A number of studies have proved that oxLDL Keywords antioxidant; endomorphins; free radical; lipid peroxidation; low-density lipoprotein Correspondence R.. Endomorphins markedly and in a
Trang 1peptides in the brain, on human low density lipoprotein oxidation
Xin Lin1, Li-Ying Xue1, Rui Wang1,2, Qian-Yu Zhao1and Qiang Chen1
1 Department of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, China
2 State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, China
It is now commonly recognized that the damaging
con-sequences of oxidative stress have been implicated in
a variety of very different human disorders, including
arteriosclerosis and diseases of the nervous system [1]
A large body of evidence indicates that the brain
appears to be particularly vulnerable to oxidative
dam-age because of its high oxygen consumption, abundant
lipid content, and relatively low antioxidant levels
[2,3] Oxidative damage may play important roles in
the slowly progressive neuronal death that is
character-istic of several different neurodegenerative disorders,
such as Alzheimer’s disease and Parkinson disease
[4,5] Reactive oxygen species (ROS) rapidly oxidize cellular lipids, resulting in the formation of numerous lipid peroxidation products in nerve cells Oxidatively modified lipids are able to react with cellular and sub-cellular membranes, leading to neuronal cell death [6,7]
Low density lipoprotein (LDL) is present in the brain and is exposed to a highly oxygenated and lipid-enriched environment, making it susceptible to free radical-mediated lipid peroxidation that can result in the formation of oxidized LDL (oxLDL) [7–9] A number of studies have proved that oxLDL
Keywords
antioxidant; endomorphins; free radical; lipid
peroxidation; low-density lipoprotein
Correspondence
R Wang, State Key Laboratory for Oxo
Synthesis and Selective Oxidation, Lanzhou
Institute of Chemical Physics, Chinese
Academy of Sciences, Lanzhou 730000,
China
Fax: +86 931 8912561
Tel: +86 931 8912567
E-mail: wangrui@lzu.edu.cn
(Received 3 December 2005, accepted
23 January 2006)
doi:10.1111/j.1742-4658.2006.05150.x
Neurodegenerative disorders are associated with oxidative stress Low den-sity lipoprotein (LDL) exists in the brain and is especially sensitive to oxi-dative damage Oxioxi-dative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases Therefore, protecting LDL from oxidation may be essential in the brain The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid pep-tides in the brain, on LDL oxidation has been investigated in vitro The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2¢-azobis(2-amidinopropane hydrochloride) (AAPH) Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobar-bituric acid reactive substances, and the relative electrophoretic mobility Low density lipoprotein oxidative modifications were assessed by evaluat-ing apoB carbonylation and fragmentation Endomorphins markedly and
in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endo-morphins In all assay systems, EM1 was more potent than EM2 and
l-glutathione, a major intracellular water-soluble antioxidant We propose that endomorphins provide protection against free radical-induced neuro-degenerative disorders
Abbreviations
AAPH, 2,2¢-azobis(2-amidinopropane hydrochloride); BHT, butylated hydroxytoluene; DNPH, 2,4-dinitrophenylhydrazine; EM1, endomorphin 1; EM2, endomorphin 2; GSH, L -glutathione; LDL, low density lipoprotein; oxLDL, oxidized LDL; REM, relative electrophoretic mobility; ROS, reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substance.
