Ch 2.1 Chemicals
Natural extracts of isochaihulactone (E- and Z-form mixture) was from
National I-Lan University. Pured E- and Z-form of isochaihulactone were synthesized
from Prof. Yulin Lam, department of Chemistry, National University of Singapore.
Molecular weight is 398.41 g/mole. Steroids, prednisolone, was chosen for positive
control (SIGMA-ALODRICHTM). Both they were dissolved in DMSO dimethyl
sulfoxide (BIOSHOP®).
Ch 2.2 Devices
CO2 incubator (Thermo Scientific Revco Elite II)
Vortex Mixer (VWR Vortexer 2)
Inverted microscopy (Optima)
Centrifuge (Aron laboratory instruments inc)
Microcentrifuge (Thermo Scientific)
Microplate reader (Bio-Tek instrument, INC.)
Electrophoresis power supply (Bio-Rad Scientific)
Mini-PROTEIN® 3 Cell (Bio-Rad Scientific)
Electrophoretic transfer cell (Bio-Rad Scientific)
pH meter (Thermo Scientific)
Autoclaver (EZ medica company)
Electronic balance (OHAUS)
Kodak Medical X-ray Processor 101
Ch 2.3 Cell culture
The murine macrophage cell line, J774A.1 obtained from ATCC (Rockville,
MD, USA), and RAW264.7, were cultured in RPMI 1640 medium (HyClone®,
Thermo) supplemented with 10% fetal bovine serum, which composed of FBS
(bioind®) and 1%, 25 mM HEPES and 1% L-glutamine. Cells were incubated at 37oC
in a humidified atmosphere containing 5% CO2 / 95% air. And the subculture is to use
the suitable medium for them, both macrophages J774A.1 and RAW264.7 with RPMI
medium in 100mm cell culture dish. Till their growth to about 80% area distribution
of 100mm cell culture dish, the cultured medium should be demediumed and added
the PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3)
in order to wash out clearly at least twice. And then the dish is added new medium
and gathering all available cells for different experimental purposes, including
keeping the cells growth (meidium changed only when cells are few), and seeding
cells of the same counts with cell-count meter into 60 mm cell culture dish. When
cells have grown for almost over 80% area of 100 mm cell culture dish, they could be
freezed with several steps. First part of step is the same as to change the medium and
gather cells, FBS with 10% DMSO is added and moved into tube. And the tube full of
cells and FBS with 10% DMSO is put in 40C, -200C, for one hour respectively. After
that, the tube should be stored in -800C refrigerator for over-night. After the day, the
tube is moved into the liquid nitrogen device. When we want to defrost cells, the tube
with cells and FBS with 10% DMSO could be got from the liquid nitrogen device,
and warm the tube in the water bath with the temperature 370C, and then pour the
liquid into a sterile tube and centrifuge in 1000 rpm for 5 minute. The supernatant
medium is taken out and added new one for cell culture.
Ch 2.4 Cell viability
Macrophages were seeded in 5*105/ml, 100 μl/well for 24 hours. The chemical
was added with specific concentration in it for 24 hours. After that, Alamar Blue®
(AbD Serotec) was added 10 μL in. Read the plate with ELISA reader in wavelength
570 nm and 600 nm after 4 hours at least. The numbers of absorption were counted
with an equation to show the cell survival percentage compared to the control, which
is added with dimethyl sulfoxide (DMSO).
Ch 2.5 Nitric Oxide NO release test
NO, an inflammatory marker, was released after macrophages stimulated by
lipolysaccharides (LPS). The Griess Reagent System (Promega®) is composed of
Sulfanilamide Solution (1% sulfanilamide in 5% phosphoric acid) and NED Solution
(0.1% N-1-napthylethylenediamine dihydrochloride in water). Fist step is to 100 l of
sample was mixed with 50 l Sulfanilamide Solution and shaking the plate for 30
minutes for well-mixed. Later, 50 l NED solution was added into the 150 l solution
and shaking for 10 minutes (light protection with aluminum foil coverage). The three
react with the nitrite group to form colored azo group and then tested with ELISA
reader in the wavelength 520 nm.
