Objective:

2

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:

2

A carbon tetrachloride (CCl

4

)-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

CCl

4

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 CCl

4

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

R

IPK1

and RIPK3 mRNA levels were measured by quantitative real-time polymerase chain reaction. PANoptosis-related proteins were detected by Western blot.

Results:

2

In CCl

4

-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:

2

TFLS might ameliorate HF by targeting ZBP1 to suppress PANoptosis

highlighting its multi-modal regulation of apoptosis

pyroptosis

and necroptosis.