| Brain death is an irreversible pathological state of the whole brain functioning failure which involves the brainstem. Literally, the brain death leads to lung injury, which brings oxygenation insufficiency giving rise to the dysfunction and injury of other organs. The exploration into the mechanism of brain death-related lung injury plays a key role in the prophylaxis and treatment of brain death related lung injury.The calcitonin gene-related peptide (CGRP) is a multi-tasking protein, such as vasodilator, myocardial strengthener, lung protector, brain reservoir, immunomodilator, etc.Researchers have demonstrated that the amount of CGRP in human body changed after craniocerebral injury and was related to the brain injury severity.Based on the hypothesis that the changes amount of CGRP in human body which participates in development of the brain death-related lung injury, this experiment adopted the method of slow continuous intracranial pressure to build a brain death model. The radioimmunoassay, RT-PCR, Western blot were used to study the changes of CGRP and endothelin-1 (ET-1) in rat blood plasma and lung tissue at several time points during brain death and the relationship between those changes with brain death-related lung injury. Given the research on the CGRP change pattern after brain death and the influence of CGRP to the lung injury, it initiates a novel solution for the prophylaxis and treatment of brain death-related lung injury. CGRP was administered intravenously during brain death modeling. Radioimmunoassay, ELISA, RT-PCR, Western blot and immunohistochemistry are to be employed to observe the amount of brain death rat's tumor necrosis factor-a (TNF-α), interleukin-1β(IL-1β), interleukine-6 (IL-6) and other inflammatory factors in the blood plasma as well as that of malondialdehyde (MDA), superoxide dismutase (SOD) competence in the lung tissue and the influence on the AQP-1 and y-GCS mRNA and proteins' expression. The lung function and morphology are simultaneously observed in order to explore the possible mechanism of CGRP's protective effect on the lung.Since that the exogenous CGRP is inactivated, metabolized quickly and short-acting and that CGRP recombinant adenovirus vector if transferred into the trachea cells is characterized by host cell genome disintegration which is a safe convenient and durable, it is theoretically a better protector for the brain death-related lung injury. This project is aimed to construct the Ad5-CGRP-EGFP and transfer it into trachea cells to observe the CGRP expression change of CGRP gene transfected ran lung tissue and also the effect of CGRP gene therapy on the lung tissue's AQP-1 and y-GCS, the amount of MDA, SOD competence, pulmonary water content, lung morphology as well as the content of TNF-α, IL-1β, IL-6 in blood plasma. The mechanism of CGRP gene therapy in the lung protection of brain death is also explored.This study intends to clarify the in vivo CGRP change pattern after brain death and it's link with the brain death-related lung injury. The CGRP gene therapy effect and mechanism on the lung injury are both studied through the inference of CGRP gene transferring, with view of throwing a light theoretically on the prophylaxis and treatment of brain death-related lung injury.This study includes the following three parts.Part one CGRP expression in brain death rat and related lung injury1 Methods 1.1 Twenty adult Wistar rats, weighted between 250 and 300g, were randomly divided into control(A) and brain-death(B) groups (n=10). Brain-death model was established in group B. The group A were placed Foley balloon catheter in intracalvarium only; no brain-death model established. Blood sample were collected at the time point of anesthesia 15 min (TO) and 0 h (T1),2 h (T2),6h (T3) and 12 h (T4) after brain-death model established. The samples of lung tissues were harvested at T4 time point.1.2 Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. Histological alterations in lung tissues were examined by HE staining. And at the same time lung index and lung water content were measured. The levels of CGRP in lung tissues were examined by RT-PCR and Western blot.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. P< 0.05 was considered statistically significant.2 Results2.1 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. MAP of group B were significantly lower at T1 and after T1 time points than that of TO time point, HR of group B were significantly faster at T1 and after T1 time points than that of TO time point (P<0.05). At the peak time point MAP and HR of group B were significant increase (P<0.05). Comparison of MAP at T1 and after T1 time points, MAP of group B, were significant lower than that of group A, comparison of HR at T1 and after T1 time points, HR of group B, were significant faster than that of group A (P<0.05).2.2 In group A, the levels of CGRP in serum was higher at T1 than that at T0, and lower at T2 than that at T1, and the difference was significant (P<0.05). In group B, the levels of CGRP was higher at than that at TO, and lower at T2-T4 than that at T0, and the difference was significant (P<0.05). The levels of CGRP in group B were higher than that in group A at T1 time points, but lower than that in group A at other time points, and the difference was significant (P<0.05).2.3 The levels of ET-1 was higher at other time points except T4 than that at TO in group A, and the difference were statistically significant (P<0.05). The levels of ET-1 was higher at other time points than that at TO in group B, and the difference was significant (P<0.05). The levels of ET-1 in group A was higher at same time points than that in group B, and the difference were statistically significant (P<0.05).2.4 The levels of mRNA of CGRP in lung tissues were significantly up-regulation in group B compared with that in group