Physalin B Suppresses Inflammatory Response to Lipopolysaccharide in RAW264.7 Cells by Inhibiting NF-κB Signaling
Journal of Chemistry
Physalin B Suppresses Inflammatory Response to Lipopolysaccharide in RAW264.7 Cells by Inhibiting NF-?B Signaling
Lang Yi 0 1
Qing Wang 1
Bingbing Xie 1
Congwei Sha 2
0 Department of Pharmacy, Guangdong Food and Drug Vocational College , Tianhe District, Guangzhou 510520 , China
1 Department of Immunology, Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine , Baiyun District, Guangzhou 510405 , China
2 Guangdong Provincial Institute of Biological Products and Materia Medica , Baiyun District, Guangzhou 510440 , China
Physalin B from Physalis angulata L. (Solanaceae) is a naturally occurring secosteroid with multiple biological activities. But its anti-inflammatory activity and mechanism remain unclear. Physalin B effects on RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) were observed in this study. 0e expression and secretion of tumor necrosis factor-? (TNF-?) and interleukin-6 (IL-6) induced by LPS were significantly inhibited by physalin B. Meanwhile, the NF-?B nuclear translocation induced by LPS was inhibited by physalin B. 0e anti-inflammatory effects of physalin B could not be inhibited by mifepristone (RU486), the blocker of glucocorticoid receptor. In conclusion, physalin B can suppress inflammatory response to LPS in macrophages by inhibiting the production of inflammatory cytokines via NF-?B signaling.
Inflammation is a fundamental pathological phenomenon
and a complex biological response participating in the
development of diverse diseases [
]. Inflammation is usually
mediated by eicosanoids and cytokines released by injured or
infected cells, especially activated immunocytes. TNF-? and
IL-6 are key inflammatory cytokines in macrophages
activated by LPS [
]. Although helpful to combat against
infection, vigorous inflammatory cytokines may lead to edema,
cellular metabolic stress, and tissue necrosis. NF-?B, an
immediate early transcriptional activator, plays a central role in
inflammatory response by binding with the promoters to
induce transcription of proinflammatory genes, such as TNF-?
and IFN-c [
]. NF-?B signaling is well known to be involved in
various diseases, including inflammation and cancers, and
thus has attracted attention as a drug target [
Physalin B, a naturally occurring secosteroid
isolated from the stems and aerial parts of Physalis angulata
L. (Solanaceae) (Figure 1), possesses a unique
13,14-seco16,24-cycloergostane skeleton, an H-ring with a C14?O?C27
bond and a cage-shaped structure, with a highly oxygenated,
complex structure similar to glucocorticoid. In addition to the
intriguing structure, there is considerable interest in the
biological activities of physalin B. In the experiments in vivo, the
physalin B anti-inflammatory effect appeared to be mostly
due to the activation of glucocorticoid receptors, which
represented novel therapeutic options for the treatment of
inflammatory diseases [
]. Physalin B was thought to have the
potential to be an effective chemotherapeutic lead compound
for the treatment of malignant melanoma [
]. It showed
strong cytotoxicity against multiple tumor cell lines [
antimitotic activity for the first cleavage [
] and inhibited the
growth of several human leukemia cells [
]. Physalin B was
considered responsible for the antimicrobial activity, at the
concentration of 200 ?g/ml, and physalin B exhibited about
85% of the inhibitory activity observed with the mixture of
physalins (pool) containing physalins B, D, F, and G, at the
same concentration [
]. Physalin B exhibited a minimum
inhibitory concentration (MIC) value (128 ?g/ml) against
Mycobacterium tuberculosis H37Rv strain [
]. ?us, physalin
B has the potential to regulate a broad range of biological
events. However, the underlying mechanisms of physalin B
remain largely unknown.
