束缚应激加重挤压伤大鼠肾损伤的内质网应激机制
摘要
目的：临床资料和法医尸检报告证实，在交通事故、刑讯逼供、虐待等事件中，非致命性机械性损伤后伤员经常发生肾损害、肾衰乃至死亡。由于机械性损伤本身不足以致人死亡，因此常常由于死亡原因不能确定而引起争议，导致案件久拖不决，给社会造成不良影响。此外，在临床实践中也经常发生受伤患者在治疗期间病情加重，致使医务人员难以应对，医患关系紧张。因此研究此类损伤引发肾损伤的确切机制是法医病理学亟待解决的科学技术问题。
损伤作为躯体应激原可引起机体应激反应；此外当事人在遭受损伤后往往会产生焦虑、紧张或抑郁等不良情绪，作为心理社会因素常引起机体超常应激反应，这种情况常被忽视。应激时，机体通过神经内分泌系统的协调作用对应激原作出整体反应，如蓝斑-交感-肾上腺髓质系统和下丘脑-垂体-肾上腺皮质轴的强烈兴奋，并伴有多种内分泌激素的改变。本室前期研究证明，束缚应激可加重挤压伤大鼠肾脏损伤，表明应激在肾损伤发生中可能发挥重要作用，但机制尚不清楚。
内质网是细胞内蛋白质折叠和成熟的场所。大量的研究表明应激激素及其代谢性产物，细胞因子，氧自由基等均可干扰蛋白质加工运输，引发内质网应激(endoplamic reticulum stress, ERS)。ERS是应激时重要的细胞反应之一，其本质上对于增强细胞对损伤的抵抗及适应能力具有重要意义，但过度强烈或持续时间过长的ERS可通过诱导凋亡和炎症反应造成组织、细胞损伤。大量研究表明，ERS可能是多种肾损伤发生的重要因素之一，但非致命性机械性损伤导致肾损伤的机制是否与ERS有关尚不明确。
挤压伤模型是公认的复制肢体软组织挫伤的动物模型，束缚应激模型是公认的复制心理社会应激的动物模型。因此，为模拟实际案情，明确应激性损伤机制，本实验将采用束缚应激加挤压伤的复合模型即应激性损伤模型，探讨束缚应激加重挤压伤大鼠肾损伤的机制是否与ERS有关，为揭示非致命性机械性损伤导致肾损伤的确切机制提供实验依据。此外，实际法医检案中，免疫组织化学(Immunohisto chemistry，IHC)染色技术的应用受到组织固定时间和死后变化等因素的影响。故本实验拟在明确ERS在应激性肾损伤中的作用机制后进一步探讨死后组织固定时间和环境温度对内质网应激相关蛋白抗原稳定性的影响，以期为非致命性机械性损伤及应激参与的死亡案件的死因鉴定提供理论依据，为建立诊断应激性损伤的指标体系提供实验技术方法。
第一部分大鼠应激性损伤模型的建立以及肾脏内质网应激的激活
目的：建立应激性损伤模型，观察大鼠肾损伤的组织形态学变化和超微结构的变化，并从整体水平探讨大鼠应激性肾损伤时，ERS是否参与束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
实验分为正常对照组，禁食水组，束缚应激组，挤压伤组，应激性损伤(即束缚应激+挤压伤复合模型)组，溶剂DMSO对照组，Salubrinal (ERS抑制剂)组，Salubrinal+应激性损伤组，每组5只大鼠。于造模成功后处死大鼠，取材。每天实验后分别称取正常组、禁食水组和束缚应激组大鼠体重，以评估是否建立束缚应激模型。采用高效液相色谱-电化学检测法(highperformance liquid chromatography coupled to electrochemical detection,HPLC-ECD)，检测血浆中NE、E的含量变化；应用Western blot方法检测应激性肾损伤时GRP78蛋白表达水平；采用透射电子显微镜(transmissionelectron microscope, TEM)技术观察大鼠肾脏的超微结构变化；采用苏木素-伊红(Hematoxylin and Eosin, HE)染色法观察大鼠后肢肌肉组织、肾脏损伤的形态学变化；采用苦味酸法检测血浆中肌酐(creatinine, Cre)的含量变化；采用二乙酰肟法检测血尿素氮(blood urea nitrogen, BUN)的含量变化。
结果：
1大鼠后肢肌肉组织病理学观察
与正常对照组相比，挤压伤组和应激性损伤组大鼠后肢肌肉组织均可见横纹肌溶解，间质出血水肿伴炎细胞浸润，该结果提示大鼠存在软组织损伤，故本实验成功的建立了大鼠挤压伤模型。
2大鼠体重的变化
正常对照组大鼠体重逐渐增加。而与正常对照组和禁食水组相比，束缚应激大鼠体重增长明显减缓(p<0.05)，该结果提示本实验成功的建立了束缚应激模型。
3大鼠血浆中NE、E浓度变化