Trang 2is cytotoxic to neurones, further suggesting that
oxLDL may not only be involved at the vascular
level of neurodegenerative diseases but also could be
more directly responsible for the degeneration of
neurones [10–12] Elevated levels of ROS and trace
metals, such as copper and iron, capable of oxidizing
LDL are present in neurodegenerative conditions
[13] Therefore, neuronal cells have to maintain an
effective antioxidation system in order to protect
themselves against ROS overload and subsequent
damage As described in various excellent reviews,
antioxidants keep the fine-tuned balance between
the physiological production of ROS and their
detoxification [14,15]
Recently, it has been observed that the female sex
hormone estrogen can inhibit the oxidation of LDL
and can attenuate the cytotoxicity of oxLDL on
neur-onal cells [14,16,17] It is believed that melatonin, a
hormone mainly secreted by the pineal gland, is an
effective free radical scavenger and antioxidant and
thus attenuates the effects of free radical-induced
neur-onal damage [18,19] There are studies investigating
the antioxidant activity of melatonin in lipid model
systems [20] and also in LDL [21,22] In addition, it
has been reported that the brain monoamines and their
metabolites can inhibit lipid peroxidation and protect
from oxidative damage in the brain [23] Recent
studies have demonstrated that enkephalins
(leu-enke-phalin, met-enkephalin) and their derivatives
(5-S-cyst-einyldopaenkephalin, 2-S-cysteinyldopaenkephalin and
[d-Ala2, d-Leu5]enkephalin) have free radicals
scaven-ging activities and the capacity to reduce ROS-induced
lipid peroxidation [24–26]
Endomorphin 1 (EM1) and endomorphin 2
(EM2), endogenous opioid peptides, have been found
in much higher amounts in the human brain [27]
The two peptides differ in one amino acid: EM1
(Tyr-Pro-Trp-Phe-NH2) and EM2
(Tyr-Pro-Phe-Phe-NH2) The major effect of the endomorphins is their
antinociceptive action [28,29] Additionally,
endomor-phins can cause vasodilatation by stimulating nitric
oxide release from the endothelium [30] and bind
to l-opioid receptors to activate G-proteins, regulate
gastrointestinal transit, respiratory system and
mem-ory [28,31–33] Endomorphins have been investigated
to modulate damage related to inflammatory diseases
of the brain [34] More recently, we have found that
endomorphins can scavenge radical, and inhibit lipid
peroxidation, DNA and protein oxidative damage
[35] It is worth to note that EM1 is more potent
than EM2 and l-glutathione (GSH) Therefore, it is
worthy to see if the antioxidant activity of
endomor-phins is also valid in LDL
The present study investigates the preventive effects
of endomorphins against Cu2+- and a water-soluble initiator 2,2¢-azobis(2-amidinopropane hydrochloride) (AAPH)-induced human LDL lipid peroxidation
in vitro, the characteristics of which have been exten-sively investigated The effects of endomorphins were compared to those of GSH It is suggested that endo-morphins and especially EM1 may provide antioxidant defence in neurodegenerative disorders
Results
Inhibition of conjugated diene formation
by EM1 and EM2
A set of representative kinetic curves of conjugated dienes formation during the peroxidation of LDL is shown in Fig 1 It is seen from Fig 1 that in the absence of exogenous antioxidants, conjugated diene formation was still inhibited for about 20 min (Fig 1, line a), demonstrating the presence of endogenous anti-oxidants in LDL) for example, a-tocopherol, carote-noids, ubiquinol-10 [36]) which can trap the initiating and⁄ or propagating radicals to inhibit peroxidation After the inhibition period, absorbance at 234 nm increased fast with time upon Cu2+-initiation, indica-ting depletion of the endogenous antioxidants and fast peroxidation of LDL The change in absorbance was inhibited by addition of EM1 (1.25–5 lm), EM2 (5 lm) and GSH (5 lm) in the inhibition period After
Fig 1 Formation of conjugated dienes during the peroxidation of LDL at pH 7.4 and 37 C, initiated with Cu 2+ and inhibited by endo-morphins and GSH [LDL] ¼ 0.2 mg proteinÆmL)1; [Cu2+] ¼ 10 l M
a, uninhibited peroxidation; b, inhibited with 1.25 l M EM1; c, inhib-ited with 2.5 l M EM1; d, inhibited with 5 l M EM1; e, inhibited with
5 l M EM2; f, inhibited with 5 l M GSH The results are representa-tive of three independent experiments.
