Figure 1 The reduction of LPS-induced TNF-α from immune cells by pentobarbital.
(A). P338D1 cells were treated with LPS (the LPS group). In the same condition,
P338D1 cells were co-incubated with pentobarbital (the LPS+8.75μg/ml and the
LPS+12.5μg/ml groups). The growth medium of untreated p338D1 cells served as the
control group. *: p < 0.05 indicates a significant difference between the LPS+8.75μg/ml
group and the LPS group，**: p < 0.01 indicates a significant difference between the
33
LPS+12.5μg/ml group the LPS group. (B). Splenocytes were treated with LPS (the LPS
group). In the same condition, splenocytes were co-incubated with pentobarbital (the
LPS+12.5μg/ml groups). The growth medium of untreated splenocytes served as the
control group; *: p < 0.05 indicates a significant difference between the LPS+Pento
group and the LPS group.
34
Figure 2 The effect of pentobarbital on the expression of TNF-α mRNA. P338D1 cells were treated with or without LPS and were co-incubated with or without pentobarbital. The mRNA levels of TNF-α were shown by RT-PCR. β-actin mRNA
levels served as an internal control to normalize the sample loading.
35
Figure 3 The effect of pentobarbital on the production of TNF-α in vivo. After LPS infusion, the levels of TNF-α (A) and nitric oxide (B) in sera of the mice were measured.
The untreated mice served as the negative control (the NS group). (*: P<0.05 indicates a significant difference between the Pento group and the NS group. +: P<0.05 indicates a
significant difference between the Pento group and the LPS group.) (C)The body
temperature of the rats was measured rectally by a digital thermometer. The untreated
mice served as the negative control (the NS group).
36
Figure 4 The effect of pentobarbital on the activities of NF-κB or AP-1 after LPS treatment. (A) pNF-κB/hrGFPor (B) pAP-1/hrGFP plasmids were transfected into Balb-3T3 cells. The untreated group was the control
group. Transfectants were treated with LPS and co-incubated with (the
LPS/pentobarbital group) or without (the LPS group) 12.5 μg/ml pentobarbital.
Transfectants were also incubated with growth medium containing 12.5 μg/ml
pentobarbital without LPS (the pentobarbital group). (*: p < 0.05 indicates a significant difference between the LPS+Pento or the Pento groups between the LPS
group.)
Figure 5
38
Figure 6 Improvement of blood biochemical factors reflecting multiple organ functions under pentobarbital anesthesia. Plasma levels of ALT (a), AST (b), LDH (c), CPK (d), BUN (e) and amylase (f) levels of rats in the Pento group, the LPS group and negative control group were measured. (*: P<0.05 indicates a significant difference
between the concentration of the Pento group and that of the NS group. +: P<0.05 indicates a significant difference between the concentration of the Pento group and that
of the LPS group.)
39
Figure 7 The protection of HEK293cells against TNF-α cytotoxicity by pentobarbital. HEK293 cells in the normal growth medium served as the control group. HEK293 cells were treated with or without TNF-α and
co-incubated without or with different dosages of pentobarbital. (*p < 0.05 indicates the
relative survival significantly differed from that of the TNF-α-treated group.)
40
Figure 8 The protection of 293 cells against apoptosis by pentobarbital under hypoxia. The normal medium cultured HEK293cells served as the control group (A). The HEK293cells were treated with 10 mM
DFO and co-incubated without (B). or with pentobarbital (C). The treated
cells were probed with Annexin V-FITC (FL-1) and PI (FL-3), and analyzed by
flow cytometer. The relative apoptosis index was calculated and shown in (D). (*: p <
0.05 indicates a significant difference between the DFO+P group and the DFO-treated
group.)
41
Part II
The Treatment of Propofol Induced the TGF-β1 Expression in Human Endothelial Cells to Suppress Endocytosis Activities of Monocytes
42
Chapter 7 Introductions (2)
7.1 Propofol
7.1.1 The chemical properties of propofol
Propofol is the most recently intravenous an aesthetics in clinical use. Propofol is
approved for the induction and maintenance of anesthesia in more than 50 countries.