Ch 2.6 Western Blotting analysis
For measurement of iNOS, proIL-1, and MAPKs phosphorylation at the protein
level, the lysate of macrophages is harvest and de-pellet to be separated by 8~10%
sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), and then
transferred electrophoretically onto a 0.2 μm microporous polyvinylidene fluoride
(PVDF) membrane (MILLIPORE, ImmobilonTM). The membranes were blocked with
5% skim milk (Anchor®) in phosphate buffer saline (PBS). The membrane was
connected with specific primary antibody and incubated with horseradish peroxidase
(HRP) Opti-ECL reagent 1 and reagent 2 mixture (Millipore) to conjugate the
secondary antibody. The fluorescent emission from the membrane was sensed with
X-ray film (BIOMAN) inside the radiographic cassette and wash it with film machine.
The membrane was re-blotted with stripping buffer for 30 minutes and conjugated the
α-actin or the protein total form as the internal control.
Ch 2.7 Enzyme-linked immunosorbent assay (ELISA)
ELISA kits were purchased from R & D system DuoSet®, including The KIT
for mouse IL-6, TNF-α, and IL-1β. 96-wells plate (Costar, tower tear®) was coated
the capture antibody for 24 hours at the room temperature and then blocking it with
reagent diluent for 2 hours at least, and the plate was well-prepared. The standard line
was determined with standard solution in indicated concentration, and the samples
and standard ones were added 100 l into the well for 2 hours for first step reaction.
Later is the aspiration step with wash buffer for about 3~5 times. The second step is
the detection antibody 100 l added into the well for 2 hours reaction. Aspiration is
necessary as the same the end of first step. The third step is the streptavidine solution
50 l added into the well for 20 minutes and then aspiration step. The fourth step is
the substrate solution for the dye 50 l added into the well for within 30 minutes
because of the color should be well-control. The fifth step is stop solution (2M
sulfonic acid) 50 l added to cut the reaction of substrate and samples. Finally the
plate was read with the ELISA reader (μ-Quant®) in wavelength 450 nm.
Ch 2.8 NF-B assay
RAW-BLUETM Cells (InvivoGen), as a mouse macrophages cell line with
transfected reporter gene of secreted embryonic alkaline phosphatase (SEAP), were
tested for the production of NF-B with the QUANTI-BlueTM reagent (InvivoGen), as
a medium for detection and quantification of SEAP. SEAP is a gene inducible by
NF-κB transcription factor (Figure 12). Cells were resistant to the selectable marker
ZeocinTM, a research used antibiotic. The 200l QUANTI-BlueTM reagent and 20l
medium are mixed and stored in 370C for light protection in 30 minutes. If the color
didn’t change significantly, check it every 15 minutes with the ELISA reader to test
the optical density for the NF-B activity test.
Ch 2.9 statistical analysis
The study use the statistical software of Sigmastat version 2.1 for analysis all
experimental data. The expression of all results is mean ± standard deviation, and uses
the one-way ANOVA analysis. P<0.05 expresses the significant difference.
Ch 2.10 Experimental flow chart
Chapter 3 Results
Ch 3.1 Cell viability investigation
To investigate the survival of murine macrophages,both J774A.1 and RAW
264.7 (concentration 0, 0.6, 1.2, 2.5, 5, 10, 20, 40 M) were treated with E or Z-form
Isochaihulactone in different doses for 24 hours and cell viability was assayed by
AlarmarBlue assay.