Trang 3the inhibition period the rate of the absorbance
increase became faster, which is close to the original
rate of propagation, demonstrating the exhaustion of
the antioxidant The kinetic data deduced from Fig 1
are listed in Table 1 It is shown in Fig 1 and Table 1
that the inhibition period tinhwas prolonged in a
dose-dependent fashion for EM1, resulting an about 1.6-,
2.8- and 4-fold longer interval than that of the control
at 1.25, 2.5 and 5 lm drug, respectively On the basis
of tinh, Rinh and Rp, the antioxidant activity follows
the sequence EM1 > GSH > EM2
Inhibition of TBARS formation by EM1 and EM2
Figure 2 shows the inhibition of thiobarbituric acid
reactive substance (TBARS) formation by EM1, EM2
and GSH during the Cu2+-induced peroxidation of LDL After 60 min incubation at 37C, 18 nmol TBARSÆmg)1 LDL protein were generated in control experiments; TBARS were instead 15.26, 11.16, 3.84 and 2.06 nmolÆmg)1 LDL protein in the presence of 0.625, 1.25, 2.5 and 5 lm EM1 It is clearly seen that EM1 significantly suppressed rate of TBARS forma-tion and increased the inhibiforma-tion period in a concentra-tion- and time-dependent manner EM2 and GSH show similar inhibitory activity Figure 3 indicates the inhibition of TBARS formation by EM1, EM2 and GSH during AAPH-induced peroxidation of LDL It was found that addition of EM1 inhibited TBARS for-mation in a dose-dependent manner EM2 and GSH also diminished the rate of TBARS formation and increased the inhibition period During both Cu2+ -and AAPH-induced peroxidation of LDL, the activity sequence is EM1>GSH>EM2
Inhibition of apoB carbonyl formation
by EM1 and EM2 Oxidative damage to protein results in the formation
of protein carbonyl groups [37] Therefore, we meas-ured the inhibitory effects of EM1, EM2 and GSH
on either Cu2+- or AAPH-induced LDL oxidation
by apoB carbonyl assay As shown in Fig 4,
Cu2+-induced apoB carbonyl formation were inhibited about 55, 41 and 35.7% at 10, 20 and 40 lm EM1, respectively At the same experimental concentrations, either EM2 or GSH were also effective During AAPH-induced LDL oxidation, the carbonyl content
Table 1 Kinetic parameters for the Cu 2+ -induced peroxidation of
LDL and its inhibition by endomorphins and GSH The reaction
con-ditions and the initial concentration of the substrates are the same
as described in the legends of Fig 1 for reactions conducted in
LDL Data are the means of three determinations.
Compounds tinh(min) Rp(10)2min)1) Rinh(10)3min)1)
EM1 (1.25 l M ) 31.9 ± 1.8** 2.3 ± 0.2 1.1 ± 0.1
EM1 (2.50 l M ) 56.6 ± 4.2** 1.9 ± 0.2 1.1 ± 0.1
EM1 (5.00 l M ) 80.6 ± 4.7** 1.5 ± 0.1 0.9 ± 0.1
EM2 (5.00 l M ) 22.5 ± 1.4 2.4 ± 0.2 3.6 ± 0.2
GSH (5.00 l M ) 54.4 ± 3.8** 2.2 ± 0.2 1.0 ± 0.1
**P < 0.01 compared with the control.
Fig 2 Inhibition of TBARS formation during the Cu 2+ -induced
per-oxidation of LDL by endomorphins and GSH at 37 C [LDL] ¼
0.2 mg protein ⁄ mL; [Cu 2+
] ¼ 10 l M a, uninhibited peroxidation; b, inhibited with 0.625 l M EM1; c, inhibited with 1.25 l M EM1; d,
inhibited with 2.5 l M EM1; e, inhibited with 5 l M EM1; f, inhibited
with 5 l M EM2; g, inhibited with 5 l M GSH Values are mean ± SE
(n ¼ 3).
Fig 3 Inhibition of TBARS formation during the AAPH-induced per-oxidation of LDL by endomorphins and GSH at 37 C [LDL] ¼ 0.5 mg proteinÆmL)1; [AAPH] ¼ 20 m M a, uninhibited peroxidation;
b, inhibited with 10 l M EM1; c, inhibited with 20 l M EM1; d, inhib-ited with 40 l M EM1; e, inhibited with 40 l M EM2; f, inhibited with
40 l M GSH Values are mean ± SE (n ¼ 3).