The commonly used brand name is Diprivan. Another commonly used name is
disoprofol. The molecular formula of propofol is C12H18O; molecular weight is 178.271
g/mol; and systematic (IUPAC) name is 2, 6-diisopropylphenol or 2, 6-Biss
(1-methylethyl) phenol (Fig.1A). The molecular models of propofol were shown in
Fig.1B [107], propofol is a water-immiscible oil but soluble in many organic solvents
like DMSO (dimethyl sulfoxide) or ethanol.
7.1.2 The pharmaceutical properties of propofol
Due to propofol is not soluble in water and cannot be injected only by itself. Initial
clinical trials were in 1977, in a form solving in cremophor EL. But cremophor
formulation produced a marked increase in plasma histamine concentration; it was
withdrawn from the market. It was subsequently reformulated as an emulsion
formulation, and suggested that the emulsion formulation may produce less discomfort
on i.v. injection based on animal test [108]. It was re-launched in 1986 by AstraZeneca
with the brand name Diprivan. The current preparation is 1% propofol, 1.2% purified
43
egg phospholipid, 2.25% of glycerol, 10% soybean oil (emulsifier). In the United States,
the products contain disodium edetate (0.005%) as a microbial growth retardant to
inhibit the growth of microorganism in the event of accidental contamination.
Propofol is a short-acting intravenous anesthetic agent used for the induction of
general anesthesia in adult patients and pediatric patients older than 3 years of age;
maintenance of general anesthesia in adult patients and pediatric patients older than 2
months of age; and sedation in medical procedure. Propofol is rapidly and extensively
distributed in the body. It crosses the blood-brain barrier quickly, and its short duration
of action is due to rapid redistribution from the CNS to other tissues, high metabolic
clearance. The elimination half-life of propofol has been estimated to be between 2–24 hours. The therapeutic concentrations of propofol in plasma is 1.5 to 6.5 μg/mL for
anesthesia [109, 110]; 0.14 to 1.92 μg/mL for sedation [111] and 0.170-0.437 μg/mL for
postoperative nausea and vomiting [112].
The side/adverse effects of propofol used as anesthetic agents clinically have been
reported according to Apnea, bradycardia, bronchospasm, erythema, hypotension and so
on [113, 114].
7.1.3 The mechanism of propofol anesthetic action
Its mechanism of anesthetic action has not been well-defined. The primary target
of propofol act which been identified is the GABAA receptor [115]. Recent research has
44
also suggested propofol activate cannabinoid receptors in the endocannabinoid system
may contribute significantly to propofol's anesthetic action [107]. There are three
classes of GABA receptors: GABAA, GABAB, and GABAС. GABAA and GABAС
receptors are ligand-gated ion channels, whereas GABAB receptors are G
protein-coupled receptors.
The GABAA receptors is formed a pentamer composed of α1β2γ2 subunits.
GABAA receptor channel is the most abundant, fast inhibitory, ligand-gated ion channel
and is found throughout the central nervous system in the mammalian brain. The
GABAA receptors mediate inhibitory neurotransmission in the central nervous system.
Under physiological conditions, the receptors are activated by GABA, but several other
compounds such as neurosteroids and barbiturates can also gate the channel. The
GABAA receptorsare a major target for drugs used for the induction and maintenanceof
general anesthesia and for the treatment of anxiety and epilepsy [116, 117].
Most volatile and intravenous anesthetics including propofol enhance the activity
of gamma-aminobutyric acid type A (GABAA) receptors and directly activate this
ligand-gated chloride ion channel and, hence, increases the chloride conductance in the
absence of its endogenous ligand, GABA. The current findings suggest that first transmembrane domain glycine 219 residue of the β2 subunit is critical for the rate at
which desensitization occurs and that both GABA and intravenous anesthetics
45
implement an analogous pathway for generating desensitization [118]. Additionally,
anesthetics are known to prolong GABA-induced Cl- channel opening, and depending
on the type of anesthetic, this potentiation of GABA-gated currents appears to alter
deactivation and/or desensitization. For example, halothane, a volatile anesthetic, has
been shown to slow the dissociation of GABA from its receptor to slow deactivation.
However, Propofol can slow both the rate of deactivation and the rate of recovery from
desensitization by stabilizing the ligand-binding structure of GABAA receptors [119,
120].