?is study observed the anti-in?ammatory underlying
mechanism of physalin B in RAW264.7 macrophages
stimulated by LPS. ?e roles of the nuclear factor kappa B
(NF-?B) and the glucocorticoid receptor in physalin B
antiin?ammatory e?ect were analyzed. ?e study might provide
evidence for physalin B as the lead structure of
2. Materials and Methods
2.1. Plant Material. Physalin B was isolated from Physalis
angulata (Physalis angulata L.). ?e herb was collected from
Puning in Guangdong Province by Mr. Wen-Biao Chen and
identi?ed by Dr. Qing-Qian Zeng. ?e specimens (GICMM
number 5) were stored in the Guangdong Institute of
Chinese Materia Medica.
2.2. Chemicals and Reagents. ?e isolated compound was
analyzed by HR mass spectrometry, and its purity was found
to be 100% for small-molecule single-crystal X-ray
diffraction analysis. LPS (from Escherichia coli 0111 : B4) was
purchased from Sigma (St. Louis, USA). ?e monoclonal
antibodies I?B (4814S), NF-?B p65 (6956S), and ?-actin
(2118S) were purchased from Cell Signaling Technology
(CST, USA). Mouse TNF-? (E09483-1643) and IL-6
(E093621643) ELISA detection kits were purchased from eBioscience
(California, USA). TRIzol (15596026) was purchased from
Invitrogen. Reverse Transcription Kit (135600) and SYBR
Green Quantitative PCR Kit (262000) were purchased from
Toyobo (Osaka, Japan). ?e ECL chemiluminescence
detection kit was purchased from ?ermo (Waltham, MA,
2.3. Isolation and Purication of Physalin B. ?e air-dried
and milled whole plants of P. angulata (8 kg) were extracted
with ethanol (85%) three times (3 ? 30 L) under immersion,
for 1 week each. After ?ltration and evaporation of the solvent
under reduced pressure, the combined crude ethanolic extract
(1067 g) was mashed and then dissolved successively with
petroleum ether, EtoAc, and n-BuOH to a?ord dried
petroleum ether-soluble (101 g), EtoAc-soluble (49 g), and
nBuOH-soluble (48 g) fractions, respectively. Accordingly, the
EtoAc-soluble extract was subjected to medium-pressure
column chromatography over silica gel (LC60A 40?63
micron) and eluted using a step gradient of a petroleum ether
and EtoAc solvent system (100 : 0, 100 : 1, 80 : 1, 50 : 1, 25 : 1,
10 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1) at a ?ow rate of 50 ml?min?1,
pressure 20 bar, to obtain ten fractions (F1?F10) based on the
TLC pro?le. Each fraction was concentrated in vacuo. Further
puri?cation of subfraction F7 (petroleum ether and EtoAc
solvent system: 5 : 1) by repeated column chromatography
over silica gel (LC60A 40?63 micron) with petroleum
etherEtoAc (100 : 0 to 1 : 1) followed by thin-layer chromatography
gave transparent crystals, physalin B, suitable for X-ray
diffraction analysis (80.0 mg).
Physalin B, colorless prismatic crystals (petroleum
etherEtoAc), mp253-254?C. HR-EI: m/z 510.1883(C28H30O9,
calcd. for 510.1884), 1H-NMR (400 MHz, acetone-d6)?: 6.92
(1H, m, H-3), 6.50(1H, s, 13-OH), 5.86(1H, dd, J 10.4, 2 Hz,
H-2), 5.61 (1H, brd, J 6 Hz, H-6), 4.56(1H, brs, H-22), 4.40
(1H, dd, J 13.6, 4.6 Hz, H-27), 3.76(1H, d, J 13.6, H-27),
1.88(3H, s, CH3-21), 1.31(3H, s, CH3-28), 1.19 (3H, s,
CH319).13C-NMR (100 MHz, acetone-d6)?: 205.3(C-1), 127.7
(C-2), 147.3(C-3), 33.3(C-4), 136.2(C-5), 124.7(C-6), 25.4
(C-7), 41.1(C-8), 34.4(C-9), 53.6(C-10), 25.2(C-11), 26.3
(C-12), 80.0(C-13), 107.8(C-14), 209.7(C-15), 56.1(C-16),
81.9(C-17), 172.7(C-18), 17.6(C-19), 81.5(C-20), 22.0(C-21),
77.6(C-22), 32.8(C-23), 31.6(C-24), 50.9(C-26), 167.5(C-26),
2.4. Cell Culture. ?e culture of RAW264.7 cells was
obtained the way we had established in our lab [
2.5. Cell Viability. RAW264.7 cells (1 ? 105/ml) were seeded
in a 96-well plate and incubated at 37?C overnight. Cells were
stimulated with various concentrations of physalin B for
24 h, and PBS was used as vehicle control. At the end of the
incubation, 10 ?l of MTT (5 mg/ml in PBS) solution was
added and incubated for an additional 2 h. After DMSO
solubilized the formazan crystals, it was measured using an
enzyme-linked immunosorbent assay (Molecular Devices,
Sunnyvale, CA) at 595 nm. ?e relative cell viability was
calculated and compared with the absorbance of the
untreated control group.