Trang 4of apoB was reduced by the addition of EM1, EM2
and GSH in a concentration-dependent manner
(Fig 5) EM1 is more potent than EM2 and GSH,
similar to that observed in the LDL oxidation
prod-ucts assay mentioned above
Inhibition of apoB fragmentation by EM1 and
EM2
Figures 6 and 7 show gel electrophoresis of apoB
trea-ted with EM1, EM2 and GSH in the presence of
10 lm Cu2+ or 20 mm AAPH at 37C for 24 h,
respectively The spot of apoB was observed in native LDL (Fig 6, lane 1), but a degradation pattern of apoB was observed when LDL was incubated with
Cu2+ (Fig 6, lane 2) Compared to the Cu2+-treated band in lane 2, a discernible increase in the intensity of bands was noted with the addition of 10, 20 and
40 lm EM1 (Fig 6A, lane 3–5) Treatment of apoB with 40 lm EM1, EM2 and GSH in the presence of
10 lm Cu2+ decreased the extent of apoB fragmenta-tion (Fig 6B) As shown in Fig 7A, when LDL was
Fig 4 Protection of apoB carbonyl formation during the
Cu 2+ -induced peroxidation of LDL by endomorphins and GSH LDL
(0.5 mg proteinÆmL)1) in NaCl ⁄ P i was incubated with 10 l M Cu 2+
and ⁄ or compounds at 37 C After incubation for 6 h, carbonyl
content was measured as described in Experimental procedures.
Values are mean ± SE (n ¼ 3) *P < 0.05 compared with GSH.
Fig 5 Protection of apoB carbonyl formation during the
AAPH-induced peroxidation of LDL by endomorphins and GSH LDL
(0.5 mg proteinÆmL)1) in NaCl ⁄ P i was incubated with 20 m M AAPH
and ⁄ or compounds at 37 C After incubation for 6 h, carbonyl
content was measured as described in Experimental procedures.
Values are the mean ± SE (n ¼ 3) *P < 0.05 compared with GSH.
B
Fig 6 SDS ⁄ PAGE of apoB fragmentation induced by Cu 2+ and inhibited by endomorphins and GSH LDL (1.5 mg proteinÆmL)1) was incubated with 10 l M Cu 2+ and ⁄ or compounds in NaCl ⁄ P i
(pH 7.4) for 24 h at 37 C (A) apoB fragmentation was inhibited by EM1 in the presence of 10 l M Cu 2+ Lane 1, native LDL; lane 2,
0 l M ; lane 3, 10 l M EM1; lane 4, 20 l M EM1; lane 5, 40 l M EM1 (B) apoB fragmentation was inhibited by compounds in the pres-ence of 10 l M Cu 2+ Lane 1, native LDL; lane 2, 0 l M ; lane 3,
40 l M EM1; lane 4, 40 l M EM2; lane 5, 40 l M GSH.
B
Fig 7 SDS ⁄ PAGE of apoB fragmentation induced by AAPH and inhibited by endomorphins and GSH LDL (1.5 mg proteinÆmL)1) was incubated with 20 m M AAPH and⁄ or compounds in NaCl ⁄ P i
(pH 7.4) for 24 h at 37 C (A) apoB fragmentation was inhibited by EM1 in the presence of 20 m M AAPH Lane 1, native LDL; lane 2,
0 l M ; lane 3, 50 l M EM1; lane 4, 100 l M EM1; lane 5, 200 l M
EM1 (B) apoB fragmentation was inhibited by compounds in the presence of 20 m M AAPH Lane 1, native LDL; lane 2, 0 l M ; lane
3, 200 l M EM1; lane 4, 200 l M EM2; lane 5, 200 l M GSH.