Ch 3.1.a Effect of isochaihulactone on the viability of J774A.1 macrophages
The Z-form of isochaihulactone showed the obvious cytotoxicity when
they were exposed to the concentration exceeding 5μM ( Z-isochaihulactone 0
M for 100 ± 4.92, 0.6 M for 119.64 ± 2.86, 1.2 M for 113.63 ± 8.62, 2.5 M
for 124.98 ± 0.57, 5 M for 107.87 ± 2.35, 10 M for 60.04 ± 4.52, 20 M for
61.44 ± 3.69, 40 M for 50.80 ± 4.77), while E-form of isochaihulactone didn’t
effect the cell viability ( E-isochaihulactone 0 M for 100 ± 4.92, 0.6 M for
114.38 ± 8.51, 1.2 M for132.54 ± 2.09, 2.5 M for 118.58 ± 11.12, 5 M for
129.84 ± 8.82, 10 M for 123.50 ± 10.74, 20 M for 126.77 ± 3.55, 40 M for
112.27 ± 6.28) (Figure 13)
Ch 3.1 b Effect of isochaihulactone on the viability of RAW264.7
macrophages
This result is similar to J774A.1 that the Z-form of isochaihulactone
showed the obvious cytotoxicity when they were exposed to the concentration
exceeding 5 μM (Z-isochaihulactone 0 M for 100±1.39, 0.6 M for 104.80 ±
9.30, 1.2 M for 104.46 ± 12.65, 2.5 M for 105.59 ± 3.65, 5 M for 92.93 ±
7.35, 10 M for 22.19 ± 4.90, 20 M for 26.92 ± 5.33, 40 M for 29.92 ± 4.45),
while E-form of isochaihulactone didn’t effect the cell viability
( E-isochaihulactone 0 M for 100 ± 1.39, 0.6 M for 107.37 ± 9.68, 1.2 M for
109.15 ± 2.37, 2.5 M for 105.55 ± 8.15, 5 M for 103.27 ± 8.64, 10 M for
106.78 ± 2.69, 20 M for 98.18 ± 3.64, 40 M for 96.41 ± 7.25 ) (Figure 14)
Ch 3.2 Anti-inflammatory activity
There’s a lot of inflammatory markers, including proIL-1, Nitric oxide (NO),
inducible nitric oxide synthase (iNOS), cytokine IL-6, TNF-α, and IL-1β. The
experiment was to investigate the expression of those inflammatory markers with E-
and Z-form, from different concentration range 6~40 μM.
Ch 3.2.a E-isocha. inhibited the IL-1 secretion and proIL-1 expression in
LPS-stimulated J774A.1 macrophages
Macrophages J774A.1 was treated Z-isochihulactone ( 0.6, 1.2, 2.5, 5 M )
and E-isochaihulactone ( 0.6, 1.2, 2.5, 5, 10, 20 M ) for investigation on the
IL-1 production after LPS stimulation for 24 hours. Z-isochaihulactone has
little effect for IL-1 production (control for 10.5 ± 5.20 pg/ml, LPS for 330.68
± 20.45pg/ml, 0.6 M for 351.93 ± 30.1 pg/ml, 1.2 M for 353.81 ± 25.86
pg/ml, 2.5 M for 311.93 ± 27.43 pg/ml, 5 M for 306.31 ± 24.63 pg/ml)
(Figure 15), while E-isochaihulactone appears obvious efficiency when
concentration > 10 M, even more better inhibition for 20 M (0.6 M for
357.56 ± 26.1 pg/ml, 1.2 M for 346.31 ± 24.46 pg/ml, 2.5 M for 289.43 ±
25.33 pg/ml, 5 M for 265.06 ± 23.43 pg/ml, 10 M for 134.43 ± 27.03 pg/ml,
20 M for 55.68 ± 10.24 pg/ml) (Figure15). With higher concentration of
E-isochaihulactone, the former of IL-1, pro IL-1 is inhibited in dose
denpendence. (Figure 16)
Ch 3.2.b E-isocha. inhibited NO release and iNOS expression in
LPS-stimulated RAW264.7 macrophages
To find out that nitrous oxide was produced by amino acid L-arginine
through the enzyme inducible Nitric Oxide synthase (iNOS) and released during
the inflammation occurrence. NO concentration (control for 0.5 ± 1.00 M, LPS
