| Hemorrhagic shock is one of the common obstetric complications,severe cases can lead to maternal multiple organ dysfunction,such as acute renal failure,disseminated intravascular coagulation(DIC),acute lung injury(ALI),acute respiratory distress sydrome(ARDS) and so on.It can lead maternal to death if the disease continues to develop.After hemorrhagic shock,blood of various organs began to redistribute that is priority supply to vital organs such as heart and brain,blood supply decreasing in skin and internal organs such as liver and kidney.However,kidney burden is to exacerbate because of increasing metabolism of the maternal and the effect of the fetus metabolism.Therefore,when hemorrhagic shock happens,renal function is more easily to be damaged.Lage number of experimental and clinical studies have found that kidney is one of the first organs to be damaged after shock.Renal injury can lead to infiltration of neutrophils, expression of cytokine and enhanced oxidative stress which can mediate inflammatory response and aggravate injuries of distant organs,such as heart,liver,lung,intestine and brain.So,it is particularly important to protect renal function after hemorrhagic shock.Nowadays,the initial treatment of hemorrhagic shock remains fluid resuscitation.Fluid resuscitation on the one hand can restore vascular volume and stabilize blood pressure which plays a positive role on successful treatment.However,a lot of animal experiments and clinical observations noted that fluid resuscitation on the other hand can lead a large number of inflammatory mediators(IL-6,TNF-α) and oxygen free radicals to release which cause the tissues and organs to suffer ischemia-reperfusion injury and give body the second hit.Kidney ischemia-reperfusion is the leading cause of acute renal dysfunction,primarily by increasing the generation of reactive oxygen species,enhancing inflammatory response and sustaining tissue hypoxia.Therefore,we should try first to reduce renal ischemia-reperfusion injury in order to protect maternal renal function following hemorrhagic shock,namely to improve renal blood flow to ensure that the premise of oxygen supply to reduce body’s inflammatory and oxidative stress.Interleukin-6(IL-6),a 26 nucleotide sequence of glycoprotein,is the main proinflammatory cytokines generated when body stress response.It increases rapidly in the serum of patients with acute kidney injury and the extent of increasing can reacts the degree of damage to organs.Tumor necrosis factor alpha(TNF-a)is firstly produced after stess and plays a critical role in inflammatory response.However,mounting evidences indicate that early production of TNF-α maybe closely associated with renal ischemial-reperfusion injury. Superoxide dismutase(SOD) acts in the first lines of defense against oxygen-derived free radicals,and the activity of SOD reflects the cellular capability of scavenging/quenching free radicals.Malondialdehyde(MDA),the degradation product of the oxygen-derived free radicals and lipid oxidation,can reflect the level of oxygen-derived free raicals in the ischemia-reperfusion injury body directly and also may help us to understand the degree of tissue ischemia-reperfusion indirectly.Blood urea nitrogen(BUN) and serum creatinine(Cr),the ultimate metabolic product of protein and muscle respectively,both excrete from kidney.Serum BUN and Cr will increase because glomercular infiltration rate decreased caused by impaired renal function.We can assess the degree of kidney damage by the elevated BUN and Cr,then compare with renal pathology,finally appraise the degree of kidney damage following hemmorrhagic shock.Estrogen mainly comes from follicular cells and granular cells,also adrenal cotex,placenta and males testicles can secret a small number of estrogen.lt can not only affect the repruductive system organs,such as ovary,uterus and breast,but also influence the cardiovascular system by regulating blood lipid and resisting oxidation.Evidences from animal experiments and clinical studies both at home and abroad indicate that estrogen has certain protective effect on renal function of hemorrhagic shock animals.However,there is no report about whether estrgen can be used to protect maternal renal function after hemorrhagic shock.As we all know that pregnancy women have a special changes in hemodynamics and neuroendocrino logy,then we want to know if exogenous estrogen can protect maternal renal function after hemorrhagic shock,how to protect and what the mechanism is.We establish the pregnant rabbit model of hemorrhagic shock to mimic clinical situation,and test the effect of estrogen on renal function,inflammatory response and oxidative stress.PARTER 1 The protective effect of estrogen on renal function after hemorrhagic shock in pregnant rabbitOBJECTIVEBlood urea nitrogen(BUN) and serum creatinine(Cr),the ultimate metabolic product of protein and muscle respectively,both excrete from kidney.Serum BUN and Cr will increase because glomercular infiltration rate decreased caused by impaired renal function.We compared