Objective To investigate the therapeutic benefits and possible mechanism of total flavonoids of Litchi chinensis Sonn. seed (TFLS) on hepatic fibrosis (HF) in vivo and in vitro. Methods A carbon tetrachloride (CCl4)-induced rat HF model and transforming growth factor β1 (TGF-β1)-stimulated hepatic stellate cell (HSC)-T6 cells were employed. For in vivo study, rats were randomly divided into 6 groups using a random number table method, including vehicle, CCl4, silybin (50 mg/kg), and low-, medium-, high-dose (25, 50, and 100 mg/kg) TFLS treatment groups. TFLS or silybin was administered daily by gavage for 8 weeks during CCl4 induction period. For in vitro study, HSC-T6 cells were divided into 4 groups, including control, TGF-β1 (20 ng/mL), TGF-β1 + Z-DNA binding protein 1 (ZBP1) siRNA (si-ZBP1), TGF-β1 + low-dose (80 µg/mL) TFLS, and TGF-β1 + high-dose (160 µg/mL) TFLS groups. Serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), hydroxyproline (HYP), total bile acid (TBA), interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. Hematoxylin-eosin and Masson’s trichrome staining were employed to conduct a histological examination of the liver tissue. Apoptosis was assessed by TUNEL. Cysteine-aspartic acid protease (CASP)-8, NLR family pyrin domain containing 3 (NLRP3), gasdermin D (GSDMD), and receptor-interacting protein kinase (RIPK) 3 were immunohistochemically detected. CASP-8, NLRP3, GSDMD, CASP-3, BCL-2, BCL-2-associated X (BAX), ZBP1, RIPK1, and RIPK3 mRNA levels were measured by quantitative real-time polymerase chain reaction. PANoptosis-related proteins were detected by Western blot. Results In CCl4-induced HF rats, TFLS improved liver function, inflammatory response, and liver tissue fibrosis (P<0.05 or P<0.01). TFLs inhibited the activation of PANoptosis initiator ZBP1, which subsequently regulated PANoptosis by increasing BAX, CASP-3, CASP-8 and decreasing BCL-2, NLRP3, GSDMD, CASP-1, RIPK1, RIPK3, and mixed lineage kinase domain-like expressions (P<0.05 or P<0.01). In vitro, TFLS inhibited TGF-β1-induced HSC-T6 cells activation and PANoptosis (P<0.05 or P<0.01). Conclusion TFLS might ameliorate HF by targeting ZBP1 to suppress PANoptosis, highlighting its multi-modal regulation of apoptosis, pyroptosis, and necroptosis.

Total Flavonoids of Litchi chinensis Sonn. Seed Improves Hepatic Fibrosis by Inhibiting PANoptosis

期刊论文
发表日期：

2025年12月9日

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Yan Tang,
Ting Wang,
Min-chao Feng,
Jun-wen Gong,
Mei-wen Tang,
Guo-zhong Chen,
Ma-ling Si,
Jian-fang Feng,
Wei-sheng Luo

摘要

Objective

To investigate the therapeutic benefits and possible mechanism of total flavonoids of Litchi chinensis Sonn. seed (TFLS) on hepatic fibrosis (HF) in vivo and in vitro.

Methods

A carbon tetrachloride (CCl₄)-induced rat HF model and transforming growth factor β1 (TGF-β1)-stimulated hepatic stellate cell (HSC)-T6 cells were employed. For in vivo study, rats were randomly divided into 6 groups using a random number table method, including vehicle, CCl₄, silybin (50 mg/kg), and low-, medium-, high-dose (25, 50, and 100 mg/kg) TFLS treatment groups. TFLS or silybin was administered daily by gavage for 8 weeks during CCl₄ induction period. For in vitro study, HSC-T6 cells were divided into 4 groups, including control, TGF-β1 (20 ng/mL), TGF-β1 + Z-DNA binding protein 1 (ZBP1) siRNA (si-ZBP1), TGF-β1 + low-dose (80 µg/mL) TFLS, and TGF-β1 + high-dose (160 µg/mL) TFLS groups. Serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), hydroxyproline (HYP), total bile acid (TBA), interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. Hematoxylin-eosin and Masson’s trichrome staining were employed to conduct a histological examination of the liver tissue. Apoptosis was assessed by TUNEL. Cysteine-aspartic acid protease (CASP)-8, NLR family pyrin domain containing 3 (NLRP3), gasdermin D (GSDMD), and receptor-interacting protein kinase (RIPK) 3 were immunohistochemically detected. CASP-8, NLRP3, GSDMD, CASP-3, BCL-2, BCL-2-associated X (BAX), ZBP1, RIPK1, and RIPK3 mRNA levels were measured by quantitative real-time polymerase chain reaction. PANoptosis-related proteins were detected by Western blot.

Results

In CCl₄-induced HF rats, TFLS improved liver function, inflammatory response, and liver tissue fibrosis (P<0.05 or P<0.01). TFLs inhibited the activation of PANoptosis initiator ZBP1, which subsequently regulated PANoptosis by increasing BAX, CASP-3, CASP-8 and decreasing BCL-2, NLRP3, GSDMD, CASP-1, RIPK1, RIPK3, and mixed lineage kinase domain-like expressions (P<0.05 or P<0.01). In vitro, TFLS inhibited TGF-β1-induced HSC-T6 cells activation and PANoptosis (P<0.05 or P<0.01).

Conclusion

TFLS might ameliorate HF by targeting ZBP1 to suppress PANoptosis, highlighting its multi-modal regulation of apoptosis, pyroptosis, and necroptosis.