Institute of Digestive Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China.
Zhoupu Hospital, Shanghai University of Medicine &Health Sciences, Shanghai, 201318, China.
BMC Complement Altern Med. 2019 Jan 7;19(1):8. doi: 10.1186/s12906-018-2424-1.
Ling-gui-zhu-gan decoction (LGZG), a classic traditional Chinese medicine formula, has been confirmed to be effective in improving steatosis in non-alcoholic fatty liver disease (NAFLD). However, the mechanism under the efficacy remains unclear. Hence, this study was designed to investigate the mechanisms of LGZG on alleviating steatosis.
Twenty four rats were randomly divided into three groups: normal group, NAFLD group, fed with high fat diet (HFD) and LGZG group (fed with HFD and supplemented with LGZG). After 4 weeks intervention, blood and liver were collected. Liver steatosis was detected by Oil Red O staining, and blood lipids were biochemically determined. Whole genome genes were detected by RNA-Seq and the significant different genes were verified by RT-qPCR. The protein expression of Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) and key molecules of glycogen and lipid metabolism were measured by western blot. Chromophore substrate methods measured glycogen phosphorylase (GPa) activity and glycogen content.
HFD can markedly induce hepatic steatosis and promote liver triglyceride (TG) and serum cholesterol (CHOL) contents, while liver TG and serum CHOL were both markedly decreased by LGZG treatment for 4 weeks. By RNA sequencing, we found that NAFLD rats showed significantly increase of PPP1R3C expression and LGZG reduced its expression. RT-qPCR and Western blot both verified the alteration of PPP1R3C upon LGZG intervention. LGZG also promoted the activity of glycogen phosphorylase liver type (PYGL) and inhibited the activity of glycogen synthase (GS) in NAFLD rats, resulting in glycogenolysis increase and glycogen synthesis decrease in the liver. By detecting glycogen content, we also found that LGZG reduced hepatic glycogen in NAFLD rats. In addition, we analyzed the key molecules in hepatic de novo lipogenesis and cholesterol synthesis, and indicated that LGZG markedly inhibited the activity of acetyl-CoA carboxylase (ACC), sterol receptor element-binding protein-1c (SREBP-1c) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), resulting in lipid synthesis decrease in the liver.
Our data highlighted the role of PPP1R3C targeting pathways, and found that hepatic glycogen metabolism might be the potential target of LGZG in preventing NAFLD.
灵龟-猪胆汁汤（LGZG）是一种经典的中药方剂，已被证实能有效改善非酒精性脂肪性肝病（NAFLD）中的脂肪变性。然而，其疗效的机制尚不清楚。因此，本研究旨在探讨 LGZG 缓解脂肪变性的机制。
将 24 只大鼠随机分为三组：正常组、NAFLD 组（给予高脂肪饮食 HFD）和 LGZG 组（给予 HFD 并补充 LGZG）。干预 4 周后，采集血液和肝脏。油红 O 染色检测肝脂肪变性，生化法检测血脂。采用 RNA-Seq 检测全基因组基因，通过 RT-qPCR 验证差异显著的基因。采用 Western blot 法测定蛋白磷酸酶 1 调节亚基 3C（PPP1R3C）和糖原及脂质代谢关键分子的蛋白表达。采用发色底物法测定糖原磷酸化酶（GPa）活性和糖原含量。
HFD 可显著诱导肝脂肪变性，并促进肝三酰甘油（TG）和血清胆固醇（CHOL）含量升高，而 LGZG 治疗 4 周可显著降低肝 TG 和血清 CHOL。通过 RNA 测序，我们发现 NAFLD 大鼠 PPP1R3C 表达显著增加，而 LGZG 降低其表达。RT-qPCR 和 Western blot 均验证了 LGZG 干预后 PPP1R3C 的变化。LGZG 还可促进肝型糖原磷酸化酶（PYGL）活性，抑制糖原合酶（GS）活性，导致肝糖原分解增加，合成减少。通过检测糖原含量，我们还发现 LGZG 降低了 NAFLD 大鼠肝糖原。此外，我们分析了肝从头合成脂肪和胆固醇合成的关键分子，表明 LGZG 可显著抑制乙酰辅酶 A 羧化酶（ACC）、固醇受体元件结合蛋白-1c（SREBP-1c）和 3-羟-3-甲基戊二酰辅酶 A 还原酶（HMGCR）的活性，导致肝脂质合成减少。
我们的数据强调了靶向 PPP1R3C 通路的作用，并发现肝糖原代谢可能是 LGZG 预防 NAFLD 的潜在靶点。
