Electronic Supplementary Material
Honokiol rescues sepsis-associated acute lung injury and lethality via the
inhibition of oxidative stress and inflammation
Te I Weng, Hsiao Yi Wu, Chia Wei Kuo, Shing Hwa Liu
Figure 1S. Effect of honokiol on nitrotyrosine and lipid peroxidation productions in
mice treated with LPS. The levels of serum nitrotyrosine and plasma malondialdehyde
(MDA) were detected in mice treated with LPS (10 mg/kg) for 6 and 24 hours,
respectively. Honokiol (HK, 2.5 and 5 mg/kg) was given 30 minutes after LPS
administration. Data are presented as means+SEM. *P<0.05 as compared with control
(vehicle). **P<0.05 as compared with LPS alone.
1
Figure 2S. Effect of honokiol on iNOS protein expression in the lungs of mice treated
with LPS. The lung iNOS protein expression was detected in mice treated with LPS
for 6 hours. Honokiol (HK, 2.5 and 5 mg/kg) was given 30 minutes after LPS
application. Lung samples were prepared and subjected to Western blot analysis for
iNOS protein expression. Quantification of protein expression was performed by
densitometric analysis. Data are presented as means+SEM. *P<0.05 as compared with
control (vehicle). **P<0.05 as compared with LPS alone.
2
Figure 3S. Effects of honokiol on LPS-induced IB degradation and NF-B-p65
protein translocation in the lungs of mice. Mice were treated with LPS (10 mg/kg) for
1 hour. Honokiol (HK, 2.5 and 5 mg/kg) was given 30 minutes after the treatment of
LPS. Lung samples were prepared and subjected to Western blot analysis for IB
degradation and NF-B p65 protein translocation. A, Cytosolic fractions were
prepared and analyzed for the content of IBprotein by Western blot. Results shown
are representative of at least three experiments. B, The nuclear extracts were prepared
for analysis of NF-B p65 protein translocation by Western blot. Quantification of
protein expression was performed by densitometric analysis. Data are presented as
means+SEM. *P<0.05 as compared with control (vehicle). **P<0.05 as compared
with LPS alone.
3
Figure 4S. Honokiol treatment ameliorates LPS-induced lung edema and histological
changes. A, Lung edema was measured as wet/dry weight ratio in mice injected
intraperitoneally with LPS for 24 h. Honokiol (HK, 2.5 and 5 mg/kg) was
administered to mice 30 minutes after induction of sepsis by LPS. Data are presented
as means+SEM (5 mice per group). *P<0.05 as compared with control (vehicle).
**P<0.05 as compared with LPS alone. B, Representative histological sections of
lungs harvested 24 h after an intraperitoneal LPS challenge in mice without or with
the treatment of honokiol. a, lung from mice treated with vehicle; b, lung from mice
treated with LPS: photograph shows marked inflammatory cell infiltration, interstitial
edema, and vascular congestion; c, lung from mice treated with LPS in the presence
of honokiol (2.5 mg/kg); d, lung from mice treated with LPS in the presence of
honokiol (5 mg/kg). Lung speciments stained with hematoxylin and eosin. Scale bar =
100 m.
4
Figure 5S. Effect of honokiol on liver damage in mice subjected to CLP. Honokiol (5
mg/kg) was administered to mice 30 min after induction of sepsis by CLP. The levels
of serum AST and ALT were detected. Data are presented as means+SEM (n=6 mice
per group). *P<0.05 as compared with control (vehicle). **P<0.05 as compared with
CLP alone.
5
Figure 6S. Effect of honokiol on bacterial clearance. The effect of honokiol on
bacterial clearance was determined by counting CFUs. Twenty-four hours after CLP
operation with or without honokiol (5 mg/kg) treatment, the intraperitoneal bacterial
counts were calculated and analyzed. Augmentin (amoxicillin and clavulanic acid;
100 mg/kg) was used as a positive control. Data are presented as means+SEM (n=5
mice per group). *P<0.05 as compared with control (vehicle). **P<0.05 as compared
with CLP alone.
6
Table 1S. The references of toxicological aspect/profile for honokiol or magnolia
extracts
Toxicological aspect/profile
Acute toxicity study (LD50) for
honokiol:
98 mg/kg administered intraperitoneally
in mice.
Acute toxicity study (LD50) for
Magnolia dealbata extract：
770 mg/kg i.p. and 2150 mg/kg p.o. in
mice.
90-day subchronic toxicity study：Rats
were administered 0, 60, 120 or 240 mg
Magnolia bark extract/kg bw/day in
the diet. No mortality, ophthalmic
abnormalities
or
treatment-related
findings in clinical observations,
hematology, coagulation or organ weight
measurements were observed. There
were no treatment-related macroscopic
or microscopic findings.
It has been observed in vivo activity of
honokiol against angiosarcomas, colon
and breast cancers in mice following i.p.
administration of 100-120 mg/kg.
Honokiol administration was well
tolerated and no weight loss or other
major side effects were observed in these
animal studies.
In the report of Hu and colleagues, the
authors have described that the toxicity
of free honokiol towards mice is strong.
However, no toxicological data for free
honokiol were shown in this study or
their previous study (Liu et al., 2008).
References
Chen CC and Teng CM. (1992) Studies on
the contents of Magnolol and Honokiaol in
Chinese Herb Formulas. Yearbook of
Committee on Chinese Medicine and
Pharmacy, Department of Health, Executive
Yuan, Taiwan.
Martínez
AL.,
et
al.
(2006)
Neuropharmacological effects of an ethanol
extract of the Magnolia dealbata Zucc.
leaves in mice. J Ethnopharmacol
106:250-255.
Liu Z., et al. (2007) Evaluation of short-term
and subchronic toxicity of magnolia bark
extract in rats. Regulatory Toxicol
Pharmacol 49:160-171.
Wolf I., et al. (2007) Honokiol, a natural
biphenyl, inhibits in vitro and in vivo growth
of breast cancer through induction of
apoptosis and cell cycle arrest.Int J Oncol.
30:1529-1537.
Chen F., et al. (2004) Honokiol: a potent
chemotherapy
candidate
for
human
colorectal carcinoma. World J Gastroenterol
10:3459-3463.
Bai X., et al. (2003) Honokiol, a small
molecular weight natural product, inhibits
angiogenesis in vitro and tumor growth in
vivo. J Biol Chem 278:35501-35507.
Hu J., et al. (2008) Liposomal honokiol, a
potent
anti-angiogenesis
agent,
in
combination with radiotherapy produces a
synergistic antitumor efficacy without
increasing toxicity.
Exp Mol Med
40:617-628.
Liu Y., et al. (2008) Enhancement of
therapeutic effectiveness by combining
liposomal honokiol with cisplatin in ovarian
carcinoma. Int J Gynecol Cancer.
18:652-659.
7