| In the recent years, atherosclerotic cardiovascular disease has become the primary reason for threatening military pilots’ health and even leading to ground. Early prevention and treatment of atherosclerosis in pilots is important for safeguarding the health of pilots, extending flying life and improving air force.Hyperlipidemia is count a great deal for atherosclerotic cardiovascular disease. Investigations from several flight forces found that the incidence of hyperlipidemia in pilots showed an increasing and younger trend. Evidence-based medicine evidence showed that endothelial dysfunction caused by hyperlipidemia is the initial factor for atherosclerosis. Existed studies demonstrated that repeated positive acceleration(+Gz) exposure could cause myocardial energy metabolism disorder in organism, elevate catecholamine level in serum, stimulate the body’s non-specific inflammatory reaction, activate platelets, and increase production of oxygen free radicals which may lead to vascular endothelial cell injury and accelerate the process of atherosclerosis with age. But whether +Gz accelerating the process of atherosclerosis and increasing the incidence of pilots’ atherosclerotic cardiovascular by aggravating hyperlipidemia-induced endothelial dysfunction remains obscure.In our work, hyperlipidemia animal models was utilized to study whether +Gz could aggravate endothelial dysfunction of hyperlipidemia rats, promote inflammatory reaction or accelerate the process of atherosclerosis, providing experimental evidences for further researching the influences of high load flight operations to the cardiovascular system of pilots with high lipid.Due to the particularity of flight, pilots consider not only the validity but also the safety of drug treatment, as well as the impact on flight safety. Multiple evidences have proved the validity and safety of TCM with multi-site and multi-target in the comprehensive treatment of hyperlipidemia. Ginkgo biloba extract(GBE) is the natural active ingredient extracted from Chinese herbal medicine Ginkgo biloba. Previous studies have found that GBE protected endothelial by antioxidant stress, regulating vasomotor, reducing the adhesion of leukocyte to endothelial cells and inhibit the activation of platelet. Meanwhile GBE has fewer side effects and no affect on cognitive. However, fewer reports have evaluated whether the GBE could protect organism from cardiovascular system damage induced by repeated +Gz acceleration.Here, a +Gz acceleration exposure animal model was used to understand the intervention effects and mechanism of GBE against +Gz acceleration induced vascular injury, provide a reference for further exploration of early drug intervention for pilots with high lipid.Part 1 Effect of repeated positive acceleration exposure on the structure and function of endothelial in hyperlipidemic ratsObjective: 1. To investigate the effect of repeated positive acceleration exposure on the function of endothelium-dependent vasodilation in hyperlipidemic rats2 To investigate the effect of repeated positive acceleration exposure on the vascular Inflammation reaction in hyperlipidemic ratsMethod: Thirty-two male Sprague-Dawley rats(180~200g) were allocated to four groups by random assignment, including a control group(Con), a repeated positive acceleration stress group(+Gz), a high lipid group(HL), and a repeated positive acceleration stress with high lipid group(HL+GG).The Con group and +Gz group were fed with common feed. The HL group and HL+Gz group were administered with high lipid feed. The +Gz group and HL+Gz group were exposed to positive acceleration through the use of a centrifuge every other day. The Con group and HL group were only fixed to the centrifuge without any exposure. The +Gz group was exposed to +10Gz acceleration with an onset rate of 1G/s, sustaining at the peak value for 30 s. The exposure was carried out 3 times per day(1 min interval) for three days a week from week 1-8.The next day of the experiment ended(fasted for 16 h), after general anaesthesia, routine disinfection, and laparotomy