for 19.20 ± 0.57 M, 2.5 M for 18.93 ± 0.73 M, 5 M for 17.98 ± 1.53 M,
10 M for 14.87 ± 2.03 M, 20 M for 9.13 ± 1.24 M) (Figure 17) and iNOS
(Figure 18) were both inhibited effectively compared to LPS stimulation by
E-isochaihulactone in dose-dependence. The steroids, prednisolone, as a positive
control, did inhibit the NO release, too. (1 M for 6.56 ± 2.03 M, 10 M for
3.18 ± 1.03 M) (Figure 17)
Ch 3.2.c E-isocha. inhibited cytokines production in LPS-stimulated
J774A.1 macrophages
To investigate if the cytokine released from murine macrophages when
they encountered the microbes’ infection, E- and Z-isochihulactone was treated
to find out the effect of released cytokines, including IL-6 (control for 0.1 ± 0.32
ng/ml, LPS for 25.10 ± 1.15 ng/ml, Z-isochaihulactone 0.6 M for 23.78 ± 1.21
ng/ml, 1.2 M for 22.99 ± 1.36 ng/ml, 2.5 M for 21.85 ± 1.43 ng/ml, 5 M for
18.13 ± 1.16 ng/ml, E-isochaihulactone 0.6 M for 23.49 ± 1.21 ng/ml, 1.2 M
for 21.92 ± 1.36 ng/ml, 2.5 M for 21.38 ± 1.43 ng/ml, 5 M for 18.72 ± 1.13
ng/ml, 10 M for 16.03 ± 1.23 ng/ml, 20 M for 9.56 ± 1.34 ng/ml) (Figure 19),
and TNF-α (control for 0.1 ± 0.27 ng/ml, LPS for 9.50 ± 0.27 ng/ml,
Z-isochaihulactone 0.6 M for 9.97 ± 0.31 ng/ml, 1.2 M for 9.16 ± 0.32 ng/ml,
2.5 M for 8.78 ± 0.35 ng/ml, 5 M for 7.00 ± 0.27 ng/ml, E-isochaihulactone
0.6 M for 9.78 ± 0.31 ng/ml, 1.2 M for 10.41 ± 0.32 ng/ml, 2.5 M for 9.78 ±
0.35 ng/ml, 5 M for 7.55 ± 0.27 ng/ml, 10 M for 6.03 ± 0.33 ng/ml, 20 M
for 4.53 ± 0.32 ng/ml)(Figure 20). It resulted that E-form of isochaihulactone
did effectively inhibit the cytokine release from murine macrophages J774A.1,
while Z-form of isochaihulactone did little effectively.
Ch 3.2.d E-isocha. inhibited cytokines production in LPS-stimulated
RAW264.7 macrophages
In order to seek if E-isochaihulactone has the same effect on other
macrophages, RAW264.7 was used for cytokine evaluation. It is design for
E-isochaihulactone treated without Z-form, and steroid, prednisolone, as a
anti-inflammatory positive control plus. After LPS stimulation for 24 hours,
IL-6 and TNF- were released from RAW264.7. That results in that
E-isochaihulactone inhibits IL6 (control for 0.1 ± 0.37 ng/ml, LPS for 5.82 ±
0.24 ng/ml, 2.5 M for 5.41 ± 0.23 ng/ml, 5 M for 5.14 ± 0.11 ng/ml, 10 M
for 4.33 ± 0.12 ng/ml, 20 M for 1.53 ± 0.05 ng/ml) (Figure 21) and TNF-
(control for 0.5 ± 0.22 ng/ml, LPS for 3.68 ± 0.47 ng/ml, 2.5 M for 3.51 ± 0.35
ng/ml, 5 M for 3.30 ± 0.58 ng/ml, 10 M for 3.17 ± 0.70 ng/ml, 20 M for
2.72 ± 0.18 ng/ml) in dose-dependence (Figure 22)
Ch 3.3 Mechanism of signal transduction pathway
According to the results above, it’s approve that E-isochaihulactone has the
anti-inflammatory activity for inhibition on cytokines and NO. To investigate the
mechanism for anti-inflammatory activity, the signal transduction pathways could be
important marker. With the ligation of LPS and TLR4 receptor complex, there are
several pathways for inflammation, including the activation of NF-B and LPS
induced phosphorylation.
