Naringenin prevents experimental liver fibrosis by blocking TGFβ-Smad3 and JNK-Smad3 pathways.
Naringenin prevents experimental liver fibrosis by blocking TGFβ-Smad3 and JNK-Smad3 pathways.
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DOI：
10.3748/wjg.v23.i24.4354
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发表时间：
2017-06-28
影响因子：
4.3
通讯作者：
Muriel P
中科院分区：
文献类型：
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作者：
Hernández-Aquino E;Zarco N;Casas-Grajales S;Ramos-Tovar E;Flores-Beltrán RE;Arauz J;Shibayama M;Favari L;Tsutsumi V;Segovia J;Muriel P
研究柚皮素（NAR）对四氯化碳（CCl 4）诱导的肝纤维化的保护作用及其分子机制。将32只雄性Wistar大鼠（120-150 g）随机分为4组：（1）对照组（n = 8），每天口服0.7%羧甲基纤维素（NAR载体）1 mL; (2)CCl_4组（n = 8）：CCl_4 + NAR组（n = 8）：NAR组（n = 8（4）NAR组（n = 8），每天给予NAR 100 mg/kg体重，连续8 wk。实验期后，在氯胺酮和甲苯噻嗪麻醉下处死动物。检测肝损伤指标丙氨酸氨基转移酶（ALT）、碱性磷酸酶（AP）、γ-谷氨酰转肽酶（γ-GTP）、还原型谷胱甘肽（GSH）、糖原含量、脂质过氧化（LPO）和胶原含量。谷胱甘肽过氧化物酶（GPx）的酶活性进行了评估。使用Masson三色和苏木精-伊红染色进行肝组织病理学检查。进行MMP-9和MMP-2的酶谱分析。Western blot检测肝组织TGF-β、α-SMA、CTGF、Col-I、MMP-13、NF-κB、IL-1、IL-10、Smad 7、Smad 3、pSmad 3和pJNK蛋白表达。NAR治疗组ALT、AP、γ-GTP和GPx酶活性升高，GSH和糖原消耗减少，LPO和胶原蛋白升高（P < 0.05）。肝组织病理学显示，当大鼠接受NAR和CCl 4时，胶原沉积减少。尽管酶谱分析显示CCl 4引起MMP-9和MMP-2明胶酶活性增加，但有趣的是，NAR给药与正常MMP-9和MMP-2活性相关（P < 0.05）。NAR的抗炎、抗坏死和抗纤维化作用可能与其抑制NF-κB活化及IL-1和IL-10的产生有关（P < 0.05）。与CCl 4处理组相比，NAR完全抑制TGF-β、α-SMA、CTGF、Col-1和MMP-13蛋白的升高（P < 0.05）。NAR抑制了Smad 3在连接区被JNK磷酸化，因为类黄酮阻断了该激酶（P < 0.05）。NAR作为自由基抑制剂具有抗氧化能力，可通过抑制NF-κB、TGF-β-Smad 3和JNK-Smad 3通路，预防CCl 4诱导的肝脏炎症、坏死和纤维化。
To study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis. Thirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson’s trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-β, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot. NAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-β, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05). NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-β-Smad3 and JNK-Smad3 pathways.