the change of serum BUN and Cr and the renal pathological channge before and after the application of estrogen,then speculated the protective effect of estrogen on renal function after hemorrhagic shock in pregnant rabbit.METHODS1.Set up hemorrhagic shock model of pregnant rabbit:Forty New Zealand pregnant rabbits were anaesthetized and femoral arteriovenous intubation,then establish hemorrhagic shock model of pregnant rabbit.The model consists of four phases:① Acute blood loss phase(0-15min),rabbits were hamorrhaged by blood withdrawal to mean arterial pressure (MAP) 40-45mmHg at an average rate of 2ml/kg/min within 15 minutes;②Shock phase(15-60min),the MAP was maintained within 40-45mmHg for 45 minutes;③Dug treatment phase(60-80min),an introvenous estrogen or fructose was given respectively to the rabbits and then wait for 20 minutes;④ Resuscitation phase(80-260min),the rabbits receives a single volume infusion of 4ml/kg normal saline to make their blood pressure reach 65mmHg and maintain 3 hours.2.Group of experiments:Forty pregnant New Zealand white rabbits at 15-25 days’ gestational age were randomly divided into four groups:Group Ⅰ (shame shock group) served as normal control,animals were underwent anesthesia and cannulation,but no hemorrhge nor fluid resuscitation.Group Ⅱ (shame shock group) is similar with Group Ⅰ excepting an intravenous estrogen was given to them at 60 minutes of the experiment.Group Ⅲ and Group Ⅳ are shock group,both groups were sentenced to bleed during the first 15 minutes,making the rabbits’ blood pressure attach to 40mmHg.then maintaining the pressure for 45 minutes and intravenous injection of estrogen and fructose were given to Group Ⅲ and Group Ⅳ respectively 20 minutes before resuscitation.3.Experimental monitoring:the animals’ MAP,heart rate (HR) and respiration rate (RR) were monitored by Pclab biological signal processing system,Blood samples were collected at 0,60,80 and 260 minutes for determination of serum BUN and Cr concentration.4.Renal pathology:Execution the animals after experiment and collecting left kidney in order to make it underwent pathology examination with HE stained.RESULTS1.Basic status:All groups were comparable in baseline measurement,such as age.body weight and gestational age (p>0.05).2.Hemodynamic changes:MAP of the rabbits before hemorrhagic shock was between (82.8～94.5)mmHg.After blood withdrawl via femoray artery,MAP decreased markedly over a 15 period (0～15min) to a comparable values of (40～45)mmHg in all of the rabbits with hemorrhagic shock.In the shame shock group,Group Ⅰ and Group Ⅱ,MAP remained stable at normotensive levels throughout the experiment. Immediatedly after estrogen infusion, the MAP of Group Ⅲ recovered to (55.9±1.3)mmHg,which was significant higher than Group Ⅳ (49.5±2.0)mmHg and remained at that level for about 10 minutes.After that,MAP was gradually decreased to(52.9±2.3)mmHg,which was still higher than the relative time point of Group Ⅳ which was (43.9±1.2)mmHg.After resuscitation,MAP of the rabbits in Group Ⅲ reached to 65mmHg within 15 minutes while Group Ⅳ needed 20 minutes.This indicated that estrogen coule maintain MAP stable.3.Changes of serum BUN level in different groups:Serum concentration of BUN in all groups was(1.26-3.06) mmol/L at T0 and no statistical difference was found between the groups (p>0.05).There is no obvious change of the serum concentration of BUN between Group Ⅰ and Group Ⅱ at different time points during the whole experiment and it indicated that estrogen had no effect on the level of serum BUN.At 60 minutes of the experiment,the concentration of serum BUN was no obvious change between Group Ⅲ and Group IV;At 80 and 260 minutes of the experiment,the level of serum BUN continued to increase in Group Ⅲ and Group Ⅳ and the level in Group Ⅳ was always higher than in Group Ⅲ,that was (5.04±0.16) mmol/L VS (3.90±0.28) mmol/L,(6.55±0.40) mmol/L vs (5.04±0.98) mmol/L repectively,and there was significant difference between the two groups (p<0.05).Above date demonstrated that estrogen can protect pregnant rabbit renal function after hemorrhagic shock and thus decrease serum BUN level.4. Changes of serum Cr level in different groups:Serum concentration of Cr in all groups was (416.37-492.67) pg/ml at T0 and no statistical difference was found between the groups (p>0.05).There is no obvious change of the serum concentration of Cr between Group Ⅰ and Group Ⅱ at different time points during the whole experiment and it indicated that estrogen had no effect on the level of serum Cr. After hemorrhagic shock,serum Cr level began to increase at 60 minutes of the experiment and the level is higher in Group IV than in Group III,that was (751.17±24.19) pg/ml vs (650.57±8.96) pg/ml,and there was significant difference between the two groups(p<0.05);The level of serum Cr began to decrease at 80 minutes of the experiment and continued to decrease until the end of the experiment,perhaps it was because resuscitation increased blood volume and decreased the concentration of serum Cr,but the level in Group