Ch 3.3.a E-isocha. had no effect on NF-B activity in LPS-stimulated
RAW-BLUETM cells
NF-B is test with macrophages RAW BLUE. E-isochaihulactone is
added in different concentration and steroid, prednisolone, as a positive control.
E-isochaihulactone slightly inhibits NF-B activation compared to positive
control. (relative optical density, control for 0.001 ± 0.02, LPS for 1.75 ± 0.04,
2.5 M for 1.66 ± 0.03, 5 M for 1.69 ± 0.03, 10 M for 1.61 ± 0.01, 20 M for
1.53 ± 0.01, prednisolone 1 M for 1.00 ± 0.02, 10 M for 0.93 ± 0.01) (Figure
23)
Ch 3.3.b E-isocha. inhibited phosphorylation of ERK1/2 in LPS-stimulated
RAW264.7 macrophages (time course study)
To find out the MAPKs activation, ERK is chosen to test the time course.
The experiment is divided into two subset for E-isochaihulactone 20 M
untreated and treated after LPS stimulation for different point of time, from 0,
10 ,20 , 30, 60 minutes. E-isochaihulactone inhibits the ERK phosphrylation for
blocking the signal transduced into nucleus. (Figure 24)
Ch 3.3.c E-isocha. inhibited phosphorylation of ERK1/2 in LPS-stimulated
RAW264.7 macrophages (dosage study)
In addition to time course study investigation, dosage study could be used
to make sure the effects. As expected, E-isochaihulactone shows the
dose-dependence inhibition for phosphorylation of ERK in concentration 5 M,
10 M, 20 M, and 40 M. (Figure 25)
Ch 3.3.d E-isocha. inhibited phosphorylation of p38 in LPS-stimulated
RAW264.7 macrophages
E-isochaihulactone inhibited phosphorylation of p38 after LPS-stimulated
in 30 minutes in concentration 5 M, 10 M, 20 M, and 40 M. (Figure 26).
What is more, the inhibition on p-p38 is obviously stronger than p-ERK.
Ch 3.3.e E-isocha. inhibited phosphorylation of JNK1/2 in LPS-stimulated
RAW264.7 macrophages
E-isochaihulactone inhibited phosphorylation of JNK1/2 after
LPS-stimulated for 30 minutes in concentration 5 M, 10 M, 20 M, and 40
M (Figure 27).
Chapter 4 Discussion and Conclusion
Ch 4 Discussion and Conclusion
Clinically prescriptive medicine of anti-inflammation has a lot of side effects.
Widely used medicine for anti-inflammation included steroids42, non-steroid
anti-inflammatory drug (NSAID)43, some other herbal medicine, and etc. Most of
them have the side effects, for examples of NSAID, acetylsalicylic acid (Aspirin®)
for Reye’s syndrome in children44, diclofenac for gastrointestinal discomfort and
tissue necrosis45, indomethacin for peptic ulcer46, and others. In addition, for
examples of steroids, prednisolone for Cushing’s syndrome and increase of blood
glucose47, dexamethasone for increased appetite and osteoporosis48,
methylprednisolone for glaucoma49, and so on. Those anti-inflammatory agents
mentioned above are common used in clinical therapy in a lot of cases of
inflammation in the clinical prescription. Because of little found new indication about
anti-inflammation, for safety of drug use, it will take more and more researches about
new compounds for anti-inflammatory effect. These new compounds usually origin
from synthesis to partition techniques, including extraction, purification, and other
techniques for isolation, especially for Chinese herbal medicine or natural products
which might have unknown chemicals.
There were a lot of researches about the Chai-Hu with different medical effects,
The common medical-used part for Chai-Hu is the root part. The ingredients included
Saikosaponin, chaihunaphthone, flavones, lignans, and isochaihulactone35-37 had been
found. Besides, polysaccharides derived from the root of Bupleurum smithii var.
parvifolium has the macrophage immunomodulatory activity, like phagocytosis50.
