Ecotoxicology and Environmental Safety 258 (2023) 114963

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Toxicological mechanism of triptolide-induced liver injury:

Caspase3-GSDME-mediated pyroptosis of Kupffer cell
Chenyang Han a, Hongyan Pei b, Yongjia Sheng a, Jin Wang a, Xiaohong Zhou a, Wenyan Li a,
Li Guo c, Yun Kong a, *, Yi Yang a, *
a
Department of pharmacy，The Second Affiliated Hospital of Jiaxing University, 314001, China
b
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China
c
Department of Center Laboratory, The Second Affiliated Hospital of Jiaxing University, China

A R T I C L E I N F O A B S T R A C T

Keywords: Aim: Triptolide (TRI) is an active diterpenoid lactone compound isolated from Tripterygium wilfordii,We focused
Triptolide on investigating the effect and mechanism of Triptolide (TRI) on liver injury.
Liver injury Methods: The toxic dose (LD50 = 100 μM) of TRI on liver Kupffer cells was explored, and network pharmaco­
Kupffer cell
logical analysis was performed to identify Caspase-3 as the target of TRI-induced liver injury. Regarding the
Pyroptosis
pyroptosis research, we examined the level of TRI-induced pyroptosis in Kupffer cells, including inflammatory
Caspase-3
cytokine detection, protein assay, microscopic cell observation and LDH toxicity test. The effect of TRI on
pyroptosis was assessed after knocking out GSDMD, GSDME and Caspase-3 in cells, respectively. We also
investigated the liver injury-inducing action of TRI at the animal level.
Results: Our experimental results were consistent with those predicted by network pharmacology, indicating that
TRI could bind to Caspase-3-VAL27 site to promote the cleavage of Caspase-3, and Cleaved-Caspase-3 induced
pyroptosis of Kupffer cells through GSDME cleavage. GSDMD was not involved in TRI’s action. TRI could pro­
mote Kupffer cell pyroptosis, elevate the inflammatory cytokine levels, and facilitate the expressions of N-GSDME
and Cleaved-Capase 3. After the mutation of VAL27, TRI could not bind to Caspase-3. Animal-level results
showed that TRI could induce liver injury in mice, while Caspase-3 knockout or Caspase-3 inhibitors could
antagonize the action of TRI.
Conclusion: We find that the TRI-induced liver injury occurs primarily through the Caspase-3-GSDME pyroptosis
signal. TRI can promote Caspase − 3 maturation and regulate kupffer cell pyroptosis. The present findings offer a
new idea for the safe use of TRI.

1. Introduction elicitation of immune injury, among which immunological liver injury

Triptolide (TRI) is an active diterpenoid lactone compound isolated
from Tripterygium wilfordii (Guan et al., 2021), which possesses signifi­
cant immunomodulatory and antiinflammatory activities and is widely
used in the clinical treatment of arthritis and immune diseases (Zhou
et al., 2021; Liu et al., 2021),TRI can also cause tissue damage in high
doses and long-term use, such as liver damage, which is common.
However, its toxicity cannot be underestimated, especially hepatotox­
icity, which is the main cause of death from it (Yuan et al., 2021).
Studies have shown that TRI can cause liver injury through a variety of
mechanisms, such as apoptosis induction, oxidative stress induction,
induction of inducible nitric oxide synthase (iNOS) expression and
has been a research focus in recent years (Mei et al., 2005; Huo et al.,
2019). A single intake of 300–1000 μG/kg TRI can cause evident liver
injury (Huang et al., 2019). In animal experiments, serum alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) were
significantly elevated in the TRI model group, and the intrahepatic
release of TNF-α and NO was increased, suggesting the occurrence of
immune injury (Huang et al., 2020a). Additionally, TRI can promote the
occurrence of inflammation by regulating interleukin (IL)− 17, IL-6 and
nuclear factor-kappa B (NF-κB) (Nan et al., 2021).
Although TRI has previously been found to promote inflammation to
cause liver injury, its exact target and mechanism of action have yet to
be further clarified. Pyroptosis is a way of inflammatory cell death,

* Corresponding authors.
E-mail addresses: kongyun@jxey.com (Y. Kong), wasd911@126.com (Y. Yang).