were performed for specimen collection. Then the serums were separated, and liver and thoracic aortas were kept. Observed the following indicators:(1)lipid metabolism indicators:TC、LDL-C、HDL-C、TG and ox-LDL;(2) morphological indicators: structure of liver tissue observed by HE staining light microscopy and endothelial ultrastructure observed by arterial tissue TEM;(3)function of endothelium-dependent vasodilation: reaction of endothelium-dependent vasodilation determined by isolated vascular rings experiment;(4) oxidative stress indicators: SOD、GSH-Px、T-AOC、GSH、MDA and NO;(5) Inflammation reaction indicators: IL-1β、IL-6、TNF-α、s ICAM-1 and ET-1;(6) Platelet activation markers: TXB2 and 6-keto-PGF1α;( 7) The protein expressions of NOX4、e NOS、LOX-1、NF-κB p-p65、NF-κB p65 and VAP-1 were measured by Western blot;(8) The protein expressions of NOX4、e NOS、LOX-1and NF-κB p65 m RNA were measured by q RT-PCR;(9) The protein expressions of e NOS and NF-κB p65 were observed by immunohistochemistry.Results: 1.Lipid Metabolism: Compared with control group, the lipid level in +Gz group had no significant difference and TC 、 LDL-C and ox-LDL in serum were increased significantly in HL and HL+Gz group(P<0.05). Compared with the HL group, LDL-C level in HL+Gz group was significantly increased(P<0.05).2. The morphology of tissues2.1 The HE staining light microscopy analysis of liver showed that the structures were intact, morphologies of hepatocyte were clear, cell lines were orderly arranged around the central venous; There no obvious pathological changes were observed in +Gz in Con group. In HL group, the structures of livers were distorted and destroyed, arrangement of liver lines was disorganized, edema could be observed and large number of hollow space was also seen in cytoplasm. In HL+Gz group, the structures of livers were destroyed, patchy necrosis were emerged, arrangement of liver lines was disorganized, edema could be observed, and large number of hollow space could also be seen in cytoplasm.2.2 TEM microscope analysis showed that in Con group, the rules of vascular endothelial cells were tightly, basement membrane intacted, no apophysis and denudation in endothelial cells, Mitochondrial structure was clear. In +Gz group, arrangement of endothelial cells was disorganized, a few of endothelial cells were shedding, mitochondrial swelling was seen. In HL group, of endothelial cells was upheaved, nucleus were shrinkage and deformation, mitochondrial was bunting, and a little of hollow space was denatured. In HL+Gz group, the shed of endothelial cells was increasing, basement membrane rupture was existing, mitochondrial was swelling obviously, cristae was dissolved, shed cellular debris were seen in the cavity.3. Function of endothelium-dependent vasodilation: Ach had a concentration-dependent vasodilation action on each group of vascular. Compared with Con group, the maximum vascular relaxation effect in +Gz group, HL group and HL+Gz group were all significantly decreased(P<0.05); Compared with HL group, the maximum vascular relaxation effect of HL+Gz was further decreased(P<0.05). SNP had a concentration-dependent vasodilation action on each group of vascular without significant differences.4. Oxidative stress indicators4.1 Oxidative stress indicators of serum: Compared with Con group, the serum SDD, GSH-Px and GSH levels of +Gz and HL+Gz group were significantly decreased(P<0.05), MDA was significantly increased(P<0.05), and the levels of GSH-Px and GSH of HL group were significantly decreased(P<0.05). Compared with HL group, the serum SDD and GSH-Px levels of HL+Gz group were further decreased(P<0.05), and MDA level was further increased(P<0.05).4.2 Oxidative stress indicators of vascular: Compared with Con group, the aorta homogenates T-AOC levels of +Gz 、 HL and HL+Gz group were significantly decreased(P<0.05), MDA was significantly increased(P<0.05). Compared with HL group, the aorta homogenates T-AOC level of HL+Gz group was further decreased(P<0.05), and MDA level was further increased(P<0.05). NO levels was decreased, but no significant