A and protein of CGRP in lung tissues were significantly down-regulation in group B compared with that in group A (P<0.05).2.5 Histological alterations:The lung injury was confirmed by HE examination in groupB. The lung Histological examination in group A was normal.2.6 In contrast to group A, lung index and lung water content were increased significantly in group B (P<0.05).Part two Effect of CGRP on the lung injury in brain death rats and its mechanisms1 Methods1.1 Seventy-five adult Wistar rats, weighted between 250 and 300g, and were randomly divided into control group (A), brain-death group (B) and CGRP treatment group (n=25). Brain-death model was established in group B and group C. The group A were placed Foley balloon catheter in intracalvarium only; no brain-death model established. In group C, CGRP 3μg/kg was injected intravenously immediately after establishment of brain-death model, followed by CGRP 6μg/kg continously injected for 12h. Blood sample and lung tissues of five rats in each group were collected at the time point of anesthesia 15 min (TO) and 0 h (T1),2 h (T2),6 h (T3) and 12 h (T4) after brain-death model established.1.2 The levels of TNF-α, IL-1βand IL-6 in the serum were measured by Enzyme-linked immunosorbent assay (ELISA). Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. The MPO and SOD activity were evaluated in the lung tissues at T4. The lung index and lung water content were measured. Histological alterations in lung tissues were examined by HE staining. The levels ofγ-GCS,AQP-1,CGRP were measured by RT-PCR and Western blot at T4.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. P value of<0.05 was considered statistically significant.2 Results2.1 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B and C, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. MAP of group B and C were significantly lower at T1 and after T1 time points than that of TO time point, HR of group B and C were significantly faster at T1 and after T1 time points than that of TO time point (P<0.05). At the peak time point MAP and HR of group B and C were significant increase (P<0.05). Comparison of MAP at T1 and after T1 time points, MAP of group B and C, were significant lower than that of group A, comparison of HR at T1 and after T1 time points, HR of group B and C, were significant faster than that of group A (P<0.05)。2.2 TNF-α:There were no significant difference for the levels of TNF-a among time points in group A. The levels of TNF-a were higher at other time points than that at T2 in group B and C, and the difference was significant (P<0.05). From T2, the level of TNF-αwas higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05)2.3 IL-1β:There were no significant difference for the levels of IL-lβamong time points in group A. The levels of IL-1βwere higher at other time points than that at T2 in group B and C (P<0.05). From T2, the level of IL-1 (3 was higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05)2.4 IL-6:The levels of IL-6 were higher at T2, T3 and T4 than that at TO and T1 in group A (P<0.05). The levels of IL-6 were higher at other time points than that at T2 in group B and C(P<0.05).From T2, the level of IL-6 was higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05) 2.5 CGRP:The levels of CGRP in serum were higher at T1 than that of TO in group A, and lower at T2 than that at T1 (P<0.05).In group B, the levels of CGRP were higher at T1 than that at T0, but lower at other time points than that at TO (P<0.05). The levels of CGRP were higher at all the time points than that at TO in group C (P<0.05). The level of CGRP was higher in group B than that in group A at T1, but lower at other time points. The level of CGRP was higher in group C than that in group A from T1, and higher than that in group B from T2 (P<0.05)2.6 ET-1:The levels of ET-1 in serum were higher at T1 and T2 than that of TO in group A (P<0.05). The levels of ET-1 were higher at other time points than that of TO in group B (P<0.05). The levels of ET-1 were higher at other time points than that of TO in group C (P<0.05). The levels of ET-1 were higher in group B and C than that in group A from T1. The levels of ET-1 were lower in group C than that in group B from T2 (P<0.05)。2.7 MDA and SOD activity:MDA activity of lung tissues in group B was higher than that in group A and C, and group C was higher than that in group A (P<0.05) SOD activity of lung tissues in group B was lower than that in group A and C (P<0.05)2.8 The mRNAs levels ofγ-GCS in group B and group C were higher than that in group A, and group C were higher than that in group B (P<0.05). The mRNAs levels of AQP-1 in group B were lower than that in group A, and group C were higher than that in group B (P<0.05). The mRNAs levels of CGRP in group B were higher than that in group A, and the mRNAs levels of CGRP in group C were lower than that in group A (P<0.05).The proteins levels of AQP-1,γ-GCS,CGRP in group B were lower than that in group A, and group C were higher than that in group A and B, and the differences were statistically significant (P<0.05).2.9 Histological alterations:The lung injury was confirmed by HE examination in group B and C, while less pathologic alterations were shown in group C. The lung Histological examination in group A was normal.2.10 In contrast to group A, lung index and lung water content were increased significantly in group B and C. But in contrast to group B, lung index and lung water content were decreased significantly in group C (P<0.05).Part three Effect of CGRP gene on lung injury in brain death rats1 Methods1.1 Fifty adult Wistar rats, weighted between 250 and 300g, and were randomly divided into control group (A), brain-death group (B), CGRP treatment group(C), empty vector group (D) and CGRP gene transfer group (E). Brain-dead model was established in group B, C, D and E. The group A were placed Foley balloon catheter in intracalvarium only; no brain-dead model established. In group C, CGRP was injected via vein immediately after establishment of brain-dead model. In group D, empty vector was transferred after establishment of brain-dead model. In group E, CGRP with enhanced green fluorescent protein gene mediated by recombinant adenovirus (Ad) vector was transinfected via trachea after establishment of brain-dead model. Blood sample and lung tissues in each group were collected at the time point of anesthesia (TO) and 12 h (T1) after brain-dead model established.1.2 The levels of TNF-α, IL-1βand IL-6 were measured by ELISA. Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. The gene transfect was accessed by fluorescence microscope. The MPO and SOD activity were evaluated in the lung tissues at T1. The lung index and lung water content were measured. Histological alterations in lung tissues were examined by HE staining. The expression of y-GCS and AQP-1 was analyzed by immunohistochemistry. The levels of y-GCS, AQP-1, CGRP were measured by RT-PCR and Western blot at T1.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. a level was set at 0.05.2 Results2.1 There was no green fluorescence in group A, B and C. Enhanced green fluorescence were seen in group D and E.2.2 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B, C, D and E, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. Than that of TO time point, MAP of group B, C, D and E were significantly lower at the time point of brain-death model established and after. Than that of TO time point HR of group B, C, D and E were significantly faster at the time point of brain-death model established and after (P<0.05). At the peak time point MAP and HR of group B, C, D and E were significant increase (P<0.05). Comparison of MAP at the time point of brain-death model established and after, MAP of group B, C, D and E, were significant lower than that of group A, comparison of HR at the time point of brain-death model established and after, HR of group B, C, D and E, were significant faster than that of group A (P<0.05)。2.3 TNF-α, IL-1βand IL-6:The levels of TNF-α, IL-1βand IL-6 in group B and D were higher than those in group E, C and A, and group E and C were higher than those in group A, group C were higher than those in group E (P<0.05)。2.4 CGRP:There were no significant differences among groups at TO. The levels of CGRP were higher in group E than those in group D, C, B and A at T1. The levels of CGRP were higher in group C than those in group D, B and A. The levels of CGRP were higher in group A than those in group D and B (P<0.05)2.5 ET-1:There were no significant differences among groups at T0. The levels of ET-1 were lower in group E than those in group D, C, B and A at T1. The levels of ET-1 were lower in group C than those in group D, B and A. The levels of ET-1 were lower in group A than those in group D and B (P<0.05)2.6 MDA and SOD activity:In the lung tissues, the MDA activity in group B and D was higher than that in group A, C and E, while group C was higher than that in group A and E, and group E was higher than that in group A (P<0.05).The SOD activity in group C and E was higher than that in group A, B and D, while group E was higher than that in group C (P<0.05)2.7 The mRNAs levels of y-GCS in group B and group C and group D and group E were higher than that in group A, and group C were higher than that in group B and D, while group E were higher than that in group C (P<0.05). The mRNAs levels of AQP-1 in group B and group D were lower than that in group A, The mRNAs levels of AQP-1 were higher in group C and E than that in group A, while group E were higher than that in group C (P<0.05). The mRNAs levels of CGRP in group B and D were higher than that in group A, and group C were lower than that in group A, while group E were higher than those in group B and D, and the differences were statistically significant (P<0.05) The protein levels of y-GCS, AQP-1 and CGRP were lower in group B and in group D than those in group A. The protein levels of y-GCS, AQP-1 and CGRP were higher in group C and E than those in group A, while group E were higher than those in group C, and the differences were statistically significant (P<0.05)2.8γ-GCS and AQP-1 immunohistochemistry:The levels ofγ-GCS and AQP-1 in group B and D were lower than those in group E, C and A, while group E and C were higher than those in group B and D, and group E were higher than those in group C (P<0.05)2.9 Histological alterations:The lung injury was confirmed by HE examination in group B and D, while less pathologic alterations were shown in group C and E. The lung Histological examination in group A was normal.2.10 Lung index and lung water:Lung index and lung water content in group B and D was higher than that in group E, C and A, while group E and C was higher than that in group A, and group E was lower than that in group C (P<0.05)Conclusion1. The CGRP level changes after brain death in rats. Lung injury after brain death has a relationship with the decreased level of CGRP, the release of TNF-a, IL-1βand IL-6, the enhanced of oxidative stress and the expression down-regulated of AQP-1.2. Exogenous CGRP has protective effect on lung injury after brain death in rats. 3. CGRP gene transfection can up-regulated the expression of CGRP in lung tissues after brain death in rats. The levels of TNF-α, IL-1βand IL-6 in serum are decreased after CGRP gene transfection in brain death rats, while the expression ofy-GCS and AQP-1 in lung tissues are increased.4. CGRP gene transfection attenuates lung injury and lung water content after brain death in rats. The mechanism may have relation with its effect of anti-inflammation, anti-oxidative stress and AQP-1 up-regulation. |
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