Trang 5incubated with EM1 in the presence of 20 mm AAPH
at 37C for 24 h, a concentration-dependent increase
in the intensity of apoB bands was observed
More-over, we investigated the protective effects of 200 lm
EM1, EM2 and GSH against 20 mm AAPH-induced
apoB fragmentation (Fig 7B) On the basis of the
intensity of bands, the protective activity follows the
sequence of EM1 > GSH > EM2
Inhibition of REM by EM1 and EM2
Using the same sample solutions as used for protein
carbonyl analysis, we performed relative
electropho-retic mobility (REM) studies Figure 8A indicates the
protective effects of EM1, EM2 and GSH against
Cu2+-induced LDL oxidation In the presence of 40 or
20 lm EM1, 10 lm Cu2+caused only a 24.5 or 49.4%
increase in REM, respectively, but in the presence of
40 lm EM2 or GSH, 10 lm Cu2+ caused a 95.4 or 66.2% increase in REM, respectively Thus, EM1 is at least twice or three times as effective as GSH or EM2 in protecting against Cu2+-induced apoB modification in LDL As shown in Fig 8B, exposure of LDL to 20 mm AAPH in the presence of an increasing concentration of EM1 (50–200 lm) resulted in a dose-dependent decrease
in REM However, EM2 and GSH were not able to prevent AAPH-induced apoB modification in LDL
Discussion
It is well established that oxidative stress is not simply
a by-product of degenerative processes or the end product of nerve cell death but may directly initiate neurodegeneration Although oxLDL has been studied primarily for its role in the development of atheroscler-osis, recent studies have identified that oxidative modi-fication of LDL is capable of eliciting cytotoxicity, differentiation, and inflammation in neuronal cells [10– 12,38] Suppression of oxidative modification of LDL
by antioxidants may be effective in preventing and treating neurodegenerative diseases [8,16] In the pre-sent study, two different initiating assays were used One is copper which is one of the major potential sources of free radical production in the brain [39]
Cu2+-induced LDL peroxidation is generally believed
to involve reductive activation of Cu2+ as the first stage The reductive activation may be accomplished
by a net transfer of one electron to produce Cu+ which is a strong pro-oxidant and can rapidly generate the ultimate initiating radicals by a Fenton-type reac-tion with peroxides or by forming an electron transfer complex with molecular oxygen Another is AAPH, a water-soluble initiator, which decomposes at physiolo-gical temperature producing alkyl radicals (R•) fol-lowed by fast reaction with oxygen to give alkyl peroxyl radicals (ROO•) to initiate LDL peroxidation (LOO•) In the presence of an antioxidant molecule,
AH, either the initiating peroxyl radical and⁄ or the propagating lipid peroxyl radical can be trapped and a new antioxidant radical, A•, produced If the A• is a stabilized radical (e.g a-tocopheroxyl radical or ascor-bate radical) which can promote the rate-limiting hydrogen abstraction reactions and undergo fast ter-mination reactions, the peroxidation would be inhib-ited [40]
The formation of lipid peroxidation products is a phenomenon common in most types of neurone damage associated with oxidative stress [7,10] Cu2+ -induced LDL peroxidation is generally monitored by
UV spectroscopy since the primary peroxidation
Fig 8 Inhibition of the increase in REM of LDL during the
Cu 2+ -induced (A) or AAPH-initiated (B) peroxidation of LDL by
endo-morphins and GSH LDL (0.5 mg proteinÆmL)1) in NaCl ⁄ P i was
incu-bated with 10 l M Cu 2+ or 20 m M AAPH and ⁄ or compounds at
37 C After incubation for 6 h, REM of LDL was measured as
described in Experimental procedures Values are the mean ± SE
(n ¼ 6) *P < 0.05 and **P < 0.01 compared with GSH, respectively.