https://doi.org/10.1016/j.ecoenv.2023.114963
Received 26 December 2022; Received in revised form 22 April 2023; Accepted 25 April 2023
Available online 30 April 2023
0147-6513/© 2023 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. Han et al.
which leads to inflammatory response through the GSDM protein-
mediated maturation of inflammatory cytokines and the development
of cell membrane pores (Bergsbaken et al., 2009). Classical pyroptosis is
mediated by Caspase 1, which cleaves GSDMD and cytokines and is
activated by proteolysis prior to pro-IL-1β and pro-IL-18 to produce their
proinflammatory forms, IL-1β and IL-18. Nonclassical signals can be
mediated by Caspase-3/4/5/11, GSDMA, GSDMB, GSDE, etc (Bergs­
baken and Cookson, 2007). Our preliminary study found that TRI could
promote pyroptosis of Kupffer cells. To further explore the liver
injury-causing mechanism and target of TRI, we performed network
pharmacology and basic experiments to further reveal its target, with a
view to providing support for its safe use.
2. Materials and methods
2.1. Cell culture
Mouse liver Kupffer cells (Procell Life Science & Technology, Wuhan,
China) were cultured using a special medium in a three gas incubator set
at 37 ◦ C with 5% CO2. Cell viability was assayed using Trypan blue.
Kupffer cells were subcultured and used for experiments after the
viability reached above 90%. We separately constructed GSDMD-,
GSDME- and Caspase-3- knockout Kupffer cells(JIMA Gene,Suzhou,
China) by CRISPR-Cas9 technology and verified them by Western-Blot,
where sgRNA-GSDMD-CAGGACCAGCCCAGCATTGGAA;sgRNA-GSDME
-GCAAGAGAGAGAACCACAATT; sgRNA-Caspase3-GAGTCTGACTGGA
AAGCCGAA.
In the TRI intervention experiment, we treated cells with gradient
concentrations of TRI (0–20–40–60–80–100–120–140–160–180–200
μM) for 24 h, and detected their viability by CCK-8 assay. The results
showed that LD50 = 100 μM at 24 h. During the research, the TRI
concentration of Kupffer cells was set to 100 μM and the time was set to
24 h.
2.2. Flow cytometry (FCM)
After 24 h of TRI treatment, the kuppffer cells were trypsinized and
adherent/suspended cells were collected. The collected cells were then
merged, washed with precooled PBS for several times and subjected to
Annexin V-FITC/PI staining as per the kit (BD Biosciences, New Jersey,
USA) instructions. FCM was used to detect the level of apoptosis, and the
results were expressed as %.
2.3. LDH assay
The cytotoxicity of TRI was detected via the LDH assay kit (Solarbio,
Beijing, China). After 24 h of Kupffer cell treatment with TRI, the LDH
release rate was determined as per the kit instructions, and the results
were expressed as %.
2.4. PI staining
The relative PI uptake rate of Kupffer cells was detected at
0–1–4–6–12–24 h. The cells were treated with 1 μg/ml PI, 120 nm NaCl,
5 mM Glucose, 1.5 mM CaCl2, 1 mm magnesium chloride and 0.1%
bovine serum albumin (BSA). The cellular absorbance was measured at
533/617 nm. PI absorption rate (%) = (ODSample-ODbackground)/
(ODmaximum-ODbackground).
PI staining of cells was performed following 24 h of TRI treatment.
After discarding the medium, the cells were washed twice with PBS,
stained using 1 μg/ml PI reagent (Beyotime Biotechnology, Shanghai,
China) for 30 min and washed twice with PBS. Positive cells, which
showed red fluorescence, were subjected to DAPI restaining and then
observed.
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Ecotoxicology and Environmental Safety 258 (2023) 114963
2.5. ELISA
The Kupffer cells were dynamically detected for inflammatory cy­
tokines. After TRI treatment, the culture medium was sampled at time
points of 1–4–6–12–24 h, centrifuged at 3000 g for 30 min, and then the
supernatant was taken for detection of IL-1β, IL-6 and TNF-α using ELISA
kit (Jiangcheng Bioengineering Institute, Nanjing, China). After incu­
bation by adding reagent as per the kit instructions, the absorbance was
measured at 450 nm with an ELISA reader (BioTek, USA), and the results
were expressed as pg/ml.
The IL-1β, IL-6 and TNF-α levels in mouse liver tissues were assayed
by ELISA kit (Jiangcheng Bioengineering Institute, Nanjing, China). The
liver tissues were cut into pieces with sterile surgical scissors, ground
with liquid nitrogen, and lysed on ice with 1.0 ml of RIPA lysate for 30
min. Then, the supernatant was collected for protein quantification as
per the kit instructions, and the results were expressed as pg/ml.
2.6. CCK-8
The Kupffer cells were seeded into 96-well plates and, 12 h later,
treated with TRI. The cellular viability was assayed at 0–1–4–6–12–24 h.
Next, 10 μl of CCK-8 reagent (Solarbio, Beijing, China) was added per
well, and incubation continued for an additional 4 h, followed by
measurement of OD value at 450 nm.
2.7. Mouse experiment
SPF C57BL/6 mice (WT) and Caspase-3-knockout mice (KO, C57BL/
6 J-Casp3em1Cya) were purchased from Cyagen Biotechnology, which
were divided into 10 mice per group. To establish a mouse model of liver
injury, 1000 μg/kg TRI was intragastrically administered once daily for
7 d. During the intervention experiment of Caspase-3 inhibitor, 160 ng
of Z-DEVD-FMK was intragastrically administered once daily in combi­
nation with TRI. The mice were killed 7 d later for detection.
2.8. ALT/AST
The mouse peripheral bloods were extracted after 7 d of consecutive
TRI administration. Tail venous bloods were collected, centrifuged, and
then the supernatants were taken for detection of ALT and AST by ul­
traviolet colorimetry (Jiangcheng Bioengineering Institute, Nanjing,
China) as per the kit instructions. The AST and ALT results were
expressed as U/L.
2.9. H&E
The mice were killed after 7 d of consecutive TRI administration.
Their liver tissues were embedded in paraffin, and prepared into 4-μm-
thick serial sections for staining as per following procedure: Xylene
deparaffinization, dehydration with gradient concentrations (100%,
95%, 80%) of alcohol, tap water rinsing for 2 min, hematoxylin staining
for 3 min, tap water rinsing for 2 min, treatment with 1% hydrochloric
acid-alcohol for 2 s, tap water rinsing for 2 min, treatment with 1%
ammonia for 20 s, staining with 0.5% eosin alcohol for 10 s, gradient
alcohol dehydration, xylene permeabilization, neutral gum sealing.
Finally, the histopathological changes of liver tissues were observed
under light microscope.
2.10. IHC and TUNEL
The paraffin-embedded liver tissue sections were deparaffinized in
xylene thrice, and soaked in anhydrous alcohol for 5 min and subse­
quently in 95–85% ethanol twice. Then, the sections were placed in
0.01 mol/L citrate buffer (PH= 6.0) and retrieved by microwave at 98 ◦ C
for 20 min, followed by cooling at room temperature for 30 min and
rinsing with distilled water. A 10-min incubation with 3% hydrogen
C. Han et al.
peroxide proceeded at room temperature to eliminate endogenous
peroxidase. After blocking with 2% BSA, the sections were incubated at
4 ◦ C with GSDMD and GSDME monoclonal antibodies (1:300 dilutions;
Abcam, USA), and further with peroxisase-labeled streptomycin
(Abcam, USA) for 15 min. After rinsing with phosphate buffer for 5 min
thrice, each section was added with freshly prepared DAB solution
(DAKO, Denmark) for visualization. The chromogenic reaction was
observed under a microscope, and then the sections were restained with