Saikosaponin from Bupleurum chinense DC. has the protections on the antioxidative
for those ROS (reactive oxygen species), like hydrogen peroxide (H2O2), superoxide
radicals (O2−), and malondialdehyde (MDA) which had high risks for health. There
were more about Nan Chai-Hu (Bupleurum scorzonerifolium) for the ingredients
found and the effects. For examples, isochaihulactone and chaihunaphthone inhibits
the CD28 costimulation, IL-2 concentration, and CD28-costimulated T cells51; With
acetone-extracted chemicals has the anti-A549 cells activity for arrest the cell cycle
and telomerase activity for cell viability assay52. In addition, saikosaponin has the anti
virus effects of coronavirus53. There was also has anti-inflammatory activity for
Chai-Hu, especially for the inhibitions on the proliferation and activity of T-cell54.
Chai-Hu has been used for a lot of disease since two thousands years ago. Most
of prescribed for medical use is the natural products, even though with different
technology for obtaining the target compound, which means, once the compound has
the isomeric character. They are mixed for components in percentage. In this study,
we found that isochihulactone, which has the E- and Z-form isomer, has the diverge
for the isomers with different effects on macrophage, especially for the cell
cytotoxicity. Z-form, but not E-form, is toxic to cells that results E-isocha. has less
limit for the higher dose to try to investigate for other medical effects because of the
cell safety. But the mechanism of the anti-inflammatory effects is still not clear, and
needs more experiments for more approval for that. If the effects are approved, it
could proceed for animal model study, even for clinical trial. For the drug use safety,
if the compound has few or free side effects to patients, it may be for prescribed
medical use instead of currently used anti-inflammatory drugs mentioned above. And
the pure E-isocha. could be obtained with chemical synthesis, and taken by patients in
a safe condition for Z-isocha. free anti-inflammatory drug. It would break the myth
for populations about the natural product is better than artificial events.
Chapter 5. Figures
Figure 1 the process of inflammation
http://www.goodpsych.com/storage/inflammation.jpg
Figure 2 nitric oxide released from macrophage
http://www.itqb.unl.pt/~Metalloproteins_Bioenergetics/macrophage.gif
Figure 3 the structure of lipopolysaccharide (LPS) http://pathmicro.med.sc.edu/fox/lps.jpg
Figure 4 The CD14/TLR4/MD2 receptor complex and signals http://www.pharmaprojects.com/contentimg/0908/TLRdiag1.gif
Innate immunity Adaptive immunity
Figure 5 macrophage plays a role in immunity
Figure 6 pathogen recognition receptors toll-like receptor
Modified from: http://www.nature.com/nri/journal/v7/n5/images/nri2079-f3.jpg
Figure 7 Bei Chai-Hu (Buplerum chinense DC) http://www.db5318.com/fengye/beicaihu2.jpg
Figure 8 Nan Chai-Hu (Buplernm scorzonerifolium Wild)
http://www.sanqi.com.tw/original-chinese-herb/chai-hu/chaihu-03.jpg
O O
O O
H
OMe
OMe OMe
O O
O O
H
OMe
MeO OMe
Figure 9 the structure of E- and Z-form isochaihulactone
Z-form E-form
O
Reaction conditions: (i) diethyl succinate, EtONa; (ii) catASium® M(S)Rh or
catASium® M(R)Rh, H2; (iii) KOH, CaCl2; (iv) NaBH4; (v) 3 M HCl; (vi) LDA,
3,4,5-trimethoxybenzaldehyde; (vii) MsCl, TEA; (viii) DBU; (ix) Ac2O, TEA; (x)
DBU.