Trang 6products of polyunsaturated fatty acids in LDL are
hydroperoxides possessing a conjugated diene structure
which shows characteristic UV absorption at 234 nm
[41] The rate of the chain propagation, Rp, the inhibited
rate of propagation by antioxidants, Rinh, and the
inhi-bition period, tinh, can be easily obtained from
spectro-photometric data In the present work, we can find from
Fig 1 and Table 1 that on the basis of tinh, Rinh and
Rp, the antioxidant activity follows the sequence
EM1 > GSH > EM2 in conjugated diene formation
Moreover, TBARS formation is also used to detect lipid
peroxidation products in LDL oxidation It is clearly
seen by comparison of Fig 1 with Figs 2 and 3 that the
antioxidant activity follows the same sequence in spite
of the activity being monitored by conjugated diene
formation or by TBARS production, and in spite of
the peroxidation being initiated by Cu2+or by peroxyl
radicals (generated by AAPH)
Increased tissue protein carbonyls have been detected
in numerous human diseases, such as rheumatoid
arth-ritis, ischaemia–reperfusion injury to heart muscle and
Alzheimer’s disease [42] Protein carbonyl formation is
a biomarker of protein oxidation and has some
advan-tages over lipid peroxidation products because the
for-mation of protein-bound carbonyl groups seems to be
a common phenomenon of protein oxidation, and
because of the relatively early formation and relative
stability of oxidized proteins [42] As shown in Figs 4
and 5, EM1 is more active than EM2 and GSH In
addition, the present study also showed that
endomor-phins inhibited not only the lipid peroxidation of LDL
but also the fragmentation of apoB of LDL and
increase in REM in a concentration-dependent manner
The results presented in this paper provide evidence
that endomorphins) endogenous opioid peptides in
the brain) are very efficient in protecting LDL
against Cu2+- and AAPH-induced oxidative
modifica-tion The inhibitory activity of EM1 is much more
effective than that of tested EM2 and GSH, a major
intracellular water-soluble antioxidant It is obvious
that these micromolar in vitro concentrations are
signi-ficantly higher than endomorphins levels normally
detected in the body Nevertheless, the following
should be taken into account Firstly, to reach a
pro-nounced and well-detectable oxidative damage in vitro,
one has to use rather high concentrations of oxidants
in vitro and, consequently, high concentrations of
endomorphins are necessary Secondly, recent studies
have demonstrated that endogenous opioid peptides
are released from cells during inflammation and stress,
and reach high levels at theses sites Finally, drugs for
effective prevention of damage or treatment may reach
pharmacological levels rather than physiological
concentrations Moreover, the general biological activ-ities of endomorphins act through l-opioid receptors, whereas the antioxidant activity of endomorphins is not dependent on opioid receptors Very probably the neuroprotective effects of endomorphins result from a combination of the different modes of action
In the case of Cu2+-induced LDL oxida-tion) where lipid peroxyl radicals are generated indi-rectly from a series of redox reactions) EM1 had no apparent copper-binding effect, as judged by both spectral study and lack of quenching of the intrinsic drug absorption by copper (data not shown) Our results indicate that EM1 can trap the lipid peroxyl radicals (LOO•) derived indirectly from copper on the surface of LDL particles and behave well as chain breaking antioxidant against Cu2+-induced LDL per-oxidation Furthermore, Cu2+-induced LDL peroxida-tion is considered to be more relevant to the in vivo situation than the AAPH-induced peroxidation, since the former most likely involves a site-specific attack of the apoB, whereas the latter produces a more-or-less random attack of free radicals in LDL Also, oxida-tively modified of LDL by Cu2+ exhibits biological properties very similar, if not identical, to those of cell-oxidized LDL In addition, a growing body of data supports a significant role for redox active metals,
