Objective: To research the expression of hypoxia inducible factor-1 alpha (HIF-1 alpha) on the apoptosis and number of T lymphocyte in Peyer’s patches after severe burn on plateau in rats. Methods: Wistar rats (n = 130) were subjected to deep thickness burn injury (30% TBSA, III degree), at two different altitudes. 60 of them were given delayed fluid resuscitation (DFR, n = 30 at each altitude) 6 h after burn at different altitude; 60 of them were carried out immediate fluid resuscitation (IFR, n = 30 at each altitude); 10 rats were subjected to 37° C warm water as sham burn (SG, n = 10). The Peyer’s patches were harvested from the ileum of rats at different time point after burn respectively. The expression of HIF-1 alpha, CD3(+) and the apoptosis and number of T lymphocyte in Peyer’s patches were detected by tissue microarray technology and immunohistochemistry. Results: The apoptosis was higher in DFR group than that in IFR group. The increase in HIF-1 alpha expression was observed mainly on cell nucleus in T lymphocytes. The expression levels of HIF-1 alpha in Peyer’s patches were much higher in DFR group and IFR group than those in SG, and they were higher at high altitude (3848 metres) than those at lower altitude (1517 metres), and also higher in DFR group compared with IFR group (all P < 0.05). The expression levels of CD3+ in Peyer’s patches were much lower in DFR group and IFR group than those in sham group, and the lowest value appeared at 12 hours after burn (all P < 0.05). Conclusion: High expression of HIF-1 alpha may induce the apoptosis of T lymphocytes in Peyer’s patches after severe burn with delayed fluid resuscitation in rats at plateau.
Hypoxia is a well-known cause of cell injury at plain, with important pathological implications in many disease processes, including cerebral ischemia and myocardial infarction [
Hypoxia-inducible factor-1α (HIF-1α), as a kind of transcriptional factor, is known to play a fundamental role in adaptive or death process in response to hypoxia [
Total of 132 Wistar rats were supplied by medical experimental animal center of Gansu Chinese Traditional Medical College. The average weight was 200 ± 30 g. Half of them were female. Animal was kept in single cage under 19˚C - 25˚C for 1 week before injury. They were fasted 12 h before experiment. Animals were anesthetized with an injection of pentobarbital sodium (40 mg/kg). The back of the rats was shaved before experiment and was immersed into 90˚C hot water for 20 s to produce a 30% total body surface area (TBSA) scald burn on the back of the rats at 1517 m and 3848 m above sea level. The experiment at 1517 m altitude was carried out in the animal experimental center of Lanzhou General Hospital in Lanzhou city, Gansu province. The experiment at 3848 m altitude was carried out in an animal experimental base of high altitude medicine in Mahanshan mountain in Yuzhong county, Gansu province, China. Animals were transported from Lanzhou (1517 m above sea level) to Mahanshan mountain (3848 m above sea level) in 1 day and the experiment was started after 3 days acclimation, finished in 10 days. All animals were randomly divided into 6 groups by table of random digit: immediate fluid resuscitation group (IFR, n = 30 at each altitude, the animals received saline injection intraperitoneally according to the Parkland formula immediately after injury for resuscitation, 40 ml/kg body weight); delayed fluid resuscitation group ( DFR, n = 30 at each altitude, the same amount of fluid was given 6h PB; and sham group (SG, n = 6 at each altitude, the back of the animals were immersed into 37˚C warm water for 20 s to imitate scald burn without saline injection).
At 6, 12, 24, 48 and 72 hours PB, animals (n = 6, at every time point in each group) were euthanized after being anesthetized with intraperitoneal injection of pentobarbital sodium (40 mg/kg). All the Peyer’s patches in the intestine (10 cm distal to the ileocecal junction) were harvested and were immediately fixed in 10% neutral buffered formalin. The samples in sham group were harvested 6 hours after sham burn.
The Peyer’s patches were then embedded in parafin and sectioned. After stained with hematoxylin and eosin, histopathological observation and photographing were carried out under optical microscope.