与正常对照组相比，束缚应激组和挤压伤组大鼠血浆中NE、E的浓度明显升高(p<0.05)。该结果提示束缚应激和挤压伤均能引发应激反应。而与挤压伤组相比，应激性损伤组大鼠血浆中NE、E的浓度明显升高(p<0.05)。与正常对照组相比，应激性损伤组大鼠血浆中NE、E的浓度分别增高了2.29倍和3.30倍。这一结果提示，应用束缚应激后挤压的方式成功建立了应激性损伤模型，双重应激原共同刺激引发了机体超常应激反应。
4大鼠肾脏组织超微结构观察
禁食水组大鼠肾小管细胞核周间隙增大，内质网高度扩张，线粒体嵴和膜融合。束缚应激组大鼠肾小管细胞核固缩，核周间隙增大，内质网扩张，线粒体嵴和膜融合，质膜内褶排列紊乱。挤压伤组大鼠肾小管扩张，线粒体嵴和膜模糊融合，粗面内质网空泡化，细胞核轻度固缩。与挤压伤组相比，应激性损伤组大鼠肾脏组织超微结构损伤最明显，肾小管细胞核周间隙增大，染色质轻度边移，核周细胞器减少，线粒体外膜基本消失，内质网扩张，质膜内褶排列紊乱，肾小管细胞膜破裂。该结果提示束缚应激加重挤压伤大鼠肾脏超微结构损伤。
5Western blot检测GRP78蛋白的表达
与挤压伤组相比，应激性损伤组大鼠肾脏GRP78的表达明显增高(p<0.05)，而给予ERS抑制剂Sal可明显抑制应激性损伤诱导的GRP78表达升高(p<0.05)。该结果提示Sal可明显抑制ERS的启动，故本实验进一步观察了应用Sal后应激性肾损伤的形态学变化。
6大鼠肾脏组织病理学观察
与正常对照组相比，挤压伤组大鼠可见肾小球肿胀充血，少量炎细胞浸润及间质充血，而束缚应激组大鼠除可见上述病理改变外，尚可见炎细胞浸润。与挤压伤组大鼠相比，应激性损伤组大鼠肾脏组织病理学变化最明显，可见肾小球肿胀淤血，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。该结果表明束缚应激可加重挤压伤大鼠肾损伤。而应用ERS抑制剂Sal可明显减轻应激性损伤组大鼠肾损伤程度。而DMSO组和Salubrinal组大鼠肾脏仅可见少量的炎细胞浸润。该结果提示ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
7大鼠血浆Cre和BUN含量的变化
与正常对照组相比，束缚应激组和挤压伤组大鼠血浆中Cre和BUN的浓度明显升高(p<0.05)。该结果提示束缚应激和挤压伤均能引发肾损伤。而与挤压伤组相比，应激性损伤组大鼠血浆中Cre和BUN的浓度明显升高(p<0.05)。该结果表明束缚应激可加重挤压伤大鼠肾损伤。而应用ERS抑制剂Sal可明显减轻应激性损伤组大鼠肾损伤程度。该结果提示ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
小结：本部分实验成功建立大鼠应激性损伤模型，ERS抑制剂可减轻应激性肾损伤，初步证实ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
第二部分ERS介导的束缚应激加重挤压伤大鼠肾损伤的机制研究
实验一、ERS介导的束缚应激加重挤压伤大鼠肾损伤的凋亡机制
目的：建立应激性损伤模型，从整体水平研究ERS诱导凋亡是否参与了束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
分组同第一部分实验。应用Western blot方法检测应激性肾损伤时ERS诱导凋亡的特异性蛋白CHOP、caspase-12和凋亡终末执行分子caspase-3蛋白表达水平；采用原位末端标记技术(TdT-mediated dUTP nickend labeling, TUNEL)观察大鼠肾脏的细胞凋亡情况。
结果：
1Western blot检测CHOP、caspase-12和caspase-3蛋白的表达
与正常对照组相比，束缚应激组和挤压伤组大鼠肾脏CHOP、caspase-12和caspase-3表达均明显增高(p<0.05)。与挤压伤组相比，应激性损伤组大鼠肾脏CHOP、caspase-12和caspase-3的表达均更加显著增高(p<0.05)。而应用ERS抑制剂Sal均可明显抑制上述蛋白的变化(p<0.05)。该结果提示束缚应激加重挤压伤大鼠肾损伤的机制与ERS诱导的细胞凋亡有关。
2TUNEL法检测肾脏组织细胞凋亡情况
与对照组相比，束缚应激组和挤压伤组大鼠肾脏组织阳性细胞数目均明显增多(p<0.05)；与挤压组相比，应激性损伤组大鼠肾脏组织的凋亡细胞数更加明显增加(p<0.05)，而应用Sal可使凋亡细胞数明显减少(p<0.05)。该结果证实束缚应激加重挤压伤大鼠肾损伤的机制确实与ERS诱导的细胞凋亡有关。
实验二、ERS介导的束缚应激加重挤压伤大鼠肾损伤的炎症机制