Figure 10 the chemical synthesis for E- and Z-isochaihulactone by Yulin Lam,
Ddepartment of Cchemistry, National University of Singapore Z-isochaihulactone
E-isochaihulactone
Figure 11 definition of structure of cis-trans and E-Z stereoisomerism The chlorine is the priority element for dichloroethene cis-form (A) and trans-form (B); the fluorine and ethyl group are the priority elements for fluoromethylpentene Z-form (C) and E-form (D)
A B C D
Figure 12 RAW BLUE cells with reporter gene for NF-B assay
Modified from: http://www.autogenbioclear.com/D/pics/HekblueIL1b_assay.jpg
Figure 13 Effect of isochaihulactone on the viability of J774A.1 macrophages
Murine macrophages J774A.1 were seeding in 5*105/ml in 96-well plate, well/100
l and treated with isochaihulactone E-form ( ) and Z-form ( ), respectively, in 24 hours with the Alamar Blue reagent at least 4 hours to show the cell proliferation. ＃ p < 0.05 v.s isochai. 0 M.
Figure 14 Effect of isochaihulactone on the viability of RAW264.7 macrophages
Murine macrophages RAW264.7 were seeding in 5*105/ml in 96-well plate,
well/100 l and treated with isochaihulactone E-form ( ) and Z-form ( ), respectively, in 24 hours. With the Alamar Blue reagent at least 4 hours to show the cell proliferation. ＃ p < 0.05 v.s isochai. 0 M.
Figure 15 E-isocha. inhibited the IL-1 secretion in LPS-stimulated J774A.1
macrophages
Murine Macrophages J774A.1 were seeding in 5*105/ml in 96-well plate, well/100l and pre-treated for 30 minutes with E-form and Z-form of
isochaihulactone, respectively. And then LPS was treated for 24 hours. The cell medium was harvested and tested the IL-1β production with ELISA. ＃ p <
0.05 v.s LPS stimulated only.
Figure 16 E-isocha. inhibited proIL-1 expression in LPS-stimulated J774A.1
macrophages
Murine macrophages J774A.1 were seeding pre-treated with E-form of
Isochaihulactone from 2.5μM~20μM for 30 minutes, respectively, and then
treated with lipopolysaccharide (LPS) 1μg/ml for 6 hours. Harvest the cell lysate to test the expression of pro IL-1 with western blotting. ＃ p < 0.05 v.s LPS
stimulated only.
Figure 17 E-isocha. inhibited NO release from RAW264.7 macrophages
Murine macrophages RAW264.7 were pre-treated with E-form of
Isochaihulactone and Prednisolone for 30 minutes, respectively. LPS was treated
in 24 hours and the medium was harvested to test NO concentration with Griess Reagent. ＃ p < 0.05 v.s LPS stimulated only.
Figure 18 E-isocha. inhibited iNOS expression in LPS-stimulated RAW264.7
macrophages
Murine macrophages RAW264.7 were pre-treated with E-form of
Isochaihulactone for 30 minutes, respectively. LPS was treated in 24 hours and
the cell lysate was harvested to test the expression of iNOS and actin, as an internal control with Western blotting. ＃ p < 0.05 v.s LPS stimulated only.
Figure 19 E-isocha. inhibited IL-6 production in LPS-stimulated J774A.1
macrophages
Murine Macrophages J774A.1 were pre-treated for 30 minutes with E-form
and Z-form of Isochaihulactone, respectively. And then LPS was treated for 24
hours. The cell medium was harvested and tested the IL-6 production with ELISA. ＃ p < 0.05 v.s LPS stimulated only.
Figure 20 E-isocha. inhibited TNF- production in LPS-stimulated J774A.1
macrophages
Murine Macrophages J774A.1 were pre-treated for 30 minutes with E-form
and Z-form of Isochaihulactone, respectively. And then LPS was treated for 24
hours. The cell medium was harvested and tested the TNF-α production with ELISA. ＃ p < 0.05 v.s LPS stimulated only.
Figure 21 E-isocha. inhibited IL-6 production in LPS-stimulated RAW264.7
macrophages
Murine Macrophages RAW264.7 were pre-treated for 30 minutes with E-form
Murine Macrophages RAW264.7 were pre-treated for 30 minutes with E-form