The terminal deoxynucleotidyl transferase (TdT)-mediated d-UDP-biotin nick end labeling (TUNEL) method was used to detect the apoptosis of lymphocytes in Peyer’s patches with modification [
The expression of HIF-1α and CD3+ protein from the Peyer’s patches were determined by Strept-Actividin-Biotin-Complex (SABC) method. The paraffin sections (4 µm) were dehydrated in xylene and graded ethanol series, and added in order with primary antibody [HIF-1α (rabbit polyclonal antibody, Wuhan Boster Co., China), CD3+ (rabbit anti-rat monoclonal antibody, Wuhan Boster Co.)], after incubated in a humid box overnight, biotinylated secondary antibody (rabbit anti-rat IgG, Wuhan Boster Co.) added and incubated for 20 min at 37˚C, SABC reagents and DAB solution (Wuhan Boster Co.) added. For negative control, the sections were treated with PBS instead of primary antibody. Immunohistological quantitative analysis was carried out as following: ten sample sections from each group were chosen for analysis. 6 high power visual fields were observed randomly. The Dpcontrollor 7.0 photographing system and the pro-plus 5.0/Ipp5.0 Image analysis system were used to determine the absorbance (A value) of HIF-1α and CD3+ protein.
Results are expressed as means ± s. Group comparisons were carried out by variance analysis, using SPSS 10.0 medical statistical software. P-value less than 0.05 was considered significant.
The construction of Peyer’s patches in SG at 1517 m was very clear. There was no inflammatory cell infiltration. The construction of Peyer’s patches was also clear in IFR group, only some germinal center of some follicle enlarged. There was light inflammatory cell infiltration 6 - 12 h PB. Inflammatory cell infiltration was observed in DFR group, apoptotic lymphocytes distributed in the germinal center singularly or areataly. The pathological changes aggravated gradually after 6 - 12 h PB.
The construction of Peyer’s patches in SG at 3848m was clear too, but both the B cell zone and T cell zone enlarged and the volume of Peyer’s patches increased too. There was a little inflammatory cell infiltration in IFR group, single apoptotic cell distributed in the germinal center, the membrane of apoptotic cell shrinked, the neuclous karypyknosis and margination of chromatin and round and ellipse apoptotic body were observed. The above pathological changes alleviated 48 - 72 h PB.
Apoptotic cells were observed in Peyer’s patches at two altitudes in experimental groups. The apoptotic cell number increased gradually 6 - 12 h PB and reached the zenith at 12 h PB. The nucleus of apoptotic cell showed brown color, the chromosome condensed, and distributed in the germinal center of Peyer’s patches clusterly (
altitude | group | 6 h PB | 12 h PB | 24 h PB |
---|---|---|---|---|
1517 m | SG (n = 6) | 11.36 ± 2.1 | ||
IFR (n = 18) | 15.92 ± 0.52a | 19.47 ± 0.63a | 17.52 ± 0.34a | |
DFR (n = 18) | 20.09 ± 0.81ab | 25.68 ± 1.26ab | 21.43 ± 0.70ab | |
3848 m | SG (n = 6) | 14.28 ± 0.88c | ||
IFR (n = 18) | 17.92 ± 0.76ac | 24.42 ± 1.51ac | 21.73 ± 0.78ac | |
DFR (n = 18) | 24.06 ± 1.33abc | 28.90 ± 1.91abc | 27.17 ± 1.56abc |
Notice: a: vs SG at the same altitude, P < 0.05; b: vs IFR at the same altitude, P < 0.05; c: vs corresponding group at low altitude, P < 0.05; Blank means no data.
Quantitative analysis of lymphocyte apoptosis rate after 6 - 24 h PB showed: 1) the apoptosis rate in DFR group was higher than that in IFR group at the same altitude 6 - 24 h PB (P < 0.05). The highest apoptotic rate in experimental group was at 12 h PB (P?0.05). 2) The apoptosis rate in both DFR and IFR group at high altitude was higher than that in low altitude (P?0.05) (
The HIF-1α expression increased both in IFR and DFR group at 6 - 72 h PB, compared with that in SG at the same altitude. The positive expression mainly distributed in the nucleus of lymphocytes, showing a buffy colour granules (
Compared with the CD3+ expression at the same altitude in SG, the CD3+ expression decreased both in IFR and DFR groups, mainly distributed on the cell membrane of T lymphocyte, showing brow-yellow color. The quantitative analysis showed that the CD3+ expressions at both altitudes in DFR and IFR groups at different time point were lower than that in SG (P value all less than 0.05). The CD3+ expressions in DFR group at low altitude and in DFR group at 6 h, 12 h, 24 h, 48 h, 72 h PB at high altitude were all lower than that in IFR group at the same altitude. The CD3+ expressions both in DFR and IFR groups from 6 h to 72 h PB were all lower than that in corresponding low altitude groups (P < 0.05).