目的：建立应激性损伤模型，从整体水平研究ERS诱导的炎症反应是否参与了束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
分组同第一部分实验。应用Western blot方法检测应激性肾损伤时单核细胞趋化蛋白-1(Monocyte chemoattractant protein-1, MCP-1)的蛋白表达水平；肾组织固定石蜡包埋后，连续切片，采用IHC法观察大鼠肾脏的GRP78和MCP-1的表达情况。
结果：
1Western blot检测大鼠肾脏MCP-1蛋白的表达
与正常对照组相比，束缚应激组和挤压伤组大鼠肾脏MCP-1表达均明显增高(p<0.05)。与挤压伤组相比，应激性损伤组大鼠肾脏MCP-1的表达更加明显增高(p<0.05)，同时，应用Sal可明显抑制MCP-1的表达(p<0.05)。该结果提示束缚应激加重挤压伤大鼠肾损伤的机制与ERS诱导的MCP-1相关的炎症反应有关。
2IHC法检测大鼠肾脏GRP78和MCP-1的表达
与正常对照组相比，连续切片染色可见应激性损伤组大鼠肾脏组织相同部位同时存在明显增高是GRP78和MCP-1的阳性表达。该结果为ERS诱导与MCP-1相关的炎症反应提供了形态学上的证据。
小结：束缚应激加重挤压伤大鼠肾损伤与ERS有关，ERS一方面通过激活CHOP和caspase-12凋亡通路诱导肾脏细胞凋亡，另一方面通过促进MCP-1的表达加重肾脏炎症反应。
第三部分固定时间和环境温度对ERS标志性蛋白GRP78和CHOP蛋白抗原稳定性的影响
实验一、GRP78和CHOP在大鼠应激性肾损伤中的持续表达
目的：由于当事人常经历非致死性创伤后一段时间内才发生死亡，故建立应激性损伤模型，从整体水平研究应激性肾损伤后一段时间内GRP78和CHOP的表达水平变化。
方法：
实验分为正常对照组和应激性损伤后0h,1d,3d,5d及7d组，每组5只大鼠。分别于造模后相应时间点处死大鼠，取材。采用IHC法观察大鼠肾脏的GRP78和CHOP的表达情况。
结果：
IHC半定量分析GRP78和CHOP在大鼠应激性肾损伤的持续表达
应激性损伤后0h-7d大鼠肾脏GRP78和CHOP的表达水平均随时间的延长逐渐升高，在第3天达峰值，之后有所下降，但与对照组相比，均有统计学意义。该结果表明应激性损伤后一段时间内应用IHC技术仍能检测到GRP78和CHOP的表达。实验二、固定时间对GRP78和CHOP蛋白抗原稳定性的影响
目的：研究固定时间对大鼠应激性损伤的肾组织中GRP78和CHOP抗原稳定性的影响。
方法：
实验分为正常对照组和应激性损伤组。每组5只大鼠，于造模后3d处死大鼠，取肾脏分别甲醛固定1d、3d、5d及7d后脱水，石蜡包埋。应用HE染色法观察大鼠肾脏组织形态学的变化；采用IHC发观察固定时间对大鼠肾脏的GRP78和CHOP抗原稳定性的影响。
结果：
1固定不同时间对肾脏HE染色结果的影响
固定时间对两组肾脏组织的HE染色结果没有明显影响。与正常对照组相比，应激性损伤组大鼠肾损伤明显加重，均可见肾小球肿胀淤血，肾小囊闭塞，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。
2固定时间对肾脏GRP78和CHOP抗原稳定性的影响
固定不同时间(3~7d)对肾脏GRP78和CHOP的抗原稳定性未见明显影响。与正常对照组相比，应激性损伤组肾脏GRP78和CHOP表达均明显增高(p<0.05)。
实验三、环境温度对GRP78和CHOP蛋白抗原稳定性的影响
目的：研究环境温度对大鼠应激性损伤后离体肾组织中GRP78和CHOP抗原稳定性的影响。
方法：
实验分为正常对照组和应激性损伤组。每组5只大鼠，于造模后3d处死大鼠，取肾脏：①在4℃下分别放置1d、3d、5d、7d、9d及11d；②在17℃及25℃下分别放置1d、3d、5d及7d后进行取材、固定、包埋。应用HE染色法观察不同环境温度下大鼠肾脏组织形态学的变化；采用IHC法观察环境稳定对大鼠肾脏GRP78和CHOP抗原稳定性的影响。
结果：
1不同环境温度下放置不同时间对肾脏组织结构的影响
不同环境温度下，随着放置时间的延长，肾脏组织自溶变化逐渐明显加重。4℃放置至第9d，17℃和25℃分别放置至第3d，均可见不同程度的肾细胞结构模糊，部分细胞核缺失，胞浆溶解，胞浆呈均质化伊红色。但正常对照组和应激组大鼠肾脏形态学变化已无法区分。
24℃和17℃保存对GRP78和CHOP抗原稳定性的影响
4℃放置1~9d，17℃放置1~3d，应激性损伤组肾脏的GRP78和CHOP的表达强度与范围均逐渐减弱，但与对照组相比，仍有一定差异(p<0.05)。