7.1.4 The effects of propofol on immune response
Previous studies have demonstrated that propofol might have immune-modulating
effects on various types of human immune cells like leukocytes, neutrophils and
macrophage.
7.1.4.a The effects of propofol on leukocytes
There are researches which had showed that propofol is able to reduce significantly
the migration of leukocytes through endothelial cell monolayers with a dose-dependent
effect. These interactions between leukocytes and endothelial cells play a critical role
during inflammatory processes to defense microorganisms. Leukocytes attack invading
microorganisms after migrating from the intravascular space into tissue. To arrive at the
extra-vascular tissue, leukocytes migrate through a monolayer of endothelial cells.
46
During this migration, leukocytes undergo morphologic changes from rounded,
relatively smooth cells to elongated, ruffled cells with pseudopodia. The results of the
investigation indicate that the influence of propofol both on leukocytes and endothelial
cell monolayers [46]. The results would imply that propofol could inhibit
leukocytes-dependent immune defense by acting directly on leukocytes.
7.1.4.b The effects of propofol on neutrophils
Neutrophils are the most numerous one of leukocytes. Neutrophils play a crucial
role in the early line of anti-bacterial host defense mechanism as a component of
nonspecific cell-mediated immunity. The neutrophi1 response to microbial invasion
consists of chemotaxis, adherence, phagocytosis, and intracellular killing. Propofol
significantly inhibited reactive oxygen species [22], chemotaxis, and phagocytosis of
neutrophils in a dose-dependent manner. A clinically relevant concentration of propofol
suppressed these neutrophils functions, this impairment observed in vitro, implying that
leads to clinical immunological suppression during medical procedures [39]. Increase in
intracellular calcium concentrations in neutrophils stimulated by
N-formyl-L-methionyl-L-leucyl-L-phenylalanine was dose-dependent attenuated by
propofol. This decreasing effect on intracellular calcium in neutrophils may be one of
the mechanisms responsible for the propofol-induced suppression of chemotaxis and
phagocytosis. Furthermore, the increase in intracellular Ca2+ of neutrophils is thought to
47
be one important pathway by which extracellular stimuli are transmitted to activate
enzymes responsible for the production of O2- (nicotineamide adenine dinucleotide
phosphate oxidase) in the cell membrane. However, the ROS produced by neutrophils
that accumulate in various organs is thought to play a critical role in the pathogenesis of
endotoxin-induced multiple organ failure. The ability of propofol to decrease neutrophi1
functions could be beneficial in particular situation.
7.1.4.c The effects of propofol on macrophages
Moreover, a clinically relevant concentration of propofol can suppress mouse
macrophage-like Raw 264.7 cells functions of chemotaxis, phagocytosis, oxidant production, and IFN- γ mRNA synthesis in concentrationand time-dependent manners,
possibly through inhibiting their mitochondrial membrane potential and adenosine
triphosphate synthesis but did not affect cell viability [121]. Another evidence was
showed that the number of phagocytic cells (ingesting at least one particle) of
patient-derived alveolar macrophage would significantly decreased in 4h after induction
of anesthesia [37]. Despite of many researches showed that propofol may impair
macrophage functions; propofol anesthesia has also been reported to induce proinflammatory cytokines, including tumor necrosis factor- α, interferon-γ (IFN- γ),
interleukin-1β, and interleukin-8 in orthopedic surgery patients [16]. However, multiple
factors could be involved in modulating macrophage functions in the surgical
48
procedures, so that the ex vivo studies did not clarify whether propofol alone could
modulate macrophage activities. Therefore, an in vitro study will be needed to verify the
role of propofol in modulating macrophage functions to rule out the contribution of
other factors. Nevertheless, there are few articles emphasizing on the human
monocytes-like cells modulated by propofol within clinical dosages. This is one of our
interests and makes our efforts to in this study.