altitude | group | 6 h PB | 12 h PB | 24 h PB | 48 h PB | 72 h PB |
---|---|---|---|---|---|---|
1517 m | SG (n = 6) | 0.133 ± 0.016 | ||||
IFR (n = 30) | 0.159 ± 0.005 | 0.195 ± 0.006a | 0.175 ± 0.003a | 0.168 ± 0.007a | 0.155 ± 0.003a | |
DFR (n = 30) | 0.201 ± 0.008ab | 0.257 ± 0.013a | 0.214 ± 0.007ab | 0.208 ± 0.006a | 0.222 ± 0.008ab | |
3848 m | SG (n = 6) | 0.166 ± 0.009 | ||||
IFR (n = 30) | 0.179 ± 0.008ac | 0.244 ± 0.015ac | 0.217 ± 0.008ac | 0.206 ± 0.008ac | 0.187 ± 0.007ac | |
DFR (n = 30) | 0.241 ± 0.013abc | 0.289 ± 0.019abc | 0.272 ± 0.016abc | 0.228 ± 0.013abc | 0.192 ± 0.050a |
a: vs SG at the same altitude, P < 0.05; b: vs IFR at the same altitude, P < 0.05; c: vs corresponding group at low altitude, P < 0.05, Blank means no data.
altitude | group | 6 h PB | 12 h PB | 24 h PB | 48 h PB | 72 h PB |
---|---|---|---|---|---|---|
1517 m | SG (n = 6) | 0.478 ± 0.097 | ||||
IFR (n = 30) | 0.327 ± 0.040a | 0.241 ± 0.024a | 0.306 ± 0.020a | 0.312 ± 0.022a | 0.355 ± 0.042a | |
DFR (n = 30) | 0.269 ± 0.011ab | 0.202 ± 0.010ab | 0.251 ± 0.013ab | 0.256 ± 0.013ab | 0.278 ± 0.067ab | |
3848 m | SG (n = 6) | 0.376 ± 0.035 | ||||
IFR (n = 30) | 0.297 ± v | 0.204 ± 0.030ac | 0.245 ±0.013ac | 0.264 ±0.009ac | 0.289 ± 0.014ac | |
DFR (n = 30) | 0.228 ± 0.007abc | 0.145 ± 0.018aBc | 0.206 ± 0.006abc | 0.234 ± 0.009abc | 0.240 ± 0.010abc |
a: vs SG at the same altitude, P < 0.05; b: vs IFR at the same altitude, P < 0.05;c: vs corresponding group at low altitude, P < 0.05, Blank means no data.
Immune cells are exposed to different oxygen tension, including hypoxia, as they develop, migrate and function in primary, secondary, and tertiary lymphoid organs with different infrastructure, vasculature, and oxygen supply [
Our previous study has demonstrated that the apoptosis rate of gut-associated lymphatic tissue (GALT) increased postburn (PB) with delayed resuscitation [
There was a little expression of HIF-1α at 3848 m altitude in sham group; this revealed that hypoxia may be involved in the induction of apoptosis. The result is in agreement with the report that hypoxia could enhance the HIF-1α accumulation and apoptosis of T cells, and inhibit T-cell proliferation [
CD3+ protein, as a marker of T lymphocytes, the trough value of CD3+ expression also appeared at 12 h PB, and the value in DFR group were all lower than that in IFR group at different altitude. And the lowest expression of CD3+ occurred at 12h PB. These results suggested that the apoptotic lymphocytes were mainly T cells. The expression of CD3+ protein was antiparalleled to the apoptosis of lymphocytes [
It is well known that hypoxia increase cellular ROS generation, probably from mitochondrial electron transport complexes [
ROS could stabilize HIF-1α and prevent the degradation of HIF-1α. The HIF-1α could be detected in T lymphocytes in sham group, because the half-life of HIF-1α was very short under normoxia. They were ubiliquitinated quickly after production, and then dissolved by protease [
Supported by: The general program of medical science and technology of Chinese PLA during the 11th “five year” plan. Fund No: (06G030).
Zhang, C., Liu, Y., Ma, M. and Zhang, S.F. (2016) The Influence of HIF-1α Expression on Apoptosis and Number of T Lymphocyte in Peyer’s Patches after Burn with Delayed Fluid Resuscitation in Rats at Plateau. Surgical Sci- ence, 7, 390-399. http://dx.doi.org/10.4236/ss.2016.79056