Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The ischemia/reperfusion-induced injury of donor
lungs is an important pathological process which influences the
survival rate of donor lungs after transplantation. It is
believed that the injury is mainly caused by inflammatory
reactions[1]. The responses of ischemia/reperfusion-induced
inflammation include the induction of adhesion molecules,
infiltration of immune blood cells, and the release of cytokines.
NF-κB is a key regulator of many inflammatory
genes[2,3]. It plays an important role in modulating the expression of
varieties of inflammatory cytokines. Our previous studies have
reported that inhibiting NF-κB reduces the expression of
inflammatory and apoptotic genes and protects endothelial
and neuronal cells from
apoptosis[4,5]. The Chinese herb
Tripterygium, which is produced in the south of China, has
the ability to control organ rejection and is highly
considered an ideal drug. Triptolide (TRI) has been reported as a
strong inhibitor of NF-κB[6,7] and protects donor hearts
against ischemia/reperfusion-induced injury in heart
transplantation[8]. NF-κB inhibitors were used during lung
preservation and reperfusion periods to observe its influence on
lung functions. This study sought to investigate the
protective effect of TRI on ischemia/reperfusion-induced injury
of transplanted rabbit lungs. As the results showed, TRI
inhibited NF-κB-mediated immunoresponses and lung injury
induced by ischemia/reperfusion.
Materials and methods
Reagents TRI and lipopolysaccharide (LPS) were
purchased from Sigma-Aldrich (St Louis, MO, USA); the ELISA
kit of intercellular adhesion molecule-1 (ICAM-1) was
purchased from Xitang Biotechnology (Shanghai, China), and
the NF-κB activity detecting kit was purchased from Boster
Biotechnology (Wuhan, China).
Animals and grouping In total, 36 healthy adult rabbits
of both sexes, weighing 2.5±0.5 kg were randomly divided
into 3 groups. In each group, 6 rabbits were prepared as
donor lungs, and the other 6 were the recipients. In group I
(control, Con), the lungs were perfused and preserved in
improved low-potassium dextran (LPD) solution and were
used as the negative control. In group II (LPS), the lungs
were perfused and preserved in LPD solution containing LPS
(50 µg/L) and were used as the positive control. In group III
(TRI), the lungs were perfused and preserved in LPD
solution containing TRI (0.5 mg/L).
Preparation of the donor lungs The rabbits were
anesthetized with 3% pentobarbital (45 mg/kg, ip). After
tracheotomy was performed, the rabbits received mechanical
ventilation at a rate of 32/min, tidal volume (VT) = 15 mL/kg,
FiO2 = 60%. After median sternotomy, the pericardium was
opened, and the pulmonary artery and ascending aorta were
separated and cannulized. Then they were heparinized by
injecting heparin (3 mg/kg) into the left auricle. After that,
the pulmonary conus were transversely cut and a catheter
was inserted via the left auricle. The hilum of right lung was
ligated, then the lung was infused with TRI or LPS with a
perfusion pressure of 60 cm H2O at 4 ºC. The infusion ended
when the effluent from the left auricle became clear. After
infusion, the left lung was retrieved as the donor lung and
soaked in the corresponding preservation solution at 4 ºC
for 4 h.
Transplantation of the donor lungs The recipient rabbits
were anesthetized and thoracotomy was also performed. The
inferior vena cava was dissociated and cannulized. Heparin
was injected into the left auricle at a dose of 3 mg/kg. The
inferior vena cava was slit at the distal end, ligated at the
proximal end, and then connected to the donor pulmonary
artery via a pump. The recipients' left auricle was connected
with that of the donor. The blood circulation was resumed
by pumping blood from the inferior vena cava into the
donors' pulmonary artery at a flow rate of 10 mL/min, and flowed
back to recipients' heart through a left auricle adaptor. The
reperfusion continued for 1 h.
Measuring the levels of
PvO2 of the donor lungs During
the circulated perfusion, the PvO2
of vein blood gas of the dissociated lungs after regurgitation was measured. Changes
of the PvO2 levels were recorded every 15 min by a blood gas
analyzing machine (Radiometer ABL5, Denmark), it lasted
for 1 h.
Measuring the wet weight of the donor lungs
After reperfusion for 1 h, the donor lungs were taken out and
weighed. They were then put into an 80 ºC oven for 48 h and
weighed again after drying. The percentage of the wet weight
was calculated according to the data. The formula was: wet
weight (%) = (total weight_dried weight)/total weight.
Measuring the activity of myeloperoxidase of the donor
lungs Detection of myeloperoxidase (MPO) activity was
performed using the Myeloperoxidase (MPO) Detection Kit
(Nanjing Jiancheng Bioengineering Institute, China.
Approximately 0.05 g of lung tissue was dissected and rinsed with
0.9% NaCl to remove blood contamination; 5% homogenate
was made according to its mass ratio. The procedure was
done according to the protocol provided by the kit.
