Piperine attenuates fibrogenesis and inflammation in hepatic fibrosis by switching STAT3 phosphorylation at Ser727
Article
Figures
Metrics
Preview PDF
Reference
Related
Cited by
Materials
摘要:
目的：胡椒（Piper nigrum）是一种兼具药食同源特性的香辛料，因其独特的辛辣风味而被广泛应用，并长期用于民间消化系统相关疾病的防治。胡椒碱（Piperine, PIP）是从胡椒中提取的一种生物碱类活性成分，具有多种药理活性，尤其在肝脏保护方面表现出良好的潜力。肝纤维化是由多种慢性肝病进展为肝硬化发展的重要病理阶段，但目前尚缺乏有效且理想的治疗药物。因此，本研究旨在探讨胡椒碱的抗肝纤维化作用及其潜在机制，为肝纤维化的治疗提供新的思路。 方法：本研究体内实验采用腹腔注射硫代乙酰胺（thioacetamide, TAA）（第1周100 mg/kg，每周3次；第2-5周200 mg/kg，每周2次）连续5周诱导C57BL/6J小鼠建立肝纤维化模型，并分别灌胃给予胡椒碱（PIP, 15, 30或60 mg/kg）或姜黄素（curcumin, Cur, 20 mg/kg）进行治疗。体外实验中，采用转化生长因子-β（Transforming growth factor-β, TGF-β, 10 ng/mL）刺激肝星状细胞（hepatic stellate cells, HSCs）2 h使其活化，并分别以胡椒碱（PIP, 3.125, 6.25 或12.5 μmol/L）或信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）抑制剂氯硝柳胺（niclosamide, 40 μmol/L）处理6 h。同时，利用TGF-β刺激激活人肝星状细胞系LX-2细胞，并通过特异性siRNA沉默STAT3基因后，再给予胡椒碱（PIP, 12.5 μmol/L）处理。 结果：在体内实验中，胡椒碱（PIP）可降低小鼠血清转氨酶水平，减少肝脏的炎性浸润、胶原沉积、肝细胞排列紊乱及肝小叶结构破坏等组织病理学变化，并减轻细胞外基质（extracellular matrix, ECM）的过度沉积，具体表现为α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）、I型胶原蛋白（collagen type I, collagen I）以及金属蛋白酶组织抑制因子1（tissue inhibitor of metalloproteinases 1, TIMP-1）与基质金属蛋白酶13（matrix metalloproteinase 13, MMP13）比值的下调。胡椒碱（PIP）还可抑制炎症细胞因子包括白细胞介素-1受体1型（interleukin-1 receptor type 1, IL-1R1）、半胱氨酸天冬氨酸蛋白酶1（cysteine-aspartic acid protease 1, Caspase-1）和白细胞介素-6（interleukin-6, IL-6）的释放以及减少髓过氧化物酶（myeloperoxidase, MPO）的表达。机制上，胡椒碱（PIP）特异性地抑制STAT3 Ser727位点的磷酸化，下调磷酸化Janus激酶2（phosphorylated janus kinase 2, p-JAK2）的表达，并上调细胞因子信号转导抑制因子3（suppressor of cytokine signaling 3, SOCS3）及活化STAT蛋白抑制剂1/3（protein inhibitor of activated STAT 1/3, PIAS1/3）的表达。体外实验中，胡椒碱（PIP）可减少活化的HSCs中ECM的沉积并抑制炎症因子释放，同时能够选择性地抑制STAT3 Ser727位点磷酸化而不是影响Tyr705位点的磷酸化，进而调节p-JAK2、SOCS3及PIAS1/3的表达，发挥类似STAT3抑制剂的作用。在活化LX-2细胞中，STAT3基因缺失放大了胡椒碱（PIP）对α-SMA、p-JAK2的抑制作用，并增强其对SOCS3和PIAS1/3的上调作用。 结论：胡椒碱（PIP）通过抑制ECM过度沉积和炎症因子的释放改善肝纤维化，其作用机制可能与靶向调控STAT3 Ser727位点磷酸化有关。本研究为开发抗肝纤维化的活性候选药物提供了实验依据，也为临床上拓展肝纤维化的治疗策略提供了新的思路。
Abstract:
Objective: Piper nigrum is a medicinal and edible spice that is popular worldwide due to its unique spicy flavor, and is considered as a folk medicine for treating digestive system diseases. Piperine (PIP) is a bioactive alkaloid derived from P. nigrum, with remarkable hepatoprotective efficacy. Hepatic fibrosis represents a critical phase in the advancement of distinct chronic liver conditions towards cirrhosis, and currently lacks effective therapeutic drugs. The study aims to investigate the hepatoprotective functions of PIP and the underlying mechanism of PIP in combating hepatic fibrosis. Methods: C57BL/6J mice with hepatic fibrosis were induced by thioacetamide (TAA) and subsequently subjected to treatment with PIP or curcumin. Immortalized rat hepatic stellate cells (HSCs) were stimulated with transforming growth factor-b (TGF-b), followed by culture with PIP or niclosamide [signal transducer and activator of transcription 3 (STAT3) inhibitor], respectively. Human hepatic stellate cell line LX-2 were activated by TGF-b and transfected with specific small interfering RNA (siRNA) to silence STAT3 gene, and afterwards cultured with PIP. The mouse AML-12 cells and macrophage-like murine cells Raw 264.7 were stimulated with lipopolysaccharide (LPS), followed by culture with PIP. Results: In vivo, PIP reduced serum transaminase levels, collagen deposition, and decreased the excessive accumulation of extracellular matrix (ECM), comprising a-smooth muscle actin (a-SMA), collagen type I (Collagen I), and tissue inhibitor of metalloproteinases-1 (TIMP-1)/matrix metalloproteinase 13 (MMP13) ratio expressions. PIP inhibited inflammatory cytokines release and myeloperoxidase (MPO) expression, including interleukin-1 receptor type 1 (IL-1R1), cysteine-aspartic acid protease-1 (Caspase-1), and IL-6. PIP specifically inhibited STAT3 phosphorylation at Ser727, downregulated phosphorylated janus kinase 2 (p-JAK2), and upregulated suppressor of cytokine signaling 3 (SOCS3) and protein inhibitor of activated STAT 1/3 (PIAS1/3) expressions. In vitro, PIP inhibited ECM deposition and