difference.5 Inflammation reaction indicators5.1 Inflammation reaction indicators of serum: Compared with Con group, TNF-α, IL-1 and IL-6 levels of +Gz,HL and HL+Gz group were apparently raised(P<0.05), the adhesion protein s ICAM-1 level of HL and HL+Gz group was significantly increased(P<0.05). Compared with HL group, the IL-1β,IL-6 and TNF-α levels of HL+Gz group were further decreased(P<0.05).5.2 Inflammation reaction indicators of vascular: Compared with Con group, the levels of IL-6 and ET-1 in +Gz,HL and HL+Gz group were significantly decreased significantly. Compared with HL group, the IL-6 and ET-1 levels of HL+Gz group were further increased(P<0.05).6 Indicators of platelet activation: Compared with Con group, the serum TXB2 levels of HL and HL+Gz group were significantly increased(P<0.05),with the 6-keto-PGF1α levels decreasing significantly, and the ratio of TXB2 to 6-keto-PGF1α was out of balance. Compared with HL group, the serum TXB2 level of HL+Gz group was further increased(P<0.05), the ratio of TXB2 to 6-keto-PGF1α was further losing balance, and there was no significant difference in the level of 6-keto-PGF1α.7. The result of western blot showed that compared with Con group, the aortic e NOS level of +Gz、HL and HL+Gz group was significantly decreased(P<0.05), in which the NOX4 and VAP-1level was significantly increased(P<0.05), the aortic LOX-1 level of HL and HL+Gz group was significantly increased(P<0.05), the p-p65/p65 level in HL+Gz group was significantly decreased(P<0.05). Compared with HL group, the e NOS level of HL+Gz group was significantly decreased, but the NOX4,LOX-1,VAP-1 and p-p65/p65 levels were all significantly increased(P<0.05).8. The result of q RT-PCR showed that compared with Con group, the aortic e NOS level of HL+Gz group was significantly decreased(P<0.05); the NOX4、LOX-1 and NF-κB p65 m RNA levels in group +Gz, HL and HL+Gz were significantly increased(P<0.05). Compared with HL group, the aortic e NOS level of HL+Gz group was significantly decreased(P<0.05) and the NOX4、LOX-1 and NF-κB p65 m RNA levels were significantly increased.9. The result of immunohistochemistry showed that e NOS expressed a strong positive staining on artery endothelial in Con group, weak positive staining in group +Gz and HL and weaker in group +Gz +HL. NF-κB p65 showed weak positive staining on aortic wall in group Con, and positive staining was increased in group +Gz and HL. NF-κB p65 expressed a strong positive staining on artery endothelial in HL+Gz group. Meanwhile in nuclear, the expression of NF-κB was significantly increased.Conclusion: 1. Repeated +Gz acceleration aggravated the dyslipidemia and oxidative stress damage in hyperlipidemia rat and it also aggravated the vascular dysfunction of endothelium-dependent relaxation. The mechanism may be that +Gz stress further up-regulated the expression of NOX4, increased the level of oxidative stress, decreased the expression of e NOS.2. Repeated +Gz acceleration exposure improved Inflammation reaction in hyperlipidemia rat and aggravated the ultrastructure of vascular endothelial and inflammatory injury. The mechanism may be that +Gz stress promoted platelet activation, further up-regulated the expression of LOX-1, activated NF-κB and increased the production of pro-inflammatory cytokine.Part 2 The protective effect of GBE to the endothelial of repeated +Gz acceleration exposure induced hyperlipidemia ratsObjective: 1 To research the protective effect of GBE to the function of vascular endothelium-dependent vasodilation in hyperlipidemia rats induced by repeated +Gz acceleration exposure.2 To research the Intervention effect of GBE to inflammatory injury of vascular in hyperlipidemia rats induced by repeated +Gz acceleration exposureMethod: Fifty male Sprague-Dawley rats(180~200g) were allocated to five groups by random assignment, including a control group(Con), a high lipid with +Gz stress group(HL+Gz), a low dosage group of GBE(GBE-L), a middle dosage group of GBE(GBE-M),and a high dosage group of GBE(GBE-H). and a repeated positive acceleration stress