Absorbency was measured (A) on a spectrophotometer at 460 nm
and calculated the activity of MPO.
Measuring the concentrations of the ICAM-1 of the
donor lungs The ELISA method was adapted to measure the
ICAM-1 by determining the absorbency at 492 nm.
Approximately 10 mg of tissue of the donor lungs was processed
following the protocol of the manufacturer. The
concentration of the ICAM-1 was calculated according to the
standard curve, which was made from a series of different known
concentrations of standard bovine serum albumin.
Measuring the expression of NF-κB of the donor lungs
Immunohistochemistry was performed using the
Strep-tavidin biotin complex kit to measure the expression of
NF-κB. The first antibody was the anti-NF-κB
(P65) mono-clonal antibody with a dilution of 1:100. Positive cells
were identified as those whose nucleus had yellow-brown
grains. Eight sections from each group were chosen, and
5 fields were observed randomly in each section. Then
the number of positive cells was counted under high-power
fields (×400). The average count of 5 fields was taken as
the result.
Observing the ultrastructure of the donor lungs
After reperfusion, 1 cm3 of tissue from the left lung was dissected
and fixed in 4% glutaraldehyde to make electron microscopic
samples. The changes in the ultrastructure were observed
by using the H600 transmission electron microscope (Hitachi,
Japan).
Statistical analysis The experimental data were shown
as mean±SD. An analysis of these data was performed with
ANOVA using the SPSS 12.0 statistical package (SPSS,
Chicago, IL, USA). P-values of less than 0.05 were accepted
as statistically significant.
Results
Effect of TRI on the levels of
PvO2 in the donor lungs As
the time of reperfusion was prolonged, the levels of
PvO2 in the donor lungs of all groups decreased. In group I, the
levels PvO2 decreased starting at 30 min after reperfusion
and decreased continuously thereafter. In group II,
significant decreases of the levels
PvO2 were observed 15 min after reperfusion. On the other hand, although the levels of
PvO2 were also reduced during reperfusion in group III, these
changes were less intense than those in groups I and II
which had statistical significance at the end of reperfusion
(P<0.01; Figure 1).
Effect of TRI on pulmonary edema of the donor lungs
Pulmonary edema of the donor lungs in group II was higher
than that in group I (P<0.05). In contrast, in group III,
pulmonary edema was significantly lower compared to group I
(P<0.05; Figure 2).
Effect of TRI on MPO activity in the donor lungs
The MPO activity in group II was significantly higher than that
in group I (P<0.01), while in group III, it was lower than that
in group I (P<0.01; Figure 3).
Effect of TRI on NF-kB activity in the donor lungs
The NF-κB immunoreactivity in group II was significantly higher
than that in group I (P<0.01), while in group III,
NF-κB immunoreactivity was lower than that in group I
(P<0.01; Figure 4).
Effect of TRI on the expression of ICAM-1 in the donor
lungs The levels of ICAM-1 in group II significantly
increased compared with that in group I (P<0.01). However,
compared with that in group I, the levels of ICAM-1 in group
III decreased significantly (P<0.01; Figure 5).
Effect of TRI on the ultrastructural changes in the
donor lungs In group I, the microvilli of the type II pneumocyte
were still dense, and the nucleus remained normal. Most of
the mitochondria cristae in the cytoplasm were satiated and
some mitochondria swelled occasionally. The endoplasmic
reticulum did not enlarge. The osmiophilic multilamellar body
was kept intact and the vessle structure integrity was
maintained (Figure 6A). In group II, the microvilli of the type II
pneumocyte were sparse. The nuclear chromatin was
marginated and the mitochondria swelled significantly; their
cristae were short and scarce. The morphology of some
mitochondria changed and appeared vacuole-like. The
osmium-philic multilamellar body developed poorly, and the vascular
endothelial cells also swelled. The endoplasmic reticulum
enlarged slightly (Figure 6B). In group III, the microvilli of
the type II pneumocyte and the nuclear structure were normal.
The mitochondria in the cytoplasm swelled slightly. The
osmiumphilic multilamellar body was intact. The microvilli
of the type I pneumocyte and the nuclear structure were also
normal; the mitochondria and endoplasmic reticulum enlarged
slightly (Figure 6C).