325℃保存对GRP78和CHOP抗原稳定性的影响
放置1~3d，应激性损伤组肾脏GRP78的表达强度与范围均逐渐减弱，但与对照组相比仍明显增高(p<0.05)。
放置1d，与对照组相比，应激性损伤组肾脏CHOP的表达强度仍明显增强(p<0.05)，但随放置时间延长，其肾脏CHOP的表达强度与范围均逐渐减弱，放置3d~7d与对照组相比无明显差异。
小结：
1GRP78和CHOP抗原稳定性较好，在大鼠应激性损伤后肾脏组织固定7d以内，均可检测到GRP78、CHOP的表达。
2GRP78和CHOP抗原稳定性较好，大鼠应激性损伤后肾脏组织在4℃放置1-9d、17℃和25℃放置1-3d内均可检测到GRP78的表达。CHOP抗原稳定性稍弱于GRP78，大鼠应激性损伤后肾脏组织在4℃放置1-9d、17℃和25℃放置1d内仍能检测到CHOP的表达。
综上，本实验成功建立了大鼠束缚应激和挤压伤复合模型即应激性损伤模型，观察了损伤后大鼠肾脏的病理学变化；检测了肾脏中内质网应激、细胞凋亡及炎症因子表达情况；以及组织固定时间和环境变化对肾脏中GRP78和CHOP抗原稳定的影响，成功地建立了诊断应激性肾损伤的指标体系：
(1)明确的机械性损伤(交通伤、地震、矿难、刑讯逼供等)的体表征象。
(2)肾脏的组织学变化为肾小球肿胀淤血，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。
(3)通过TUNEL可证实肾脏存在细胞凋亡，电镜下也可见肾小管细胞核周间隙增大，染色质轻度边移等凋亡现象。
(4)肾脏ERS特异性蛋白GRP78和CHOP的表达明显增高。但该指标体系尚有待于在实际检案中进行验证。
Objective: Clinical data and forensic autopsy reports confirm thatnon-lethal mechanical trauma, such as soft tissue injuries, traffic accidentinjuries, and confessions obtained by torture, often lead to kidney injury, renalfailure, and even death. Because this kind of injury itself is not sufficient tocause death, it is not yet clear how this kind of damage causes renal injury,which challenges clinicians and confuses clinicians and legal medical experts.Hence, there is an urgent need in forensic pathology to determine themechanism(s) underlying kidney injury following non-fatal mechanicaltrauma.
The damage as a physical stressor caused a stress response; and after thedamage the injured organism would have a bad mood, such as anxiety,helplessness, or depression, which can cause an extraordinary psychologicalstress response, that was often ignored. In response to stress, through thecoordinating role of neuroendocrine system the body made a overall reaction.For example, the hypothalamo-pituitary-adrenal axis and the sympatheticnervous system (SNS) are excited strongly, which results in significantchanges in kinds of endocrine hormones. Our previous studies havedemonstrated that restraint stress can aggravate the kidney damage in crushinjury rats, suggested that stress played an important role in happening ofkidney injury, but the mechanism(s) is not clear.