with high lipid group(HL+Gz).The rats of con group were fed with common feed. The HL+Gz, GBE-L、GBE-M and GBE-H group were administered with high lipid feed and exposed to positive acceleration through the use of a centrifuge every other day. The Con group was only fixed to the centrifuge without any exposure. The peak value of +Gz was set up to+10Gz, with an onset rate of 1G/s, sustaining at the peak value for 30 s. The exposure was carried out 3 times per day(1 min interval) for three days a week from week 1-8.The rats of intervention group were prophylactically administered by gavage at about 8:00 am(before exposure) at a dose of 10 m L/kg(body weight) for 8 weeks. The low, middle and high dose groups of GBE were administered at a daily dose of 50 mg/kg, 100 mg/kg and 200 mg/kg(equal to 10 m L/kg), respectively. The control group and HL+Gz group were administered at a daily dose equal aqueous solution. The next day of the experiment ended(fasted for 16 h), after general anaesthesia, routine disinfection, and laparotomy were performed for specimen collection. Then the serums were separated, and liver and thoracic aorta were kept. Indicators needed to observe were as part1.Result: 1.Lipid Metabolism: Compared with HL+Gz group, the serum levels of TC and LDL-C in GBE-L,GBE-M and GBE-H group were raised apparently and ox-LDL level of GBE-M and GBE-H were significantly decreased in HL and HL+Gz group(P<0.05).Compared with the GBE-L, the levels of TC、LDL-C and ox-LDL in GBE-M and GBE-H were further increased(P<0.05).2. The morphology of tissues2.1 The HE staining light microscopy analysis of liver showed that the structures were intact, morphologies of hepatocyte were clear, cell lines were orderly arranged around the central venous; In HL+Gz group, the structures of livers were destroyed, patchy necrosis were emerged, arrangement of liver lines was disorganized, edema could be observed, and large number of hollow space could also be seen in cytoplasm. Compared with HL+Gz, the damages of GBE-L, GBE-M and GBE-H were relieved, edema reduced, number of hollow space was also decreased.2.2 TEM microscope analysis showed that in Con group, the rules of vascular endothelial cells were tightly, basement membrane intacted, no apophysis and denudation in endothelial cells, Mitochondrial structure was clear. In HL+Gz group, sheds of endothelial cells were increased, basement membrane rupture was existing, mitochondrial was swelling obviously, cristae was dissolved, shed cellular debris were seen in the cavity. In GBE-L group, the arrangement of endothelial cells was rupture with shedding, part of cristae was dissolved. In GBE-M and GBE-H groups, endothelial cells were swelled, a few of mitochondria were edema and cristae was dissolved.3. Function of endothelium-dependent vasodilation: Ach had a concentration-dependent vasodilation action on each group of vascular. Compared with HL+Gz group, the maximum vascular relaxation effect in the GBE-M and GBE-H groups were both apparently sinked(P<0.05). SNP had a concentration-dependent vasodilation action on each group of vascular without significant differences.4. Oxidative stress indicators4.1 Oxidative stress indicators of serum: Compared with HL+Gz group, the serum GSH-Px and GSH levels of GBE-L group were apparently increased(P<0.05), SOD, GSH-Px and GSH was significantly increased in GBE-M and GBE-H(P<0.05), MDA level was significantly increased(P<0.05). Compared with GBE-L, the serum levels of SOD 、 GSH-Px and GSH in GBE-M and GBE-H group were were significantly increased(P<0.05) and MDA level was significantly increased(P<0.05).4.2 Oxidative stress indicators of vascular: Compared with HL+Gz group, the aorta homogenates T-AOC levels of GBE-M and GBE-H group were apparently raised(P<0.05), MDA was apparently sinked(P<0.05) and there was no significant difference for NO. Compared with GBE-L group, the aorta homogenates T-AOC level of GBE-H group was significantly decreased(P<0.05), and MDA level was further increased(P<0.05). NO levels was decreased, there were no significant differences for NO and