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

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.

Chinese medicine; PANoptosis; Z-DNA binding protein 1; hepatic fibrosis; total lavonoids of Litchi chinensis Sonn. seed.