Discussion
Lung transplantation is becoming an effective means for
the treatment of lung diseases in the terminal stage. The
protection of the functions of donor lungs is one of the
critical factors that determine the success of lung
transplantation[9,10]. Most deaths occur within 30 d after
transplan-tation. The main cause of death during the first
postoperative month is non-specific graft failure secondary to
ischemia/reperfusion-induced lung
injury[11,12]. Many
attempts have made to reduce injury, including improvement
of preservation solutions, adequate setting of flushing
solutions, reperfusion pressure and ventilation, inhaled
nitric oxide, administration of prostaglandins,
complementary inhibitors, the antagonists of the platelet-activating
factor, and surfactant enhancers[13]. Generally, donor lungs
can be safely preserved for 4_6 h. Among those that died
after lung transplantation, 15%_20% died from lung injury
induced by ischemia/reperfusion[14]. Lung injury induced
by ischemia-reperfusion is characterized by increased
pulmonary vascular resistance, decreased oxygenation capacity,
worsened compliance, and edema
formation[15]. Multiple mechanisms are involved in ischemia/reperfusion-induced
lung injury. It is generally believed that the injury is a
process of inflammatory reaction, which includes the activation
of endothelial, the expression of adhesion molecules, and
also the adherence, aggregation, and activation of leucocytes
and platelets, as well as the release of free radicals. In these
inflammatory reactions, the transcription factor
NF-κB is the key regulator which controls the expression of many genes
involved in inflammation, such as the activation of the
endothelium and adherence of molecules. Therefore,
NF-κB has become a new target for anti-inflammatory treatments[16].
Tripterygium extracts have been used widely to treat
inflammatory and autoimmune diseases such as rheumatoid
arthritis and immune complex nephritis in
China[17, 18]. In organ transplantation, both clinical and experimental studies
have demonstrated that Tripterygium extracts effectively
prolong allograft survival[19]. TRI is an active ingredient
extracted from Tripterygium
wilfordii multiglycoside[20]. It
is a colorless, needle-like crystal, with a relative molecular
weight of 360 g/mol; its molecular formula is
C2OH24O6. It has complex chemical compositions and diverse
pharmacological actions. It is TRI that makes Tripterygium
wilfordii multiglycoside an effective immunosuppressant. The
immunosuppressive effects of TRI can be partially attributed to
its potent inhibition of T cell activation and interleukin-2
production[21]. Remarkably, the inhibitory effect of TRI on T
cell activation has been shown to be more potent than that
exhibited by cyclosporine[22] and Tacrolimus
(FK-506)[23].
MPO is a specific enzyme in the cytoplasm of neutrophils,
which induces the activation and aggregation of neutrophils.
The changes in MPO in the lung can reflect the levels of
neutrophil aggregation in the lung after reperfusion. This
study also found that the levels of
PaO2 decreased after reperfusion, the activities of MPO increased, and the
structure was damaged in the donor lungs. These pathological
changes in the donor lungs were exacerbated by LPS
treat-ment. In contrast, these pathological changes in the
TRI-treated group were improved, suggesting that TRI can
protect donor lungs against ischemia/reperfusion-induced injury.
The present study further demonstrated that the
ischemia/reperfusion-induced elevation in NF-κB and the
NF-κB target gene ICAM-1 significantly decreased by TRI. These
results suggest that TRI may reduce injury of the donor
lungs induced by ischemia/reperfusion through inhibiting
the activity of NF-κB and reducing the expression of
ICAM-1. Along with other studies[24,
25], TRI may have a wide application in organ transplantation as an
anti-inflammatory and immunosuppressant. However, TRI also has
toxicity, just as many Chinese medicines do. It affects the
digestive system, genital system, hematopoietic system, and
so on. Thus, it is better to extract its effective monomer
component to study its metabolic process so as to raise its
pharmacodynamic action and reduce its toxicity.
References
1 Peralta C, Fernández L, Panés J, Prats N, Sans M, Piqué JM,
et al. Preconditioning protects against ischemia-reperfusion through
blockade of tumor necrosis factor-induced P-selection
up-regulation in the rat. Hepatology 2001; 33: 100_13.
2 Azzolina A, Bongiovanni A, Lampiasi N. Substance P induces
TNF-alpha and IL-6 production through NF kappa B in
peritoneal mast cells. Biochem Biophys Acta 2003; 1643: 75_83.
3 Jiang B, Xu S, Brecher P, Cohen RA. Growth factors enhance
interleukin-1 beta-induced persistent activation of nuclear
factor-kappa B in rat vascular smooth muscle cells. Aterioscler
Thromb Vasc Biol 2002; 22: 1811_6.
4 Qin ZH, Wang YM, Nakai M, Chase TN. Nuclear
factor-κB contributes to excitotoxin-induced apoptosis. Mol Pharmacol
1998; 53: 33-42.
5 Qin ZH, Chen RW, Wang YM, Nakai M, Chuang DM, Chase TN.
NF-κB nuclear translocation up-regulates c-Myc and p53 during
N-methyl-D-aspartate receptor-mediated apoptosis. J Neurosci
1999; 19: 4023_33.
6 Hu K, Liu Z, Liu D. Triptolide inhibits vascular endothelial
growth factor expression and production by endothelial cells.