inflammatory cytokines release in activated HSCs. PIP selectively suppressed p-STAT3 at Ser727 without affecting Tyr705, and modulated p-JAK2, SOCS3, and PIAS1/3 expressions in activated HSCs, function as a STAT3 inhibitor. PIP inhibited LPS induced M1 polarization of RAW 264.7 macrophages. PIP decreased Cleaved Caspase 3 expression and apoptotic cells in LPS induced AML 12 hepatocytes. STAT3 deficiency amplified regulation of PIP on a-SMA and p-JAK2, as well as its upregulation on SOCS3 and PIAS1/3 in activated LX-2 cells. Conclusion: PIP improved hepatic fibrosis via inhibiting ECM excessive deposition and inflammatory secretion. Switching STAT3 phosphorylation at Ser727 might be the underlying targets for PIP against hepatic fibrosis, which provide an effective candidate and therapeutical strategy for hepatic fibrosis.Objective: Piper nigrum is a medicinal and edible spice that is popular worldwide due to its unique spicy flavor, and is considered as a folk medicine for treating digestive system diseases. Piperine (PIP) is a bioactive alkaloid derived from P. nigrum, with remarkable hepatoprotective efficacy. Hepatic fibrosis represents a critical phase in the advancement of distinct chronic liver conditions towards cirrhosis, and currently lacks effective therapeutic drugs. The study aims to investigate the hepatoprotective functions of PIP and the underlying mechanism of PIP in combating hepatic fibrosis. Methods: C57BL/6J mice with hepatic fibrosis were induced by thioacetamide (TAA) and subsequently subjected to treatment with PIP or curcumin. Immortalized rat hepatic stellate cells (HSCs) were stimulated with transforming growth factor-b (TGF-b), followed by culture with PIP or niclosamide [signal transducer and activator of transcription 3 (STAT3) inhibitor], respectively. Human hepatic stellate cell line LX-2 were activated by TGF-b and transfected with specific small interfering RNA (siRNA) to silence STAT3 gene, and afterwards cultured with PIP. The mouse AML-12 cells and macrophage-like murine cells Raw 264.7 were stimulated with lipopolysaccharide (LPS), followed by culture with PIP. Results: In vivo, PIP reduced serum transaminase levels, collagen deposition, and decreased the excessive accumulation of extracellular matrix (ECM), comprising a-smooth muscle actin (a-SMA), collagen type I (Collagen I), and tissue inhibitor of metalloproteinases-1 (TIMP-1)/matrix metalloproteinase 13 (MMP13) ratio expressions. PIP inhibited inflammatory cytokines release and myeloperoxidase (MPO) expression, including interleukin-1 receptor type 1 (IL-1R1), cysteine-aspartic acid protease-1 (Caspase-1), and IL-6. PIP specifically inhibited STAT3 phosphorylation at Ser727, downregulated phosphorylated janus kinase 2 (p-JAK2), and upregulated suppressor of cytokine signaling 3 (SOCS3) and protein inhibitor of activated STAT 1/3 (PIAS1/3) expressions. In vitro, PIP inhibited ECM deposition and inflammatory cytokines release in activated HSCs. PIP selectively suppressed p-STAT3 at Ser727 without affecting Tyr705, and modulated p-JAK2, SOCS3, and PIAS1/3 expressions in activated HSCs, function as a STAT3 inhibitor. PIP inhibited LPS induced M1 polarization of RAW 264.7 macrophages. PIP decreased Cleaved Caspase 3 expression and apoptotic cells in LPS induced AML 12 hepatocytes. STAT3 deficiency amplified regulation of PIP on a-SMA and p-JAK2, as well as its upregulation on SOCS3 and PIAS1/3 in activated LX-2 cells. Conclusion: PIP improved hepatic fibrosis via inhibiting ECM excessive deposition and inflammatory secretion. Switching STAT3 phosphorylation at Ser727 might be the underlying targets for PIP against hepatic fibrosis, which provide an effective candidate and therapeutical strategy for hepatic fibrosis.
关键词:
细胞外基质;肝纤维化;胡椒碱;胡椒;信号转导与转录激活因子3
Keywords:
Project Supported:
The authors express their gratitude to Department of science and technology of Jilin Province (No. 20250205012GH), National Natural Science Foundation of China (Nos. 82560813, 81973555). Science Foundation of Jilin Province (No. YDZJ202501ZYTS270).