hematoxylin, sealed and observed under an Olympus-BX51 microscope.
During TUNEL staining, the tissue sections were added dropwise with
TUNEL reagent, incubated for 30 min, washed with TBST, and then
observed microscopically.
2.11. Western-blot and pull-down
Following 24 h of treatment with TRI, the suspended and adherent
Kupffer cells were collected, lysed on ice with 1.0 ml of RIPA buffer
(Beyotime Biotechnology, Shanghai, China) for 30 min, and then
centrifuged at 10,000 g for 15 min. The supernatant was taken for
protein quantification using BCA kit (Beyotime Biotechnology,
Shanghai, China). The volume of protein solution was replenished with
5x loading buffer to 20 μl. After boiling for 8 min, electrophoresis was
performed at 80 V and subsequently at 120 V. Next, gel was removed,
and the proteins were electrotransferred onto PVDF membranes at a 300
mA constant current for 0.5–2 h. Thereafter, the PVDF membranes were
blocked with 5% skimmed milk powder for 2 h, incubated with TBST-
diluted monoclonal primary antibody, washed twice in TBST, and
further incubated with horseradish peroxidase-labeled goat anti-rabbit
secondary antibody (Abcam, USA), followed by chemiluminescence
detection. OD was analyzed via Image Pro-Plus 6.0, and the results were
expressed as OD comparisons between the target protein and the in­
ternal reference GAPDH. The proteins in tissues were detected by
consulting the aforementioned method.
Pull-down assay for TRI-Biotin binding to Caspase-3. The SUMO-
labeled Caspase-3 protein was expressed in E. coli overnight. After in­
duction with β-D-thiogalactopyranoside, the OD600 was 0.7–0.8. Cells
were suspended in Tris-HCl and NaCl, and then collected, ultra­
sonicated, centrifuged and purified on Ni column. On the column, His-
SUMO-Caspase-3 protein was washed twice with 500 mM of imid­
azole, while His-SUMO tag was cleaved with SUMO protease (ULP-1)
and subjected to Ni column purification. The purified protein was
separated by SDS-Page and verified by Coomassie blue staining. Re­
combinant protein (15 μg) was bound to Biotin-labeled TRI for pull-
down with streptavidin beads (Sigma, Massachusetts, USA). The beads
were blocked in 5% BSA in Tris buffer, while protein was precleared
with beads that were subsequently discarded. Following incubation with
50 μM of Biotin-MAF, the magnetic beads were washed thrice in Tris
buffer, and then boiled using the buffer and β-mercaptoethanol. The
recombinant G protein magnetic beads were incubated with Caspase-3
antibody (Abcam, Massachusetts, USA). After washing with Tris
buffer, Caspase-3 was detected according to the aforementioned
Western-Blot procedure, while biotin was detected using the horseradish
peroxidase-conjugated antibiotin antibody (CST, Boston, USA).
2.12. Network pharmacology
During the analysis of TRI and liver injury targets, a combination of
PubChem database (https://pubchem.ncbi.nlm.nih.gov/), HERB data­
base (http://herb.ac.cn/), Gene Card database (www.genecards.org/),
STRING (https://string-db.org/), Cytoscape 3.7.1 software and Meta­
scape Database (https://metascape.org/) were used.
(1) Acquisition of active constituent and action targets: The CAS
number of active constituent was obtained based on PubChem
database, and the targets of its action were derived based on
HERB database.
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Ecotoxicology and Environmental Safety 258 (2023) 114963
(2) Construction of "drug-action target" network: Cytoscape 3.7.1
was utilized to visualize the "drug-action target" interaction
network diagram.
(3) Taking the intersection of drug and disease targets: Disease tar­
gets were collected via the Gene Card database. To eliminate the
interference of subsequent experiments by targets lowly associ­
ated with disease, those whose relevance scores ≥ median were
selected as the disease targets. The potential targets of the drug
for treating acute hepatic injury were acquired by taking the
intersection of drug action targets with screened disease targets.
(4) PPI network analysis: The action targets of drug for disease
treatment were input into the STRING database for PPI network
analysis, where the confidence level was set at 0.7. Target
screening was performed, and data were derived. The results
were imported into Cytoscape 3.7.1 to reflect the intensity of
target interaction according to the Degree value, and the corre­
sponding targets with Degree values of ≥ twice the median were
defined as the core targets. The PPI network diagram was created
by deciding the edge thickness according to the magnitude of
combined score.
(5) Construction of "drug-disease-target" network: The "drug-disease-
target" interaction network diagram was created via Cytoscape
3.7.1, and the degree of "drug-disease-target" interaction was
obtained via the network analyzer function, thereby clarifying
potential targets through which can the drug treat acute hepatic
injury, as well as the intensity of their interaction.
(6) GO enrichment analysis: Utilizing the Metascape database, a
comprehensive enrichment analysis was conducted on gene
ontology (GO) terms, including GO Biological Process, GO Mo­
lecular Function and GO Cellular Component.
2.12.1. Statistical methods
All measurement data were expressed as x ±SD, and analyzed and
processed via SPSS 17.0. After homogeneity test for variance, compar­
ison between two sets of data was made by two independent samples t-
test. Three or more sets of data were analyzed by one-way ANOVA, and
the subsequent pairwise comparison between groups was made by LSD
method. All of the above tests were two-sided, and the differences were
considered statistically significant when P < 0.05.
3. Results
3.1. TRI induced pyroptosis of Kupffer cells
We treated Kupffer cells with gradient concentrations of TRI, and
found that cell viability was weakened significantly as the concentration
rose, with LD50 = 100 μM during a 24-h treatment duration [Fig. 1A].
Accordingly, we adopted a TRI amount of 100 μM and a processing time
of 24 h during the experiment. TRI could induce Kupffer cell damage, as
manifested by significantly higher cell mortality than that in the DMSO
group [Fig. 1B]. TRI induced pyroptosis of Kupffer cells, and the LDH
release rate was significantly higher than that in the DMSO group
[Fig. 1C]. Meanwhile, the rate of PI uptake rose significantly over time
[Fig. 1D]. Detection of inflammatory cytokines also revealed that TRI
could induce the release of Kupffer inflammatory cytokines. In DMSO
group, the cytokine levels did not change significantly over time, while
in TRI group, the IL-6, IL-1β and TNF-α levels were significantly upre­
gulated, showing higher values than those in the DMSO group [Fig. 1E-
G]. TRI could also promote the opening of Kupffer cell membrane pores,
as manifested by the significantly stronger fluorescence intensity in PI
staining compared to the DMSO group [Fig. 1H]. I detected the
pyroptosis-related proteins, finding that N-GSDMD and Cleaved-Caspase
1 were almost unexpressed, while N-GSDME and Cleaved-Caspase-3
were significantly upregulated, TRI group, in particular, exhibited
significantly higher levels of expression compared to the DMSO group
C. Han et al. Ecotoxicology and Environmental Safety 258 (2023) 114963

