表达又明显下调，差异有统计学意义（均P < 0.05）。 结论 龙胆苦甙可缓解大鼠NASH相关肝损伤，其机制可能与龙胆苦甙上调肝组织LC3Ⅱ表达从而促进了自噬有关。
关键词： 龙胆苦甙; 微管相关蛋白1轻链3Ⅱ; 微管相关蛋白1轻链3Ⅰ; 自噬; 非酒精性脂肪性肝炎
丁洁 , 刘思奇 , 王艺颖 , 王霖 , 施承宏 , 王芳 , 常国楫 , 华丽娟 , 陈华憶 , 李生浩 , 王晴晴 . 龙胆苦甙上调大鼠LC3Ⅱ表达促进自噬缓解非酒精性脂肪性肝炎[J]. 实用医学杂志, 2023 , 39(16) : 2022 -2028 . DOI: 10.3969/j.issn.1006-5725.2023.16.003
Objective To clarify the effect of gentiopicroside on alleviating nonalcoholic steatohepatitis （NASH） in rats， and to explore the role of LC3II gene and autophagy in this process. Methods Establishment of SD rat NASH model was established and assigned to normal control group， model control group， gentiopicroside group， gentiopicroside+autophagy inhibitor 3-methyladenine group， metformin positive control group， 8 rats in each group. After 4 weeks of treatment， mRNA and protein expression of LC3Ⅱ were detected， LC3Ⅰ in liver tissue was assessed by q-PCR and Western blot， HE staining was performed for liver histopathology， the therapeutic effect of gentiopicroside on NASH in rats was evaluated with serum lipids （CHOL and TG）， alanine aminotransferase and inflammatory factor （IL-1β and TNF-α） changes. Results （1）In the normal control group， the structure of hepatic lobule was normal， without fatty change and inflammatory damage； In the model control group， the structure of hepatocyte cord was disordered， with severe liver steatosis and inflammatory damage， and spot and flaky necrosis was observed； The liver steatosis and inflammatory damage were alleviated in gentiopicroside group and metformin positive control group； Compared with gentiopicroside group， liver steatosis and inflammatory damage in gentiopicroside+3?MA group were significantly aggravated， and the structure of hepatic lobule was disordered and necrosis was increased. （2）The ALT， CHOL， TG， IL-1β， TNF-α levels of NASH model control group were significantly higher than those in the normal control group， the ALT， CHOL， TG， IL-1β， TNF-α levels of gentiopicroside group， metformin positive control group were reversed to some extent， while gentiopicroside+3?MA group the level of ALT， CHOL， TG， IL-1β， TNF-α is higher than that of gentiopicroside group， the difference was statistically significant， all P < 0.05. （3）The expression of LC3Ⅱ mRNA and protein in the liver tissue of rats in the model control group was significantly lower than that in the normal control group. This trend was reversed in the gentiopicroside group and the metformin positive control group， while the expression of LC3ⅡmRNA and protein in the gentiopicroside+3-MA group was significantly lower， the difference was statistically significant， all P < 0.05. Conclusion Gentiopicroside could alleviate NASH-related liver injury in rats. Its mechanism may be related to GPS up-regulating the expression of LC3Ⅱ in liver tissue and promoting autophagy
| 1 | ZHOU J， ZHOU F， WANG W， et al. Epidemiological Features of NAFLD From 1999 to 2018 in China［J］. Hepatology， 2020，71（5）：1851-1864. |
| 2 | SUMID Y， YONEDA M， SEKO Y， et al. Role of vitamin E in the treatment of non-alcoholic steatohepatitis［J］. Free Radic Biol Med， 2021，177：391-403. |
| 3 | UDOH U S， RAJAN P K， NAKAFUKU Y， et al. Cell Autophagy in NASH and NASH-Related Hepatocellular Carcinoma［J］. Int J Mol Sci， 2022，23（14）：7734. |
| 4 | FILALI-MOUNCEF Y， HUNTER C， ROCCIO F， et al. The ménage à trois of autophagy， lipid droplets and liver disease［J］. Autophagy， 2022，18（1）：50-72. |
| 5 | JIN M， FENG H， WANG Y， et al. Gentiopicroside Ameliorates Oxidative Stress and Lipid Accumulation through Nuclear Factor Erythroid 2-Related Factor 2 Activation［J］. Oxid Med Cell Longev，2020，2020：2940746. |