Cu, as key modulators of the pathogenic pathways that underlie neurodegenerative disorders and oxLDL-mediated neuronal damage in vivo, would depend on the availability of Cu [39,43]
Recently, we have reported that endomorphins can directly scavenge galvinoxyl radicals and AAPH-derived alkyl peroxyl radicals [35] The difference of EM1 and EM2 is primarily Trp and Phe at position 3 Trp does not possess phenolic hydrogens and only has
an indole ring, similar to melatonin ) a hormone mainly secreted by the pineal gland) which has been reported to be able to protect from oxidative damage
in the central nervous system [18] However, free amino acids such as Trp and Phe cannot react with galvinoxyl radicals (data not shown) Therefore, it is suggested that both the indole ring on the Trp as well
as the side chain on the indole nucleus are essential for the antioxidant activity of EM1 The most active hydrogen of EM1 might be H-10 on the Trp, which is
an allylic hydrogen It is well known that allylic hydro-gens are very active and easily abstracted by free radi-cals In addition, conjugation with -NH on the indole shall further weaken the C–H-10 bond The mechanis-tic details are worthy of further study
Lipid peroxidation is increased in neurophathologi-cal conditions such as Alzheimer’s and Parkinson disease [10] Recent studies have reported that oxLDL
Trang 7is cytotoxic to neurones and application of
antioxi-dants may attenuate neurone death Thus, inhibition
of LDL peroxidation by antioxidants becomes an
attractive therapeutic strategy to prevent and treat
neurodegenerative diseases This has led to a great deal
of research devoted to the prevention of lipid
peroxi-dation in LDL by antioxidants [8,17] However, the
great pharmacological disadvantages of many
antioxi-dants are their very limited passage through the
blood–brain barrier [14] Therefore, the existence of
antioxidants in the brain protective systems or with
much better blood–brain barrier permeation may be
essential The recently demonstrated powerful
antioxid-ant activities of certain amines and imines may be the
starting point for developing neuroprotective
anti-oxidants [44] Our data demonstrate that
endomor-phins may inhibit the formation of oxLDL and thus
minimize subsequent oxLDL-induced toxicity We
pro-pose that the neuroprotective activity of endomorphins
may provide new insights into therapeutics of
neuro-degenerative diseases and a new understanding for
oxidative stress in the brain
Experimental procedures
Chemicals
EM1 and EM2 were synthesized in our laboratory [45] The
purity of the compounds was determined by HPLC
(> 95%) and their structures were verified by MS and
amino acid analysis Agarose, 2,4-dinitrophenylhydrazine
(DNPH), thiobarbituric acid (TBA) and GSH were from
Sigma (St Louis, MO) AAPH and butylated
hydroxytolu-ene (BHT) were from Aldrich (Milwaukee, WI) Sudan
Black B, Acrylamide and bis-acrylamide were from BBI
(Markham, CA) All other chemicals were of the highest
quality available
LDL isolation
Blood collected into the anticoagulant EDTA (final
concen-tration 3 mm) was taken from healthy volunteers LDL
(1.019–1.063 gÆmL)1) was isolated from the plasma by a
discontinuous density gradient centrifugation procedure as
described elsewhere [41] at 140 000 g for 6 h using a
HITA-CHI 55P-72 ultracentrifuge in KBr solution at 4C in the
presence of EDTA (100 lm) The isolated LDL fraction
was then dialysed with phosphate buffer (NaCl⁄ PipH 7.4)
containing 100 lm EDTA to prevent oxidation during
dialysis EDTA was removed by dialysis with NaCl⁄ Piprior
to the oxidation experiments The concentration of protein
was determined by the method of Lowry et al [46] LDL
was stored in the dark at 4C and used within 1 week
Determination of conjugated dienes The ability of endomorphins to inhibit Cu2+-mediated LDL oxidation was evaluated in quartz cuvettes through continuous spectrophotometric monitoring of absorbance increase at 234 nm, reflecting conjugated diene formation during peroxidative processes [41] LDL (0.2 mg pro-teinÆmL-1) was incubated at 37C using a Shimadazu (Kyoto, Japan) model UV-260 spectrophotometer Oxida-tion was initiated by 10 lm CuSO4 EM1, EM2 and GSH were added to inhibit the peroxidation The absorbance at