2.6. Detection of TNF-? and IL-6 Release. RAW264.7 cells
were treated with positive drug (1 ?M dexamethasone) or
di?erent concentrations of physalin B (20, 10, and 5 ?M) for
2 h and then stimulated with 1 ?g/ml LPS for 8 h. PBS was
used as the control. After the incubation, the culture
supernatant was collected. Concentration of in?ammatory
cytokines (TNF-? and IL-6) was analyzed by ELISA.
2.7. Real-Time PCR for TNF-? and IL-6 Assay. RNA was
extracted from RAW264.7 cells by TRIzol method. cDNA
synthesis and the quantitative PCR were obtained as
described in the previous study of our lab [
2.8. Western Blotting for I?B? and NF-?B p65 Protein Assay.
For Western blotting assay, we used KeyGEN Nuclear and
Cytoplasmic Protein Extraction Kit to extract NF-?B p65
and I?B? protein and BCA protein assay kit to measure the
protein concentrations. Protein samples (50 ?g) were
fractionated by 10% SDS-PAGE and transferred onto PVDF
membranes (Bio-Rad). Nonspeci?c reactivity was blocked
by 5% BSA for 2 h at room temperature, followed by primary
antibodies for anti-mouse NF-?B p65, I?B?, GAPDH diluted
to 1 : 1000 and then by goat anti-mouse HRP-conjugated
secondary antibody at 1 : 15000. ?e speci?c proteins were
detected by exposing membranes to Kodak X-Omat ?lms,
and densitometric analysis was performed by using the
Quantity One to scan the signals.
2.9. Statistical Analysis. Results were showed as mean ?
SEM. One-way analysis of variance (ANOVA) and Tukey
multiple comparison tests were used to analyze the data.
P < 0.05 was considered statistically signi?cant. All analyses
were performed using SPSS 13.0 for Windows.
3. Results and Discussion
3.2. E?ect of Physalin B on the mRNA and Protein Levels
of TNF-? and IL-6. In order to evaluate the e?ect of physalin
B on LPS stimulation, ELISA was used for determining
the levels of in?ammatory cytokines IL-6 and TNF-?. LPS
(1 ?g/ml) signi?cantly increased the mRNA and protein
levels of TNF-? and IL-6. However, dexamethasone (1 ?M)
signi?cantly inhibited the mRNA and protein levels of
TNF-? and IL-6 (P < 0.01). Physalin B decreased TNF-?
and IL-6 mRNA and protein levels signi?cantly at 5, 10,
and 20 ?M in a concentration-dependent manner (P < 0.05
or P < 0.01) (Figure 3).
3.1. Cell Viability. In order to determine the working
concentration of physalin B, RAW264.7 cells were treated
with 12.5, 25, and 50 ?M physalin B. We found that physalin
B at 50 ?M can markedly reduced the cell viability of
RAW264.7 cells compared with control (P < 0.01), but
others did not show any signi?cant di?erence (Figure 2).