IV was always higher than that in Group III that was (702.38±9.06) pg/ml vs (591.80±12.16) pg/ml, (647.44±20.28) vs (555.10±17.39) pg/ml repectively,and there was significant difference between the two groups (p<0.05).Above date demonstrated that estrogen can protect pregnant rabbit renal function after hemorrhagic shock and thus decrease serum Cr level.5.Kidney pathology in different groups:Kidney tissue in Group IV was damaged most severesly,including edema,degeneration and necrosis in a wide range of renal tubular epithelial,contraction in glomerular capillary loops,lymphosytic infiltration in renal interstitium;However,damage was obviously lighter in Group III than that in Group IV and there was no significant injury in Group I and Group II.This indicated that estrogen can alleviate renal injury after hemorrhagic shock in a certain extent.CONCLUSIONEstrogen has certain protective effect on pregant renal function after hemorrhagic shock,so as to decrease the level of serum BUN and Cr and alleviate the renal pathological injury.PARTER 2 Effect of estrogen on inflammatory cytokines in pregnant rabbit with hemorrhagic shockOBJECTIVEInterleukin-6 (IL-6) is the main proinflammatory cytokines generated when body protecting itself from stress response.It increases rapidly in the serum of patients with acute kidney injury and the extent of increasing can reflect the degree of damage to organs.Tumor necrosis factor alpha (TNF-a)is firstly produced after stess and plays a critical role in inflammatory response.However,mounting evidences indicate that early production of TNF-αmaybe closely associated with renal ischemial-reperfusion injury. We compared the change of serum IL-6 and TNF-αlevel after the application of estrogen,then speculated the probably protective mechanism of estrogen on renal function after hemorrhagic shock in pregnant rabbit.METHODS1.The shock model and experimental groups are the same as capter 1 which had been described in detail.2.Experimental monitoring:the animals’ MAP,heart rate (HR) and respiration rate (RR) were monitored by Pclab biological signal processing system,Blood samples were collected at 0,60,80 and 260 minutes for determination of serum IL-6 and TNF-α concentration.RESULTS1.Changes of serum IL-6 level in different groups:Serum concentration of IL-6 in all groups was (84.62～98.79) ng/L at T0 and no statistical difference was found between the groups (p>0.05).There is no obvious change of the serum concentration of IL-6 between Group Ⅰ and Group Ⅱ at different time points during the whole experiment and it indicated that estrogen had no effect on serum IL-6 level.At 60 and 80 minutes of the experiment,the concentration of serum IL-6 was no obvious change between Group Ⅲ and Group Ⅳ;At 260 minutes of the experiment,the level of serum IL-6 began to increase in Group Ⅲ and Group Ⅳ and the level in Group Ⅳ was higher than that in Group Ⅲ,that was (110.35±.79) ng/L vs (98.25±0.85) ng/L and there was significant difference between the two groups (p<0.05).Above date demonstrated that estrogen can decrease serum IL-6 level and protect pregnant rabbit renal function after hemorrhagic shock.2. Changes of serum TNF-αlevel in different groups:Serum concentration of TNF-α in all groups was (153.37～201.56) ng/L at T0 and no statistical difference was found between the groups (p>0.05).There is no obvious change of serum TNF-α concentration between Group I and Group II at different time points during the whole experiment and it indicated that estrogen had no effect on serum TNF-αlevel.At 60 minutes of the experiment,the concentration of serum TNF-αwas no obvious change between Group Ⅲ and Group Ⅳ;At 80 minutes of the experiment,the level of serum TNF-abegan to increase in Group Ⅲ and Group Ⅳ and the level in Group Ⅳ was higher than that in Group Ⅲ,that was (263.93±7.77) ng/L vs (216.97±18.61)ng/L and there was significant difference between the two groups (p<0.05).At 260 minutes of the experiment,serum TNF-α began to decrease,perhaps it was because resuscitation increased blood volume so as to make serum TNF-αlevel decrease,but the level in Group Ⅳ was still higher than that in Group Ⅲ that was(241.01±6.32) ng/L vs (203.55±7.02) ng/L.Above date demonstrated that estrogen can decrease serum TNF-α level and protect pregnant rabbit renal function after hemorrhagic shock.CONCLUSIONThe application of estrogen can reduce serum IL-6 and TNF-αlevel that increased after hemorrhagic shock.This indicated that estrogen can decrease inflammatory cytokines level,thereby protect renal function against ischemia-reperfusion injury.PARTER 3 Effect of estrogen on oxygen free radicals in pregnant rabbit with hemorrhagic shockOBJECTIVEStudies have found that the change of serum MDA and SOD level can directly reflect the extent of oxidative stress and the degree of tisse ischemia-reperfusion injury.Moreover,existing dates have confirmed that oxidative stress can mediate the kidney ischemia-reperfusion injury.We have already know that estrogen could alleviate the degree of oxidative stress in unpregnant