The endoplasmic reticulum (ER) is the site of protein synthesis and foldingin cell. Lots of studies have confirmed that processing and transport ofproteins can be disturbed by stress hormones, toxic metabolites, cytokines,oxygen radicals and so on, then ER stress (ERS) is activated. ERS is one ofthe importante cell response in stress, that is necessary for cells resisting injuryand adaptive capacity. But excessive ERS not only induces apoptosis, but also activates inflammatory signaling pathways, which can cause tissue and celldamage. Numerous studies have showed that ERS may be one of theimportant factors in a variety of kidney injury, but whether or not ERS isrelated with kidney injury caused by non-lethal mechanical trauma isunclear.
The crush injury model is an accepted animal model to simulate softtissue injuries, while the restraint stress model is an accepted animal model toreproduce psychological stress. Thus, in order to simulate the actual case andclear injury mechanisms, the present study established a stress injury modelconsisting of restraint stress and crush injury to determine whether or notERS is related with the mechanism of that restraint stress can aggravatekidney damage caused by crush injury. Hoping to provide experimentbasis for kidney injury caused by non-lethal mechanical trauma.Additionally, in actual forensic cases, the use of immunohistochemical (IHC)indicators was affected by the tissue fixation time, postmortem changes and soon. So after clearing the mechanisms of ERS in stressful kidney injury, thisstudy further discussed the effects of tissue fixation time and environmentaltemperature on the stability of ERS related proteins, which would providetheoretical basis and experimental methods for setting up diagnosisindicator system of stressful injury.