3.3. ?e E?ect of GR Antagonist on the Inhibition of TNF-?
and IL-6 Expression by Physalin B. To analyze whether the
anti-in?ammatory e?ect of physalin B relied on the
glucocorticoid receptor (GR), we chose the GR selective antagonist
mifepristone (RU486). Our study showed that RU486 did not
inhibit the e?ect of physalin B on the levels of TNF-? and IL-6
mRNA and protein (P > 0.05) in contrast to dexamethasone
3.4. ?e E?ects of Physalin B on I?B and NF-?B/p65 Protein
Level. Stimulation with LPS caused I?B decreasing in
cytoplasm and NF-?B p65 increasing in nucleus. However,
when the cells were pretreated with physalin B, the LPS e?ect
on I?B and NF-?B p65 was reversed. Our data revealed that,
in the cells cotreated with LPS and physalin B, the
LPSinduced p65 was suppressed. ?ese results demonstrated
that physalin B could inhibit NF-?B activation.
Physalins share a unique 13,14-seco-16,24-cycloergostane
skeleton, with a highly oxygenated and complex structure.
Type B physalins, such as physalin B, have an H-ring with
a C14?O?C27 bond and a cage-shaped structure. ?e AB-ring
of physalins that is commonly found in plant steroids was
suggested to be involved in biological activities. For instance,
Ma and coworkers suggested that the A-ring of physalin A
could form a covalent bond with cysteine residues of IKK?
]. In contrast, little attention was paid to the contribution
of the cage-shaped right-sided structure in Type B physalins
(e.g., physalin B). Masaki et al. [
] hypothesized that the
unique partial structure would play an important role in the
Also, physalin B has a similar glucocorticoid structure.
Glucocorticoids have anti-in?ammatory activities with
many adverse e?ects, such as osteoporosis, metabolic
diseases, high blood pressure, and so on.
From our results, physalin B signi?cantly inhibited the
mRNA expression and secretion of TNF-? and IL-6 in
macrophages induced by LPS at the concentrations without
obvious cytotoxicity (Figures 2 and 3). In addition, the
results showed that RU486 inhibited the anti-in?ammatory
e?ects of dexamethasone, but not physalin B in RAW264.7
cells (Figure 4). It suggests that physalin B does not require
GR for the anti-in?ammatory activity in vitro, which is
di?erent from Vieira?s research [
]. Physalin B, although with
secosteroidal chemical structure, did not act through the
glucocorticoid receptor in macrophages, which means its
structure group di?erent from glucocorticoid is the active core.
NF-?B, an immediate early transcriptional activator,
participates in in?ammatory responses and acute phase
through increasing the expression of immediate early
in?ammatory genes by binding with the promoters, including
TNF-?, IFN-c, NOS II, ICAM, and so on [
]. I?B? is the
main regulator of NF-?B, and NF-?B combined with I?B? is
]. When I?B? is degraded in cytoplasm,
NF?B can translocate to the nucleus and transcriptional
activation is activated. From the results (Figure 5), the decreased
I?B? protein level in cytoplasm and the increased NF-?B
p65 protein level in nucleoprotein induced by LPS were
reversed by physalin B. ?ese results provide evidence that
physalin B exerts anti-in?ammatory e?ect by inhibiting
Physalin B, a naturally occurring secosteroid from Physalis
angulata L., can inhibit in?ammatory response in LPS-induced
macrophages by inhibiting NF-?B activation in vitro. Its
antiin?ammatory e?ect is independent on the glucocorticoid
receptor. Our study suggests that physalin B could be a potential
new therapeutic agent against in?ammation.
0e data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
0e authors declare that none of the authors has any kind of
conflicts of interest related to the present work.
Lang Yi, Qing Wang, and Bingbing Xie performed the
cellbased assay experiments. Yanjun Yang and Congwei Sha
performed the isolation and purification of physalin B. Yanjun
Yang drafted the manuscript. Yanjun Yang and Yan Dong
supervised and coordinated the study and revised the
0e authors acknowledge the financial support from the project
supported by the Natural Science Foundation of Guangdong
(S2013010013484) and Science and Technology Project of
Guangdong (2014A020210016 and 2011B031700072).
Hindawi Publishing Corporation
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