Fig. 1. TRI induced pyroptosis of Kupffer cells A: TRILD50 experimental results. LC50 = 100 μM B: FCM results (n = 3). TRI induced apoptosis, as manifested by
higher cell mortality than that in the DMSO group. Inter-group comparison, **P < 0.05. C: LDH release rate (n = 3). TRI group exhibited significantly higher rate of
LDH release compared to the DMSO group. Inter-group comparison, **P < 0.05. D: Relative uptake rate of PI (n = 3). With the prolongation of time, the PI uptake rate
was significantly elevated, which was higher than that in the DMSO group. Inter-group comparison, **P < 0.05. E-G: ELISA (n = 3). The inflammatory cytokine levels
in TRI medium increased significantly in a time-dependent manner. Inter-group comparison, **P < 0.05. H: PI staining (n = 3). The PI fluorescence intensity in the
TRI group was significantly stronger than that in the DMSO group. I-J: Relative protein expression (n = 3). N-GSDMD and Cleaved-Caspase 1 were almost unex­
pressed, while N-GSDME and Cleaved-Caspase-3 were significantly upregulated. TRI group, in particular, exhibited significantly higher expression levels than the
DMSO group. Inter-group comparison, **P < 0.05.

Fig. 2. Network pharmacological analysis of TRI and liver injury A: Interaction analysis of TRI and liver injury targets. B, E, F: Analysis and scoring of targets, among
which Caspase-3 was highly likely to interact with TRI. C-D: Construction of PPI and drug-target networks.

