Piperine, major ingredient of Piper nigrum, attenuates fibrogenesis and inflammation in hepatic fibrosis by switching STAT3 phosphorylation at Ser727

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. 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- β (TGF- β ), 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- β 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. In vivo , PIP reduced serum transaminase levels, collagen deposition, and decreased the excessive accumulation of extracellular matrix (ECM), comprising α -smooth muscle actin ( α -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 α -SMA and p-JAK2, as well as its upregulation on SOCS3 and PIAS1/3 in activated LX-2 cells. 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.