Part1: The establishment of rat stressful injury model and the activationof renal ERS
Objective: To establish a rat stressful injury model, and to observe thepathologic, ultrastructure changes and to research whether or not restraintstress could aggravate kidney damage caused by crush injury through ERS.
Methods:
The rats were randomly divided into the following8groups (n=5pergroup): control (Con); food and water deprivation (Dep); restraint stress (RS);crush injury (CI); stressful injury (SI); Solvent (dimethyl sulfoxide) control(DMSO); and solute (salubrinal [ERS inhibitor]) control (Sal); Sal+SI. Therats were sacrificed after the end of experimental procedures. On the first day before stress and on the end of daily stress the body weights were recorded inCon, Dep and RS groups, to evaluate whether or not the RS model isestablished. Plasma levels of NE and E were evaluated by high performanceliquid chromatography coupled to electrochemical detection (HPLC-ECD);western blot was used to analysis the GRP78protein level in rat renal stressfulinjury; ultrastructure changes in rat kidney were evaluated by transmissionelectron microscope (TEM); pathologic findings in rat hind limb muscles andrenal injury were evaluated by Hematoxylin and Eosin staining (HE); plasmalevel of creatinine (Cre) was evaluated by picric acid method, and blood ureanitrogen (BUN) was evaluated by diacetyl monoxide's reagent colorimetricmethod.
Results:
1Pathologic findings in hind limb muscles
Rhabdomyolysis, interstitial hemorrhage, edema, and an abundance ofinflammatory cell were observed in the hind limb muscles of the crush andstressful injury groups, compared with the normal histologic structures of thecontrol group. These results suggested that the rats had a soft tissue injury.Therefore, the present experiment successfully established the rat crush injurymodel.
2The changes of rat body weights
Body weight of rats increased gradually in Con group. The weight gain inRS group was slowed down compaired with the control and Dep groups(p<0.05). Therefore, the present study successfully established the rat restraintstress model.
3Catecholamine changes in rat plasma samples
The mild increase in NE and E concentrations following RS and CIsuggested that a stress response was induced. The most significant changes inNE and E concentrations were noted in the SI group, compaired with the CIgroup. The NE and E concentrations in SI group were2.29and3.30timesrespectively higher than these of Con group, suggested that the rat stressfulinjury model was successful established though using restraint stress and crush injury, inducing extraordinary strong stress response caused by double attack.
4Ultrastructure changes in rat kidney
Dep group: the nuclear-week gap was widened, ERs were highlyenlarged, mitochondria crest and membrane were fused. RS group: the nucleiwere pyknotic, nuclear-week gap became widened, ERs were enlarged,mitochondria crest and membrane were fused, the plasma membraneinfoldings were arranged irregularly. CI group: tubules were dilated, ERs werevacuolation, mitochondria crest and membrane were fused. The nuclei weremild pyknotic. SI group, the pathological became more serious as comparedwith crush injury group. The nuclear-week gap was widened, nuclearchromatin located mainly in the nuclear membrane. Some mitochondria withruptured outer membrane, enlarged ER, disordered plasma membraneinfoldings, ruptured cell membrane and cast were observed. Those resultssuggested that restraint stress could aggravate kidney ultrastructure damagecaused by crush injury.
5Western blot analysis of the GRP78protein level
The GRP78protein levels were significantly increased in the SI group ascompared with the CI group (p<0.05). Moreover, the GRP78protein level wassignificantly decreased by an ERS inhibitor, Sal (p<0.05). Those resultssuggested that Sal could obviously suppress the activation of ERS. So thepresent study further observed the pathologic changes in the rat renal stressfulinjury with the Sal treatment.