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[Fig. 1I-J]. Thus, I speculated that TRI mediated the Kupffer cell
pyroptosis through Caspase-3-GSDME.
3.2. Network pharmacological analysis of TRI and liver injury
The interaction between TRI and liver injury targets revealed the
presence of 55 coincidental protein targets between the two [Fig. 2A].
Further, targets were bound to TRI and scored. The results showed that
nine proteins, including AKT1, Caspase-3 and CTNNB1, had strong
binding to TRI, especially Caspase-3, which exhibited a high possibility
of binding to small molecules [Fig. 2B, G]. After constructing the PPI
network, I considered that Caspase-3 was highly likely to be the action
target of TRI [Fig. 2C-E]. Hence, through network pharmacological
analysis, I considered Caspase-3 as the action target of TRI.
3.3. GSDMD knockout did not affect the TRI-induced pyroptosis
I first knocked out GSDMD, a classical pyroptosis executive protein,
in Kupffer cells, and found that the cell viability was unaffected after
GSDMD knockout. Cell viability differed insignificantly between NC and
GSDMD-/-, although TRI could still induce cell damage. Meanwhile, the
cell viability in TRI+GSDMD-/- was significantly downregulated
[Fig. 3A]. The LDH release rate in TRI+GSDMD-/- was also higher than
that in NC and GSDMD-/- [Fig. 3B]. The relative uptake rate of PI
increased significantly over time in TRI+GSDMD-/-, while changed
insignificantly in NC and GSDMD-/-, showing low PI uptake levels
[Fig. 3C]. Detection of inflammatory cytokines also revealed that TRI
could promote the upregulation of IL-6, IL-1β and TNF-α in a time-
dependent manner and was unaffected by GSDMD. Meanwhile, insig­
nificant cytokine expressions were noted in NC and GSDMD-/- [Fig. 3D-
F]. IL-1 β Maturity may not depend on GSDME signals and may be
related to the activation of inflammasome. TRI could facilitate the
opening of cell membrane pores, as manifested by the significantly
stronger fluorescence intensity of PI than that in the NC and GSDMD-/-
[Fig. 3G]. Assay of pyroptosis-related proteins found knockout of
GSDMD, which affected neither the N-GSDME nor the Cleaved-Caspase-
3 expression [Fig. 3H-I].
3.4. Knockout of GSDME or Caspase-3 could inhibit the TRI-induced
pyroptosis
I performed GSDME knockout on Kupffer cells, and found that the
TRI-induced pyroptosis was inhibited. TRI could still induce the decline
of cell viability, while insignificant viability downregulation was noted
in GSDME-/- and NC cells [Fig. S1A]. However, the LDH release rate in
TRI-GSDME-/- differed insignificantly from that in NC and GSDME-/-
[Fig. S1B]. The relative uptake rate of PI in TRI-GSDME-/- and NC
differed insignificantly from that in GSDME-/- [Fig. S1C]. No significant
changes were observed in the levels of inflammatory cytokines in TRI-
GSDME-/-, which differed insignificantly from those in NC and GSDME-/-
[Fig. S1D-F]. After PI staining, insignificant alterations were observed in
the PI fluorescence intensity between the three groups of cells, and TRI
could induce neither the cell membrane pore opening nor the PI uptake
[Fig. S1G]. Protein assay revealed that the GSDME knockout did not
influence the cleavage or expression of Cleaved-Caspase-3 [Fig. S1H-I].
TRI acted on Caspase-3 instead of on GSDME.
Caspase-3 knockout was performed on Kupffer cells, and the results
showed that the TRI-induced pyroptosis could also be inhibited after the
knockout of Caspase-3. TRI could still induce the decline of cell viability,
while insignificant viability downregulation was noted in GSDME-/- and

Fig. 3. Knockout of GSDMD did not affect the TRI-induced pyroptosis A: CCK-8 assay (n = 3). TRI could induce cell damage, so that the cell viability in
TRI+GSDMD-/- was significantly downregulated, while the GSDMD knockout did not affect the cell viability. *P < 0.05 vs. NC; #P < 0.05 vs. GSDMD-/-. B: LDH
release rate (n = 3). TRI induced pyroptosis of Kupffer cells, leading to significantly higher rate of LDH release than that in the DMSO group. *P < 0.05 vs. NC;
#
P < 0.05 vs. GSDMD-/-. C: Relative uptake rate of PI (n = 3). TRI could induce the opening of cell membrane pores and promote the uptake of PI, as manifested by
the significantly increased rate of PI uptake over time. *P < 0.05 vs. NC; #P < 0.05 vs. GSDMD-/-. F: ELISA assay (n = 3). TRI could induce the release of Kupffer
inflammatory cytokines, so that the IL-6, IL-1β and TNF-α levels were significantly upregulated over time. *P < 0.05 vs. NC; #P < 0.05 vs. GSDMD-/-. G: PI staining
(n = 3). The fluorescence intensity of PI in TRI was significantly stronger than that in NC and GSDMD-/-. H-I: Relative protein expression (n = 3). GSDMD knockout
affected the expression of neither N-GSDME nor Cleaved-Caspase-3, and TRI could still induce the expression of these two proteins. *P < 0.05 vs. NC; #P < 0.05
vs. GSDMD-/-.

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NC cells [Fig. S2A]. The LDH release rate in TRI-GSDME-/- differed
insignificantly from that in NC and GSDME-/- [Fig. S2B]. The relative
uptake rate of PI in TRI-GSDME-/- and NC differed insignificantly from
that in GSDME-/- [Fig. S2C]. No significant changes were observed in the
levels of inflammatory cytokines in TRI-GSDME-/-, which differed
insignificantly from those in NC and GSDME-/- [Fig. S2D-F]. After PI
staining, insignificant alterations were observed in the fluorescence in­
tensity of PI between the three groups of cells, and TRI could induce
neither the cell membrane pore opening nor the PI uptake [Fig. S2G].
Protein assay also revealed that the Caspase-3 knockout influenced the
cleavage and expression of GSDME [Fig. S2H-I]. the current result, I
could further prove that TRI acted on Caspase-3, rather than on GSDME.
Although our results demonstrate that TRI works through GSDME
pyroptosis, when GSDME or Caspase-3 is knocked out, TRI is likely to
inhibit cell viability through other signaling pathways. This requires
further exploration.
3.5. Caspase-3-KO could inhibit the TRI-induced liver injury in mice
Wild-type (WT) and Caspase-3-knockout (KO) mice were used for
research. After intragastric TRI administration for 7 d, I found that the
ALT and AST levels differed insignificantly between WT and KO mice.
TRI-WT mice exhibited significantly upregulated levels of ALT and AST,
which were higher than those in WT and KO mice. TRI-KO mice
exhibited lower levels compared to the TRI-WT mice [Fig. 4A-B]. Ac­
cording to the tissue damage observations, evident cell damage and le­
sions appeared in the TRI-WT mouse livers during H&E staining assay,
and the TUNEL-positive rate increased significantly. In KO and WT mice,
TUNEL was negatively expressed, while in TRI-KO mice, cell damage
was markedly ameliorated and the TUNEL-positive rate was reduced
[Fig. 4C].
Protein assay revealed that TRI promoted the expression of N-
GSDME, although producing insignificant effect on N-GSDMD and
Cleaved-Caspase 1. The Tri-KO mice exhibited lower N-GSDME level
compared to the Tri-WT mice [Fig. 4D-E]. IHC results showed that TIR