7.1.5 The effects of propofol on inflammation induced by LPS.
In addition to its action on immune cells by itself, propofol also have been shown
that it has anti-inflammatory and antioxidative effects on LPS-activated Raw 264.7 cell
[122] and endotoxemia rats [123]. In response to LPS stimulation, propofol has also been shown to inhibit INF-γ mRNA synthesis in macrophages. During inflammation,
macrophages destroy invaded microorganisms through a series of reactions, including
chemotaxis, phagocytosis, oxidant synthesis, and cytokine release. Dysfunction of these
activities will affect host macrophage-mediated immunity. Nitric oxide is an active
oxidant that contributes to the physiology and pathophysiology of macrophages. It has
been shown that propofol reduces nitric oxide biosynthesis in LPS-activated
macrophages by down-regulating the expression of inducible nitric oxide synthase [124].
The present study also shows that propofol, at a therapeutic concentration, has anti-inflammatory and antioxidative effects on the biosyntheses of TNF-α, IL-1β, IL-6,
49
and NO in LPS-activated Raw 264.7. Furthermore, propofol enhances LPS-stimulated TNF-α and IL-1β but reduces the LPS-stimulated IL-8 expression [19, 21]. However,
propofol also reduces the expression density of LPS-stimulated CD14 whereas
unaffecting HLA-DR [18].
According above, two arguments can be made in regards to the propofol effect on
the immune activity. There is no acceptable mechanism yet in regards to the effect of
propofol in the immune regulation and inflammation induced by LPS (Table 1).
7.1.6 The effects of propofol on endothelial cell
Being an intravenous agent, the effects of propofol on endothelial cell also should
be concerned. Propofol could reduce endotoxin-induced endothelial damages by
extracellular and intracellular mechanisms. By extracellular mechanism, propofol could
offers significant protection against endotoxin-induced pulmonary microvessel
endothelial cell injury by scavenging the active oxygen species released in the
extracellular space. By intracellular mechanism, propofol directly reduced the
LPS-enhanced iNOS mRNA and prevent LPS-induced cell barrier dysfunction by reducing transcription factor NF- κB protein levels. Propofol also directly protect the
endothelial cells reduces apoptosis by suppressing caspase-3activity and recover the
function in hydrogen peroxide-stimulated human umbilical vein endothelial cells [92,
125-128].
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7.2 TGF-β
7.2.1 Τhe family TGF-β
TGF-β purified from platelets, placenta, and kidney was subsequently named
TGF-β1, to distinguish it from two other highly homologous isoforms, TGF-β2 and
TGF-β3, which are interchangeable in a variety of biological assay, but which are
encode by distinct genes. TGF-β1 is the predominant isoform in most cells and tissues.
7.2.2 The biochemistry characteristic of TGF-β
Each of three mammalian isoforms of TGF-β are encodes as 390-442 amino acid
precursor proteins that contain s signal sequence and are processed proteolytically by
furin [129], a member of the mammalian convertase family of endoproteases. In mammalian cells, the presence of pre-domain is required for the proper folding and
secretion of TGF-β. TGF-β is secreted in noncovalent association with its own
pro-domain (latency- associated peptide, LAP) in a “latent” form unable to bind
receptors until activated. LAP contain N-linked glycosylation sites which be mutated would prevent the cellular secretion of TGF-β. The most critical posttranslational
modification of TGF-β is the proteolytic processing at an RXXR site by the
endoprotease furin resulting in release of this fragment with the remainder of the
biologically active 112 amino acid C-terminal domain and the subsequent noncovalent
association of the precursor called the latency-associated peptide, LAP (Fig.2). Secreted
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TGF-β is either in the form of “small” (100 kDa) latent complex consisting only of LAP
and mature TGF-β or a tertiary “large” (220 kDa) latent complex complex in which a
secretory glycoprotein, the latent TGF-β binding protein (LTBP) is covalently bound to
LAP through a disulfide bond from the third eight-cysteine repeat of the latter to the
N-terminal-most cysteine in LAP [130]. LTBPs are important both in section of TGF-β and in targeting it to extracellar matrix.
7.2.3 Activation of latent TGF-β
Activation of latent TGF-β has been studied intensively in vivo, where extremes of
pH, heat, protease or chaotropic agents have all been shown to release active TGF-β from the latent complex. These mechanisms include (1) a protease-independent
mechanism involving the binding of thrombospondin, a component of platelet α
granules and of extralcelluar matrix [131]; (2) proteolytic activation via
transreglutaminase and plasmin.plasminogen activator [132]; (3) binding of RGD sequence of the LAP of the latent complex to the αvβ6 [133]; (4) uptake and activation
of Fc receptors on macrophage of complexes of TGF-β bound to immunoglobulin [134];
(5) invasion of Candidiasis stimulates hepatocytes and monocytes to secrete active TGF-β, although the mechanism is unclear [135].