| 6 | XIAO H， SUN X， LIN Z， et al. Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism［J］. Acta Pharm Sin B， 2022， 12（6）： 2887-2904. |
| 7 | LIU Q， CHENG L， MATSUURA A， et al. Gentiopicroside， a Secoiridoid Glycoside from Gentiana rigescens Franch， Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress［J］. Oxid Med Cell Longev，2020，202：9125752. |
| 8 | ZHANG N， JIANG Y， MU F， et al. Gentiopicrin exerts anti-rheumatic effect in human fibroblast-like synoviocytes via inhibition of p38MAPK/NF-κB pathway［J］. Cell Mol Biol （Noisy-le-grand）， 2019， 65（6）： 85-90. |
| 9 | VAN HERCK M A， VONGHIA L， FRANCQUE S M. Animal Models of Nonalcoholic Fatty Liver Disease-A Starter's Guide［J］. Nutrients， 2017，9（10）：1072. |
| 10 | 王鑫蕾，倪晓晴，苏建友，等.AKT/FOXO1信号通路在限热卡高脂饮食改善肥胖小鼠肝脏胰岛素抵抗中的作用［J］.实用医学杂志，2020，36（16）：2199-2204. |
| 11 | KIM Y W， KIM J Y， PARK Y H， et al. Metformin restores leptin sensitivity in high-fat-fed obese rats with leptin resistance［J］. Diabetes， 2006， 55（3）： 716-724. |
| 12 | QIAN H， CHAO X， WILLIAMS J， et al. Autophagy in liver diseases： A review［J］. Mol Aspects Med， 2021，82：100973. |
| 13 | XIE Z， KLIONSKY D J. Autophagosome formation： core machinery and adaptations［J］. Nat Cell Biol， 2007，9（10）：1102-1109. |
| 14 | KIM J， KUNDU M， VIOLLET B， et al. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1［J］. Nat Cell Biol， 2011， 13（2）： 132-141. |
| 15 | HANDA P， MALIKEN B D， NELSON J E， et al. Reduced adiponectin signaling due to weight gain results in nonalcoholic steatohepatitis through impaired mitochondrial biogenesis［J］. Hepatology， 2014， 60（1）： 133-145. |
| 16 | QUAN H Y， KIM D Y， KIM S J， et al. Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway［J］. Biochem Pharmacol，2013， 85（9）：1330-1340. |
| 17 | WANG S， LI X， GUO H， et al. Emodin alleviates hepatic steatosis by inhibiting sterol regulatory element binding protein 1 activity by way of the calcium/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mechanistic target of rapamycin-p70 ribosomal S6 kinase signaling pathway［J］. Hepatol Res，2017，47（7）： 683-701. |
| 18 | KABEYA Y， MIZUSHIMA N， UENO T， et al. LC3， a mammalian homologue of yeast Apg8p， is localized in autophagosome membranes after processing［J］. EMBO， 2000，19（21）：5720-5728. |
| 19 | CHERRA S J 3rd， KULICH S M， UECHI G， et al. Regulation of the autophagy protein LC3 by phosphorylation［J］. Cell Biol， 2010，190（4）：533-539. |
| 20 | WANG X， DE CARVALHO RIBEIRO M， IRACHETA-VELLVE A， et al. Macrophage-Specific Hypoxia-Inducible Factor-1α Contributes to Impaired Autophagic Flux in Nonalcoholic Steatohepatitis［J］. Hepatology， 2019， 69（2）： 545-563. |
| 21 | 朱稀雯. NASH中自噬通过TRAF6介导的泛素化抑制Kupffer细胞NLRP3炎症小体激活机制的研究［D］. 重庆：重庆医科大学，2017. |
| 22 | 罗莉川.“脂肝方”对NASH大鼠肝细胞线粒体选择性自噬及其碎片降解机制的影响［D］. 南宁：广西中医药大学，2017. |
| 23 | 谭玉柱.蜘蛛香化学成分及生物活性研究［D］. 成都：成都中医药大学， 2017. |
| 24 | ZHANG Q L， XIA P F， PENG X J， et al. Synthesis， and anti-inflammatory activities of gentiopicroside derivatives［J］. Chin J Nat Med， 2022， 20（4）： 309-320. |
| 25 | 屈肖肖，乔庆阳，陈宁，等. 自噬对激素性股骨头坏死大鼠的作用效果分析［J］. 实用医学杂志，2023，39（3）：285-288，297. |
| 26 | ORRES S， SEALéS P， GARCíA-RUIZ C， et al. Mitochondria and the NLRP3 Inflammasome in Alcoholic and Nonalcoholic Steatohepatitis［J］. Cells， 2022，11（9）：1475. |
| 27 | BIASIZZO M， KOPITAR-JERALA N. Interplay Between NLRP3 Inflammasome and Autophagy［J］. Front Immunol， 2020，11：591803. |
| 28 | ZHANG Q L， XIA P F， PENG X J， et al. Synthesis， and anti-inflammatory activities of gentiopicroside derivatives［J］. Chin J Nat Med， 2022，20（4）：309-320. |
| 29 | WAN Z， LI H， WU X， et al. Hepatoprotective effect of gentiopicroside in combination with leflunomide and/or methotrexate in arthritic rats［J］. Life Sci，2021，265：118689. |
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