234 nm was measured every 5 min against appropriate ref-erence cuvettes for the duration of the experiment Every experiment was repeated three times and the results were reproducible within 10% experimental deviation
Determination of TBARS The formation of TBARS was used to monitor lipid per-oxidation [47] LDL (0.2 or 0.5 mg proteinÆmL)1) was incubated at 37C in NaCl ⁄ Pi, pH 7.4 The peroxidation was initiated by either 10 lm Cu2+ or 20 mm AAPH and inhibited by EM1, EM2 and GSH The reaction mixture was gently shaken at 37C and aliquots of the reaction mixture were taken out at specific intervals to which a tricholoroacetic acid⁄ TBA ⁄ HCl stock solution (15% w⁄ v trichloroacetic acid; 0.375% w ⁄ v TBA; 0.25 m HCl) was added, together with 0.02% w⁄ v BHT This amount of BHT completely prevents the formation of any nonspecific TBARS, as well as preventing decomposi-tion of AAPH during the subsequent boiling The solu-tion was heated in a boiling water bath for 15 min After cooling, the precipitate was removed by centrifugation TBARS in the supernatant was determined at 532 nm Results were calculated as nmol TBARSÆmg LDL pro-tein)1, using a molar extinction coefficient of
156 000 m)1Æcm)1 [47]
Determination of apoB carbonylation ApoB carbonyls were measured spectrophotometrically with the use of the carbonyl specific reagent DNPH as pre-viously reported [48] Briefly, LDL (0.5 mg proteinÆmL)1) was incubated with either 10 lm Cu2+ or 20 mm AAPH, with or without different concentration of EM1, EM2 and GSH After incubation for 6 h at 37C, 0.5 mL 10 mm DNPH in 2 N HCl was added to 1 mL of the incubation mixture and incubated at room temperature for 1 h Fol-lowing addition of 0.5 mL 20% tricholoroacetic acid, the samples were incubated on ice for 10 min and centrifuged
at 4000 g for 10 min Protein pellets were washed three times with 3 mL ethanol⁄ ethyl acetate (1 : 1, v ⁄ v) and dis-solved in 6 m guanidine (pH 2.3) The peak absorbance at
370 nm was used to quantify protein carbonyls
Trang 8Determination of apoB fragmentation
The measurement apoB fragmentation was performed by
vertical electrophoresis as described previously [49] using
a 7.5% SDS⁄ PAGE at a constant current of 20 mA for
90 min Oxidation of LDL (1.5 mg proteinÆmL)1) in
NaCl⁄ Pi was initiated by either 10 lm Cu2+ or 20 mm
AAPH and inhibited by EM1, EM2 and GSH After
incubation for 24 h at 37C, 1 mm EDTA or 0.02%
(w⁄ v) BHT was added to the reaction mixture to prevent
further oxidation, respectively Then, the samples were
mixed with an equal volume of 2· SDS ⁄ PAGE sample
buffer (100 mm Tris⁄ HCl pH 6.8, 4% SDS, 20% glycerol,
10% b-mercaptoethanol, 0.01% Bromophenol blue),
heated at 100C for 5 min and loaded onto a 7.5%
acrylamide SDS-containing gel The gels were stained
with 0.05% Coomassie Brilliant Blue R-250 and
photo-graphed
Determination of REM
The negative surface charge of apoB was determined by
agarose gel electrophoresis as described previously [49]
LDL (0.5 mg proteinÆmL)1) was incubated with either
10 lm Cu2+ or 20 mm AAPH, with or without different
concentration of EM1, EM2 and GSH After incubation
for 6 h at 37C, the samples were examined by
electro-phoresis at 100 V for 30 min in 50 mm barbital buffer
(pH 8.6) on 0.5% agarose gels and stained with Sudan
Black B The REM was defined as the ratio of migrating
distance of oxidized LDL to that of native LDL
Statistical analysis
Results are expressed as mean ± SE For most
experi-ments, mean values were compared using Student’s t-test
to evaluate statistical differences In the figures, symbols *
and ** indicate P < 0.05 and P < 0.01, respectively
Acknowledgements
We thank the National Natural Science Foundation of
China (no 20372028), the Ministry of Science and
Technology (no 2002CCC00600 and 2003AA2Z3540),
the Teaching and Research Award Program for
Out-standing Young Teachers, and Specialized Research
Fund for the Doctoral Program in Higher Education
Institution of the Ministry of Education of China for
financial support
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