6Pathologic changes in the kidney
Compared with the normal histologic structures of kidney in Con group,swelling and congestive glomeruli, minimal inflammatory cell infiltration andinterstitial hyperemia were observed in the CI group. In addition to the abovechanges, inflammatory cell infiltration was observed in the RS group. Thepathologic changes became more serious in SI group compared with the CIgroup. Swelling and congestive glomeruli, dropped epithelial cell of renaltubule, lots of inflammatory cell infiltration and interstitial hyperemia wereobserved in the SI group, suggesting that restraint stress could exacerbate rat kidney injury caused by a crush injury. Meanwhile, as an ERS inhibitor, Saltreatment alleviated the kidney injury obviously induced by stressful injury,and a few of inflammatory cells infiltration were observed in DMSO groupand Sal group, suggested that ERS might be one of mechanisms throughwhich restraint stress exacerbated rat kidney injury caused by crush injury.
7Cre and BUN changes in rat plasma samples
The mild increase in Cre and BUN concentrations following RS and CIsuggested that a kidney injury was induced. The most significant changes inCre and BUN concentrations were noted in the SI group, compaired with theCI group (p<0.05), suggested that restraint stress could exacerbate rat kidneyinjury caused by a crush injury. Meanwhile, Sal treatment alleviated thekidney injury obviously induced by stressful injury (p<0.05), suggested thatERS might be one of mechanisms through which restraint stress exacerbatedrat kidney injury caused by crush injury.
Summary: In the present part study, a rat stressful injury model wassuccessful established. The present study found that ERS inhibitor couldreduced the renal stressful injury, and preliminary confirmed that restraintstress could aggravate kidney damage caused by crush injury, and itsunderlying mechanism may be involved with ERS.
Part2: Restraint stress aggravates rat kidney injury caused by a crushinjury through ERS
Experiment1Restraint stress aggravates rat kidney injury caused by acrush injury through apoptosis induced by ERS
Objective: To establish rat stressful injury model, and to researchwhether or not restraint stress aggravates rat kidney injury caused by a crushinjury through apoptosis induced by ERS
Methods:
The rats were randomly divided into groups the same as in part1.Western blot were used to analysis the apoptosis specific proteins induced byERS (CHOP and caspase-12) and an executioner of apoptosis (caspase-3)protein levels in rat renal stressful injury. TdT-mediated dUTP nick end labeling (TUNEL) was used to observe apoptotic renal cells.
Results:
1Western blot analysis of the CHOP, caspase-12, and caspase-3proteinlevels
The CHOP, caspase-12and caspase-3protein levels were significantlyincreased in the RS and CI groups as compared with the control group(p<0.05).Moreover, the levels of the above proteins were most markedly increased in SIgroup, compared with the CI group. While the above changes weresignificantly inhibited by Sal (p<0.05), suggesting that the mechanism of thatrestraint stress aggravates rat kidney injury caused by a crush injury wasrelated with apoptosis induced by ERS.
2Localization of apoptosis by the TUNEL assay
Apoptosis was observed in renal tubules after RS and CI compared withthe control group (p<0.05). The greatest numbers of apoptotic renal tubularepithelial cells were found in SI group, meanwhile Sal decreased the numberof apoptotic cells (p<0.05), confirmed that restraint stress aggravates ratkidney injury caused by a crush injury was indeed related with apoptosisinduced by ERS.
Experiment2Restraint stress aggravates rat kidney injury caused by acrush injury through inflammation induced by ERS
Objective: To establish rat stressful injury model, and to researchwhether or not restraint stress aggravates rat kidney injury caused by a crushinjury through inflammation induced by ERS
Methods:
The rats were randomly divided into groups the same as in part1.Western blot were used to analysis the Monocyte chemoattractantprotein-1(MCP-1) protein levels in rat renal stressful injury. The kidneys werefixed, then embedded in paraffin. Serial sections of kidney were immunohisto-chemically stained for the expressions of GRP78and MCP-1.
Results:
1Western blot analysis of the MCP-1protein level
The MCP-1protein levels were increased significantly in the RS and CIgroups, compared with the control group (p<0.05). Moreover, the MCP-1protein level was most significantly increased in SI group compared with theCI group (p<0.05). And Sal treatment inhibited the MCP-1expression inducedby stressful injury (p<0.05), suggested that ERS induced MCP-1expressionincreased is one of mechanism by which restraint stress aggravates rat kidneyinjury caused by a crush injury.