MDA.5 Inflammation reaction indicators5.1 Inflammation reaction indicators of serum: Compared with HL+Gz group, IL-6, TNF-α and s ICAM-1levels of GBE groups were significantly decreased(P<0.05), IL-1β level in GBE-M and GBE-H groups were significantly decreased(P<0.05). Compared with GBE-L group, IL-1β, TNF-α and s ICAM-1levels of GBE-M and GBE-H groups were further decreased(P<0.05), level of IL-6 in GBE-H was further decreased(P<0.05). Compared with GBE-M group, the level of TNF-α in GBE-H was further decreased(P<0.05).5.2 Inflammation reaction indicators of vascular: Compared with HL+Gz group, the levels of IL-6 in GBE groups were significantly decreased significantly(P<0.05), ET-1 levels in GBE-M and GBE-H were significantly decreased(P<0.05). Compared with GBE-L group, the IL-6 and ET-1 levels of GBE-M and GBE-H groups were further decreased(P<0.05).6 Indicators of platelet activation: Compared with HL+Gz group, the serum TXB2 levels in each GBE groups was apparently raised(P<0.05),with the 6-keto-PGF1α levels decreasing significantly. Compared with GBE-L group, the serum6-keto-PGF1αlevel of GBE-H group was significantly increased(P<0.05), the unbalanced ratio of TXB2 to 6-keto-PGF1α was adjusted by drug.7. The result of western blot suggested that compared with HL+Gz group, the aortic e NOS level of GBE groups were significantly increased(P<0.05), the aortic LOX-1 level was significantly decreased(P<0.05); the NOX4, VAP-1, NF-κB p-p65/p65 levels were significantly decreased in GBE-M and GBE-H groups(P<0.05). Compared with GBE-L group, the NOX4, VAP-1, NF-κB p-p65/p65 levels were significantly decreased in GBE-M and GBE-H group(P<0.05).8. The result of q RT-PCR suggested that compared with HL+Gz group, the aortic NOX4 m RNA levels of GBE-M and GBE-H groups were significantly decreased(P<0.05); the e NOS m RNA level was significantly increased(P<0.05),LOX-1 and NF-κB p65 m RNA levels in group GBE-L and GBE-H groups were significantly decreased(P<0.05). Compared with GBE-L group, the aortic NOX4、LOX-1 and NF-κB p65 m RNA levels of GBE-M and GBE-H groups were significantly decreased(P<0.05) and the e NOS level was significantly increased(P<0.05).9. The result of immunohistochemistry showed that e NOS expressed a strong positive staining on artery endothelial in Con group, weak positive staining in groups GBE and weaker in group HL+Gz. NF-κB p65 also showed less expressions on aortic wall but increased in group HL+Gz and significantly increased in nuclear and downward in GBE groups.Conclusion: 1 Dyslipidemia and oxidative stress injury of repeated +Gz acceleration exposure induced hyperlipidemic rats could be dose-dependently relieved by GBE, which could also ease the disfunction of endothelium-dependent vasodilatation. The mechanism may be that GBE down-regulated the expression of NOX4, decreased the level of oxidative stress and increased the e NOS level.2 Inflammation reaction of repeated +Gz acceleration exposure induced hyperlipidemic rats could be dose-dependently reduced by GBE, which also eased the injury of ultrastructure of endothelial and inflammation. The mechanism may be that GBE reduced ox-LDL levels, antagonized platelet activation, down-regulated LOX-1 expression, Inhibited NF-κB and decreased the generation of proinflammatory factors.Part 3 Metabolomics reveals the protective effect of Ginkgo biloba extract in a positive acceleration induced rat modelObjective: 1 To research the effects of +Gz acceleration exposure to metabolic profile of serum.2 To research the protective effects of GBE to the metabolic profile of serum of +Gz acceleration exposure induced rats.Method: A total of 60 male SD rats were allowed to acclimatize for one week and then were allocated to five groups by random assignment, including a control group(CG), a model group(MG), a high dosage group of GBE(HDG), a middle dosage group of GBE(MDG) and a low dosage group of GBE(LDG). The control group and model group were administered orally at a daily dose 10 m L/kg(body weight) 0.5% carboxymethyl cellulose sodium aqueous solution(CMC-Na) from day 1 to day 14. The low, middle and high dose groups of GBE were administered intragastrically at a daily dose of 50 mg/kg, 100 mg/kg and 200 mg/kg(equal to 10 m L/kg), respectively, from days 1–14. All drugs were dissolved in a 0.5% CMC-Na aqueous solution。