Fig. 4. Caspase-3-KO could inhibit the TRI-induced liver injury A-B: ALT and AST (n = 10). Insignificant differences in ALT and AST levels were noted between WT
and KO mice. TRI-WT mice exhibited significantly upregulated levels of AST and AST, which were higher than those in WT and KO mice. Compared to the TRI-WT
mice, these levels were lower in the TRI-KO mice. *P < 0.05 vs. WT; #P < 0.05 vs. KO. C: H&E and TUNEL (n = 5). H&E staining revealed evident cell damage and
lesions in the livers of TRI-WT mice, as well as significantly elevated TUNEL-positive rate. In KO and WT mice, TUNEL was negatively expressed while in TRI-KO
mice, cell damage was markedly ameliorated and the TUNEL-positive rate decreased. D-E: Relative protein expression (n = 5). TRI promoted the expression of N-
GSDME, although producing insignificant effect on N-GSDMD and Cleaved-Caspase 1. The N-GSDME level in TRI-KO was lower than that in TRI-WT. *P < 0.05 vs.
WT; #P < 0.05 vs. KO. F: IHC (n = 5). TIR could promote the expression of GSDME, although producing insignificant effect on GSDMD. Caspase-3-KO could lower the
GSDME expression. G-H: ELISA (n = 5). TRI could promote the IL-6, IL-1β and TNF-α expression in tissues and peripheral bloods. These cytokine levels in TRI-KO
were lower than those in TRI-WT. *P < 0.05 vs. WT; #P < 0.05 vs. KO.

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could promote the expression of GSDME, although producing insignifi­
cant effect on GSDMD. Caspase-3-KO could lower the GSDME expression
[Fig. 4F]. Assay of inflammatory cytokines in liver tissues and peripheral
bloods found that TRI could promote the IL-6, IL-1β and TNF-α expres­
sion in these sites. The cytokine levels in TRI-KO were lower than those
in TRI-WT [Fig. 4G-H].
3.6. Caspase-3 inhibitor could inhibit the TRI-induced liver injury in mice
We applied FMK, a Caspase-3 inhibitor, to WT mice, in order to
clarify whether inhibiting Caspase-3 could antagonize the action of TRI.
The AST and ALT results showed that TRI could induce liver injury in
WT mice, leading to significantly upregulated ALT and AST levels
compared to those in WT mice. FMK could antagonize the action of TRI,
as manifested by significantly lower AST and ALT levels in TRI+FMK
mice than those in TRI mice [Fig. 5A-B]. H&E and TUNEL staining
revealed that FMK could relieve the severity of liver injury in mice, so
that the TUNEL-positive rate was downregulated, and the degree of cell
damage was reduced [Fig. 5C]. Protein assay revealed that FMK
inhibited the expression of N-GSDME and Cleaved-Caspase-3, although
producing insignificant effect on N-GSDMD or Cleaved-Caspase 1
[Fig. 5D-E]. However, the irreversible binding of FMK to Caspase-3

Fig. 5. Caspase-3 inhibitor could inhibit the TRI-induced liver injury in mice A-B: ALT and AST (n = 10). TRI could induce liver injury in WT mice, leading to
significantly upregulated ALT and AST levels compared to those in WT mice. FMK could antagonize the action of TRI, as manifested by significantly lower AST and
ALT levels in TRI+FMK mice than those in TRI mice. *P < 0.05 vs. WT; #P < 0.05 vs. TRI. C: H&E and TUNEL (n = 5). FMK could relieve the severity of liver injury
in mice, so that the TUNEL-positive rate was downregulated, and the degree of cell damage was reduced. D-E: FMK inhibited the expression of N-GSDME and
Cleaved-Caspase-3, although producing insignificant effect on N-GSDMD or Cleaved-Caspase 1. *P < 0.05 vs. WT; #P < 0.05 vs. TRI. F: IHC (n = 5). Although
having no effect on GSDMD, FMK could lower the GSDME expression, showing significant difference from the TRI group.G-H: ELISA (n = 5). FMK could lower the
inflammatory cytokine levels in liver tissues and peripheral bloods. *P < 0.05 vs. WT; #P < 0.05 vs. TRI.