Zhonghua Yixue Zazhi 2001; 81: 1106_9. Chinese.
7 Liu H, Liu ZH, Chen ZH, Yang JW, Li LS. Triptolide: a potent
inhibitor of NF-kappa B in T-lymphocytes. Acta Pharmacol Sin
2000; 21: 782_6.
8 He JK, Gu ZL, Yu SD, Shen ZY. Triptolide on inhibition of
reperfusion injury and prolongation of allograft survival in rat
cardiac transplants. Chin J New Drugs Clin 2001; 20: 423_6.
Chinese.
9 Luh SP, Tsai CC, Shau WY, Shiau SY, Jan JS, Kuo SH,
et al. Protective effects of inhaled nitric oxide and gabexate mesilate
in lung reperfusion injury after transplantation from
non-heart-beat donors. J Heart Lung Transplant 2002; 21: 251_9.
10 Rega FR, Wuyts WA, Vanaudenaerde BM, Jannis NC, Neyrinck
AP, Verleden GM, et al. Nebulized N-acetyl cysteine protects
the pulmonary graft inside the non-heart-beating donor. J Heart
Lung Transplant 2005; 24: 1369_77.
11 Thabut G, Vinatier I, Stern JB, Leseche G, Loirat P, Fournier M,
et al. Primary graft failure following lung transplantation.
Predictive factors of mortality. Chest 2002; 121: 1876_82.
12 Hertz MI, Taylor DO, Trulock EP, Boucek MM, Mohacsi PJ,
Edwards LB, et al. The registry of the international society for
heart and lung transplantation: nineteenth official report
— 2002. J Heart Lung Transplant 2002; 21: 950_70.
13 Perrot M, Liu M, Weddel K, Keshavjee S.
Ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med 2003; 167:
490_511.
14 Haydock DA, Trulock EP, Kaiser LR, Knight SR, Pasque MK,
Cooper JD. Management of dysfunction in the transplanted
lung: experience with 7 clinical cases. Washington University
Lung Transplant Group. Ann Thorac Surg 1992; 53: 635_41.
15 Mathias MA, Tribble CG, Dietz JF, Nguyen RP, Shockey KS,
Kern JA, et al. Aprotinin improves pulmonary function during
reperfusion in an isolated lung model. Ann Thorac Surg 2000;
70: 1671_4.
16 Schmidt C, Peng B, Li Z, Sclabas GM, Fujioka S, Niu J.
Mechanisms of pro-inflammatory cytokine-induced biophasic
NF-κB activation. Mol Cell 2003; 12: 1287_300.
17 Gao ZG, Zang AC, Bai RX, Radix, Tripterygium
wilfordii Hook F in rheumatoid arthritis, ankylosing spondylitis and juvenile
rheumatoid arthritis. Chin Med J 1986; 99: 317_20.
18 Jiang X. Clinical observations on the use of the Chinese herb
Tripterygium wilfordii Hook for the treatment of nephrotic
syndrome. Pediatr Nephrol 1994; 8: 343_4.
19 Wang J, Xu R, Jin R, Chen Z, Fidler JM. Immunosuppressive
activity of the Chinese medicinal plant Tripterygium
wilfordii. I. Prolongation of rat cardiac and renal allograft survival by the
PG27 extract and immunosuppressive synergy in combination
therapy with cyclosporine. Transplantation 2004; 70: 447_55.
20 Gu WZ, Chen R, Brandwein S, McAlpine J, Burres N. Isolation,
purification, and characterization of immunosuppressive
compounds from Tripterygium: triptolide and tripdiolide. Int J
Immunopharmacol 1995; 17: 351_6.
21 Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S,
et al. Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin- 2
expression at the level of purine-box/nuclear factor of activated
T-cells and NF-κB transcriptional activation. J Biol Chem 1999;
274: 13 443_50.
22 Lu H, Hachida M, Enosawa S, Li X, Suzuki S, Koyanagi H.
Immunosuppressive effect of triptolide in
vitro. Transplant Proc 1999; 31: 2056_7.
23 Chan M, Kohlmeier JE, Branden M, Jung M, Benedict SH.
Triptolide is more effective in preventing T cell proliferation
and interferon-gamma production than is FK506. Phytother
Res 1999; 13: 464_7.
24 He X, Verran D, Hu C, Wang C, Li L, Wang L,
et al. Synergistic effect of Tripterygium
wilfordii Hook F) (TWHF) and cyclosporin A in rat liver transplantation.
Transplant Proc 2000; 32: 2054.
25 Fidler JM, Ku GY, Piazza D, Xu R, Jin R, Chen Z.
Immunosuppressive activity of the Chinese medicinal plant
Tripterygium wilfordii III suppression of graft-versus-host disease in murine
allogeneic bone marrow transplantation by the PG27 extract.
Transplantation 2002; 74: 445_57.
|