On day 14, the rats of model group, the low, middle and high dose groups of GBE and were exposed to positive acceleration through the use of a centrifuge. Rats of control group were put in to a plexiglass box and fixed without any exposure. +Gz acceleration exposure peak value was set up to 10 Gz for 5min.The onset/offset rate was+1 Gz/s.After a 5 min exposure to acceleration, 1m L of whole blood was collected in 1.5 m L polythene centrifuge tubes from the retro-orbital venous sinus for metabolomics analysis. After, general anaesthesia, routine disinfection, and laparotomy were performed for specimen collection. blood sample was drawn from the inferior vena cava for biochemical assay.Positive acceleration(+Gz)-induced metabolic perturbations and the protective effect of Ginkgo biloba extract in a rat model were analyzed by metabolomics based on ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF MS). +Gz acceleration exposure related potential biomarkers were selected by principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA). Metabolites were identified by compared with the retention, MS and MS/MS of standard compounds.Results:1.Serum biochemical assay:Compared with the control group, levels of T-AOC、T-SOD、GR and GPx activities in model group were marked decreases(P<0.05),The MDA content was significantly increased in MG. There was no apparent difference in the low dose of GBE relative to CG.2. Serum metabolic profiling: The metabolic state of the model group was distant from the normal group; The middle dosage group of GBE(MDG) and high dosage group of GBE(HDG) were distinct from the model group(MG) but similar to the control group(CG), suggesting that the middle and high dosage of GBE has a significant effect on the recovery of the +Gz-induced the disturbed metabolism state and the high-dose was better.3. Potential biomarkers related to +Gz: Eighteen related +Gz potential biomarkers were tentatively identified by multivariate statistical analysis. Compared with control group, in the fatty acid β-oxidation pathway, the levels of palmitic acid and oleic acid were up regulated, Linoleyl carnitine and vaccenyl carnitine levels were down-regulated. The levels of(Lyso PE(0:0/18:2)), Lyso PC(14:0), Lyso PC(15:0), Lyso PC(18:1), Lyso PC(17:0), Lyso PC(20:2) and Lyso PC(20:1) in glycerophospholipid metabolism pathway were decreased. The levels of sphingolipid metabolism related sphingosine 1-phosphate and sphinganine 1-phosphate were increased; Levels of Bile acid metabolism related taurocholic acid and tauroursodeoxycholic acid were increased. The purine metabolism related allantoin and uric acid levels were increased. And the lysine metabolism related lysine was decreased.4. Effect of GBE to potential biomarkers related to +Gz: eleven of eighteen biomarkers could be regulated to normal levels by a high dosage of GBE, including Linoleyl carnitine, vaccenyl carnitine, palmitic acid and oleic acid, all of which are involved in the fatty acid β-oxidation pathway; uric acid and allantoin, which are involved in purine metabolism, lysine involved in lysine metabolism and Lyso PC(14:0), Lyso PC(15:0), Lyso PC(18:1), Lyso PC(17:0) which are involved in glycerophospholipid metabolism.Conclusion: 1. The metabolic profile serum was distinct from the normal because of +Gz acceleration exposure, involving fatty acid β-oxidation pathway, glycerophospholipid metabolism, phospholipid metabolism, bile acid metabolism, purine metabolism and lysine metabolism.2. GBE could lead the +Gz acceleration induced deflected metabolic profile close to the normal, improving the perturbations of fatty acid β-oxidation pathway, glycerophospholipid metabolism, purine metabolism and lysine metabolism and reducing the damage caused by +Gz acceleration exposure. |
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