7
C. Han et al.
inhibits the cutting of GSDME, as Caspase-3 is necessary for GSDME
cutting.According to IHC results, FMK could lower the expression of
GSDME, although having no effect on GSDMD, with significant differ­
ence from TRI group [Fig. 5F]. Assay of inflammatory cytokines found
that FMK could significantly lower the cytokine levels in liver tissues and
peripheral bloods compared to the TRI group [Fig. 5G-H].
3.7. Validation of targeting relationship between TRI and Caspase-3
The 3D structure of Caspase-3 protein used for docking was acquired
from UniProt database (UniProt ID: P70677), while the 3D structure of
small-molecule Triptolide used was acquired from PubChem database
(PubChem ID: 107985). Prior to the docking, AVOGADR 1.2.0 was used
to minimize the energy for the 3D structure of small-molecule Triptolide
under the MMFF94 field. Molecular docking herein was performed via
the AutoDock Vina 1.1.2. All receptor proteins were hydrogenated using
Open Source PyMol before the docking. Then, ADFRsuite 1.0 was
employed to convert all the processed small molecules and receptor
proteins into the PDBQT format that was necessary for docking via
AutoDock Vina 1.1.2. During the Vina-based docking, the grid box and
the PDBQT file of processed proteins and small molecules were used as
the input file. The level of detail of global search for the docking was set
to 32, while other parameter settings were kept default. Finally, the
docking conformation with highest output score was considered as the
bound conformation, which was subjected to molecular dynamics
simulation and analysis.
The docking between small-molecule Triptolide and Caspase-3 pro­
tein was explored with the aid of AutoDock Vina 1.1.2, and their binding
energy scores were derived. Negative binding energy value indicated the
possibility of binding. As is clear from the Table, the binding affinity
between Caspase-3 and Triptolide was − 4.4 kcal/mol, implying that
Fig. 6. Validation of the TRI binding to Caspase-3 A-B: Binding mode diagram of TRI and Caspase-3. The binding affinity between Caspase-3 and Triptolide was
− 4.4 kcal/mol, implying that both of them had certain binding potential. VAL27 was the site for Pro-Caspase-3 cleavage, and Cleaved-Caspase-3 was formed after
cleavaging Caspase-3. Meanwhile, the binding of TRI to VAL27 promoted the maturation and cleavage of Caspase-3. C-G: Pull-down assay. TRI-Biotin bound to
Caspase-3, rather than to GSDME. After the mutation of VAL27, TRI could no longer bind to Caspase-3, proving that Caspase-3-VAL27 was the binding site of TRI. H-I:
Model diagram of RMSD and RMSF fitting results.
8
Ecotoxicology and Environmental Safety 258 (2023) 114963
both Triptolide and Caspase-3 had certain binding potential. VAL27 was
the site for Pro-Caspase-3 cleavage, and Cleaved-Caspase-3 was formed
after cleavaging Caspase-3. The binding of TRI to VAL27 promoted the
maturation and cleavage of Caspase-3 [Fig. 6A-B]. Our Pull-down assay
results showed that TRI-Biotin bound to Caspase-3, rather than to
GSDME. After the mutation of VAL27, TRI could no longer bind to
Caspase-3, proving that Caspase-3-VAL27 was the binding site of TRI
[Fig. 6C-G].
RMSF (Root Mean Square Fluctuation) measures the average
movement of atom positions in a molecular structure within a period of
time, which is the root mean square of distance between a target
structure’s specific atom and the reference structure over the entire time
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑ ref 2
range. Its specific formula is: RMSFI = T1 N t=1 (ri (t) − ri ) , where ri
ref
stands for the position coordinates of ith atom in the reference structure,
ri (t) refers to the position coordinates of ith atom in the target structure
at time t, and T denotes the time range under computation. The units of
RMSF are length units, usually nm (10− 9 m) or Å (10− 10 m). The larger
the RMSF value, the greater the change of structural flexibility in the
corresponding area, and vice versa. As shown in the Figure, the amino
acid structural flexibility in regions 1–30 and 179–180 of Caspase-3
changed greatly, attributable perhaps to the presence of excessively
many loop zones therein [Fig. 6H].
RMSD (Root Mean Square Distance/Deviation) measures the differ­
ence between varying conformations of the same molecular structure,
which is the distance root mean square of all corresponding atoms
following position overlap of two conformations. Its specific formula is:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑N ref 2 ref
1
RMSD(t) = N i=1 (ri (t) − ri ) , where ri stands for the position
coordinates of ith atom in the reference structure, ri (t) refers to the
position coordinates of ith atom in the target structure at time t,
C. Han et al.
ref
ri (t) − ri is defined as the distance between two conformations of ith
atom, and N denotes the total atom number of molecule or the total atom
number of RMSD under computation. The distances for all correspond­
ing atoms were defined identically and then the root mean square was
calculated, thereby deriving the RMSD value, which measured the
structural difference of the same molecule in different conformations. Its
units are length units, usually nm (10− 9 m) or Å (10− 10 m). RMSD is
commonly used for analyzing the stability of overall molecular structure
of the system. The decrease of RMSD indicated that the structure tended
to be stable, and vice versa. It is clear from the results that after 4 ns, the
RMSD curve of Caspase-3-Triptolide was regionally stabilized at 0.7 nm,
the structure of complex changed little, and the simulation process
already reached equilibrium [Fig. 6I].
4. Discussion
As a novel mechanism of programmed cell death, pyroptosis is based
structurally on the formation of cell membrane pores, swelling of cells
and rupture of cell membranes, which is characterized mainly by ulti­
mate expression and release of inflammatory cytokines (Kepp et al.,
2010). Excessive inflammatory response can activate pyroptosis.
Numerous immune cells in the liver tissues are involved in pyroptosis,
which initiates an immunological defense response, and the liver plays a
significant role in preserving body metabolism and immune homeostasis