7.2.4 Cellular and tissue resource of TGF-β expression
Most cultured cells express at least one of the TGF-β isoforms. In many
52
mammalian cell types, all three isoforms are expressed significantly, while in other cell types one isoforms predominates. Immunoactive TGF-βs 1, 2, and 3 are present in
four-cell embryos through to the blastocyst stage and continue to be expressed in all tissues throughout development. For example, TGF-β1 mRNA is expressed in the
overlying epithelial cells. In contrast, TGF-β2 mRNA is often expressed in epithelium
of morphogenetically active tissues and epithelial cells that are undergoing
differentiation such as those in alveoli and palate, as well as superbasal keratinocytes.
TGF-β express widespread in the adult; most tissues express all three isoforms
with some differences in the expression level. The most abundant source of TGF-β1 in
humans are platelets, bone, and spleen; TGF-β2 predominates in fluids such as the
aqueous and vitreous of the eye, breast milk, and amniotic fluid.
7.2.5 The eliciting stimuli of TGF-β
The main regulators of TGF-β expression in vivo are changes in levels of steroid
hormone (either by addition of exogenous steroids or by manipulations to alter
endogenous levels), as well as cellular injury, stress, or viral/parasitic infections.
Hormone ablation or replacement strongly affects TGF-β expression. Retinoids have
strong modulating effects on the expression of TGF-β isoform in vivo [136]. For
example, β-Carotene treatment of patients with cervical intra-epithelial neoplasia leads
to an increase in immunoreactive TGF-β1 in these cells [137].
53
7.2.6 Signal transduction pathway of TGF-β
The signals of cellular responses to TGF-β are through specific
TGF-β transmembrane receptor type I and type II Ser/Thr kinase receptor. The signal
pathway initiated by TGF-β binding to the TGF-β type II receptor. After ligand
binding, TGF-β type II receptor recruits and phosphorylates the TGF-β type I receptor,
also termed activin receptor-like kinase (ALK family) [138]. This results in a conformation change and activation of the type I receptor which can propagate the
signal inside the cell to the nucleus by the phosphorylation of specific effectors [139].
TGF-β binds and transducts the signal through ALK-5 in most cells but through both
ALK-5 and ALK-1 in endothelial cells. The specific effectors that play an important role in TGF-β signal transduction form the membrane receptor to the nucleus are
called Smads. Smads (R-Smads) are recruited and phosphorylated after activation of
ALK. The activation of ALK5 induced the phosphorylation Smad2 and Smad3, and
activation of ALK1 induced the phosphorylation Smad1 and Smad5. Subsequently
Smad4 forms heteromeric complexes with R-Smads and these complexes then
translocate to the nucleus and regulated the target gene expression by collaboration
with transcription factors (TFs) and cofactors (coactivators or corepressors) (Fig3).
The end of signaling pathway is also important, and there are at least two identified
mechanism for the termination of Smad signaling. One is that dephosphorylation of
54
R-Smads by unidentified phophatases. The other is ubiquitination and protease-
dependent degradation of the activation R-Smads.
There are many TGF-β signaling pathway specific inhibitors at present (Table 3).
We choose SB 431542 hydrate to inhibit the TGF-β signaling pathway in this study.
SB 431542 inhibits the acitivity of (TGF-β1) activin receptor-like kinases (ALKs). It
is a selective and potent inhibitor of the phylogenetically related subset of ALK-4,
ALK-5, and ALK-7. Phosphorylation of Smad2 by ectopically expressed
constitutively active ALK-4, ALK-5, and ALK-7 in transfected NIH 3T3 cells is completely abolished by SB 431542 at 10 μm. In addition, the compound inhibits
TGF-β−induced Smad3 phosphorylation and nuclear localization. .
7.2.7 The biological function of TGF-β
TGF-β is a typically multi-functional protein. It may have one or several effects
TGF-β is a typically multi-functional protein. It may have one or several effects