animals with hemorrhagic shock,now,we want to describe its protective effect against oxidative stress in pregant animals.Therefore,we selected serum MDA and SOD level as observation index.METHODS1.The shock model and experimental groups are the same as capter 1 which had been described in detail.2.Experimental monitoring:the animals’ MAP,heart rate (HR) and respiration rate (RR) were monitored by Pclab biological signal processing system,Blood samples were collected at 0,60,80 and 260 minutes for determination of serum MDA and SOD level.RESULTS1.Changes of serum MDA level in different groups:Serum concentration of IL-6 in all groups was (3.86～4.5) mmol/L at TO and no statistical difference was found between the groups (p>0.05).Serum MDA level had no obvious change between Group Ⅰ and Group Ⅱ at different time points during the whole experiment and it indicated that estrogen had no effect on serum MDA level.At 60 minutes of the experiment,serum MDA level had no significant change between Group Ⅲ and Group Ⅳ;At 80 and 260 minutes of the experiment,serum MDA level began to continously increase in Group Ⅲ and Group Ⅳ and the level in Group Ⅳ was always higher than that in Group Ⅲ,that was (5.09±0.37) mmol/L vs (4.78±0.35) mmol/L, (6.29±0.17)mmol/L vs (5.47±0.17)mmol/L respectively and there was significant difference between the two groups (p<0.05).Above dates demonstrated that estrogen can decrease serum MDA level and protect pregnant rabbit renal function after hemorrhagic shock.2. Changes of serum SOD level in different groups:Serum SOD level in all groups was (178.9～198.24) U/L at TO and no statistical difference was found between the groups (p>0.05).Serum SOD level had no obvious change between Group Ⅰ and Group Ⅱ at different time points during the whole experiment and it indicated that estrogen had no effect on serum SOD level.At 60 minutes of the experiment,serum MDA level had no significant change between Group Ⅲ and Group IV;At 80 and 260 minutes of the experiment,serum MDA level began to continously decrease in Group Ⅲ and Group Ⅳ.However,there was no significant difference between Group Ⅲ and Group Ⅳ at 80 minutes of the experiment(p>0.05),both groups had significant difference compared with Group Ⅰ and Group Ⅱ respectively(p<0.05).At 260 minutes of the experiment the level in Group Ⅳ was lower than that in Group Ⅲ,that was (121.64±1.90) U/L vs (151.61±3.35) U/L and there was significant difference between the two groups (p<0.05).Above dates demonstrated that estrogen could increase serum SOD level,thus enhanced pregnant rabbits’ abilily to resist lipid peroxidation and protected cells against injury by oxygen free radicals and protected pregnant rabbits renal function against ischemia-reperfusion injury.CONCLUSIONEstrogen could protect pregant rabbit renal function from ischemia-reperfusion injury through enhancing pregnant rabbits’ ability to resist lipid peroxidation,restraining the rate and intensity of lipid peroxidation effectively,increasing serum SOD level and decreasing serum MDA level relatively and reducing the production of oxygen free radicals.SUMMARY1.In normal pregancy,the rabbit’s blood volume is increasing,blood is relative dilution and in high coagulation status which is similar with human.Combining the characteristics of human pregnancy with hemorrhagic shock,we set up pregnant rabbits model with hemorrhagic shock that could successfully simulate the pathophysiologic process of maternal hemorrhagic shock.2.We successfully set up hemorrhagic shock model in pregnant rabbits that can mimic human maternal hemorrhagic shock.Our research found that MAP of rabbits in E2SG can rise more quckly and it is higher than that in FSG after hemorrhagic shock.Also, MAP of rabbits in E2SG can reach to 65mmHg more quckly than that in FSG.It indicated that estrogen can increase MAP of pregnant rabbits in hemorrhagic shock and make it stable.3.Serum BUN and Cr level increased after hemorrhagic shock,both were relatively lower after the application of estrogen than that of fructos solution(p<0.05).Also,renal pathological injuries induced by hemorrhagic shock were lighter after the application of estrogen than that of fructos solution.These demonstrated that estrogen could protect pregnant rabbit renal function after hemorrhagic shock,decrease serum BUN and Cr level and alleviate renal pathological damage.4.The application of estrogen could relatively decrease serum IL-6,TNF-αand MDA level which were continously increasing after hemorrhagic shock and increased serum SOD level that was decreased after hemorrhagic shock.It indicated that estrogen could protect pregnant rabbit renal function after hemorrhagic shock through reducing inflammatory madiators and strengthening the body’s ability to resist lipid peroxidation.5.This study demonstrated that estrogen could protect renal function after hemorrhagic shock in pregnant rabbit by alleviating inflammatory response and oxidative stress which warrant further investigation and generalization. |
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