(Aglietti et al., 2016; Rayamajhi et al., 2013). Pyroptosis exerts its role
primarily by relying on caspase protease, and activation of Caspases
1/4/5 can promote the interleukin maturation and activation (Yang
et al., 2015). Casapase proteins can also promote the cleavage of GSDM
proteins (Bibo-Verdugo et al., 2020; Naji et al., 2016), which form
N-GSDMs to play a role in opening the cell membrane pores (Teng et al.,
2020). Kupffer cells, as a kind of liver-specific macrophages, function
crucially in liver injury. A study by Pelegrin found that LPS and ATP can
induce pyroptosis of J774a.1 macrophages, activate P2×7 receptor, and
exert their effects after substantial IL-1β expression (Pelegrin and Sur­
prenant, 2007). Rare earth oxides like Mirshafiee can promote the
activation of NLRP3 bodies and Caspase 1 in Kupffer cells, thereby
facilitating the occurrence of pyroptosis (Mirshafiee et al., 2018). Recent
research has shown that the Caspase-3 activation can induce
GSDME-dependent pyroptosis in addition to apoptosis. When cells
highly express GSDME, activation of Caspase-3 can induce
GSDME-dependent pyroptosis, but when cells lowly express GSDME,
apoptosis is induced instead (Zheng et al., 2019). As a member of the
Gasdermin protein family, GSDME is composed of cytotoxic GSDME-N
and GSDME-C (Xixi, 2018). Upon stimulation by external pathogenic
factors or death-inducing signals, cells promote cleavage of GSDME into
GSDME-N fragments by activating Caspase-3, which then bind to
phospholipid molecules on the cell membranes to form GSDME pores,
thereby leading to the release of inflammatory IL-1β and IL-18 cytokines
until swelling and rupture of cells (Huang et al., 2020b).
For many years, it has been a clinical consensus that long-term or
large-dose application of TRI, a major active constituent of Tripterygium
wilfordii, can induce liver injury. I further explored the role and mech­
anism of TRI in liver injury. Our results demonstrated that TRI could
induce pyroptosis of Kupffer cells and promote the maturation and
release of inflammatory cytokines. According to TEM observations, TRI
did cause the opening of Kupffer cell membrane pores. However, note­
worthy was that TRI primarily promoted the expression of N-GSDME
and Cleaved-Caspase-3, while producing no effect on N-GSDMD or
Cleaved-Caspase 1. Suggestively, TRI was associated chiefly with the
activation of nonclassical Caspase-3-GSDME signal. Network pharma­
cological analysis revealed that Caspase-3 had a strong targeting rela­
tionship with TRI. To further clarify the involvement of GSDMD and
GSDME in TRI-induced liver injury, I separately knocked out GSDMD
and GSDME. The results showed that the knockout of GSDMD did not
affect the action of TRI, with Kupffer cells still being pyroptotic.
9
Ecotoxicology and Environmental Safety 258 (2023) 114963
Contrastively, after GSDME knockout, TRI could no longer induce the
Kupffer cell pyroptosis, which lowered the levels of inflammatory cy­
tokines and inhibited the opening of cell pores. However, noteworthy
was that TRI still promoted the expression of Cleaved-Caspase-3. This
indicated that GSDME knockout failed to affect the Caspase-3-
cleavaging effect of TRI, which were independent of each other; be­
sides, GSDME was also not the action target of TRI. Combined with the
network pharmacological results, I identified Caspase-3 as the major
action target of TRI. VAL27 was the cleavaging site of Caspase-3, and
after cleavage of Caspase-3, Cleaved-Caspase-3 was formed, which
further interacted with GSDME to form N-GSDME (Huang et al., 2020b).
In our study, N-GSDME was also insignificantly expressed after knocking
out Caspase-3, and TRI was unable to enhance the cleavage of GSDME.
Through Pull-down assay, I verified that Caspase-3 was the major action
target of TRI, and that VAL27 was the binding site. After the mutation of
VAL27, TRI could no longer bind to Caspase-3. The fitting results of
RMSD and RMSF also indicated that the TRI-Caspase-3 binding model
had low energy and high stability.
During animal experiments, we used WT and Caspase-3-KO mice.
The results showed that TRI could induce liver injury in mice and elevate
ALT and AST levels. However, in Caspase-3-KO mice, the action of TRI
was antagonized to a certain extent and liver damage was ameliorated.
Notably, TRI activated Caspase-3-GSDME in tissues and elevated the
levels of N-GSDME and Cleaved-Caspase-3, although producing insig­
nificant effect on GSDMD-Casapase 1. These findings were consistent
with the results of Kupffer cell experiments. When Caspase-3 inhibitor
was used, the action of TRI was also inhibited and the severity of liver
injury in mice was relieved.
5. Conclusion
Through network pharmacology and other means, we revealed the
mechanism and targets whereby TRI induced liver injury. The binding of
TRI, a Caspase-3 activator, to VAL27 site promoted the maturation and
cleavage of Cleaved-Caspase-3, which further promoted the cleavage of
GSDME to form N-GSDME, ultimately resulting in Kupffer cell pyrop­
tosis. Caspase-3-GSDME is the major signal of TRI-induced liver injury.
The findings of this study provide a new reference for the prevention and
treatment of TRI-induced liver injury, which also offer a new idea for the
rational use of TRI in clinical settings.
CRediT authorship contribution statement
Chenyang Han, Hongyan Pei: Experimental operation and data
acquisition. Yongjia Sheng, Jin Wang, Xiaohong Zhou: Experiment
design and data statistical analysis. Wenyan Li, Li Guo: Project fund
support. Yun Kong, Yi Yang: Article writing and revision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgement
Thank you for the support of ZheJiang Provincial Natural Science
Foundation.
Data availability statement
The data that support the findings of this study are available from the
C. Han et al.
corresponding author upon reasonable request.
Fund
ZheJiang provincial Natural Science Foundation [LYY20H280005].
Ethical approval and consent to participate
The study approvaled with Ethics Committee.
Consent for publication
All authors approval published the article.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2023.114963.
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