2IHC analysis of GRP78and MCP-1distribution
GRP78and MCP-1were abundantly expressed in the SI kidneycompared with the control group. We also found that in serial sections, theMCP-1-positive cells also had positive expressions of GRP78. Those resultsprovided a morphological basis for the inflammation related with MCP-1caused by ERS.
Summary: Restraint stress can aggravate kidney damage caused by crushinjury, which was attributed to ERS. ERS not only leads to initiation of therenal apoptotic processes promoted by CHOP and a caspase-12-dependentpathway, but also increase expression of MCP-1to aggravate the renalinflammatory response.
Part3: The effects of the fixed time and environmental temperature onthe antigen stability of ERS markers, GRP78and CHOP
Experiment1The continuous expressions of GRP78and CHOP in ratrenal stressful injury
Objective: Because the injured organism often died within a period oftime after the nonfatal injuries occurred, the aim of the present study is toobserve the expressions of GRP78and CHOP within a period of time after ratstressful injury.
Methods:
Animals were randomly divided into six experimental groups forsampling at0h,1,3,5and7days after SI and a control group (n=5per group).The kidneys were quickly removed and fixed in10%formalin. IHC stainingwas used to observe the expressions of GRP78and CHOP after stressful injury in rat kidneys.
Results:
Semi-quantitative evaluation of IHC staining of GRP78and CHOPcontinuous expressions in the rat renal stressful injury
Within0h-7d after the stressful injury, with the extension of time, theexpressions of GRP78and CHOP all gradually increased (p<0.05), and on the3days it up to a peak. Those results suggested that within a week afterstressful injury, GRP78and CHOP still could be detected by IHC staining.
Experiment2The effects of the fixed time on the antigen stability ofGRP78and CHOP
Objective: To observe the effects of fixed time on the antigen stability ofGRP78and CHOP in rat kidney stressful injury.
Methods:
Animals were randomly divided into a control group and four SI groups(n=5per group). The rats were sacrificed on the3rd day after the end of theexperimental procedures. The kidneys were quickly removed and fixed in10%formalin for different fixation times (1,3,5and7days) following by paraffinembedding. Pathologic changes in kidney were evaluated by HE staining; IHCwere used to observe the effects of fixed time on the antigen stability ofGRP78and CHOP in rat kidney.
Results:
1Pathologic changes in the kidney after stressful injury in samples fixedwithin a week
Fixation time had no significant effect on the kidneys in both of the twogroups. Swelling and congestive glomeruli, narrowed renal capsule,substantial inflammatory cells infiltration, narrowed renal capsule, droppedepithelial cell of renal tubule and interstitial hyperemia were observed in theSI group.
2The stabilities of GRP78and CHOP for fixed within a week
Kidney fixation over3–7days did not adversely affect the levels ofGRP78and CHOP. Compared with the control group, GRP78and CHOP levels were significantly higher in kidney samples from the S1group(p<0.05)。Experiment3The effects of environmental temperature on the antigenstability of GRP78and CHOP
Objective: To observe the effects of environmental temperature on theantigen stability of GRP78and CHOP in rat isolated kidney after stressfulinjury.
Methods:
Animals were randomly divided into two experimental groups (n=5pergroup): control group and stressful injury group. Rats were sacrificed on the3rd day after the end of the experimental procedures.①The kidneys werequickly removed and stored at4°C for sampling at1,3,5,7,9and11days;②The kidneys were quickly removed and stored at17°C and25°C forsampling at1,3,5, and7days respectively. And then the kidneys were fixedin10%formalin for paraffin embedding. Pathologic changes in kidney wereevaluated by HE staining; IHC were used to observe the effects ofenvironmental temperature on the antigen stability of GRP78and CHOP in ratkidney.
Results:
1Pathologic changes in the kidney after stressful injury in samples forstored at different environmental temperature
As kidney storage became more prolonged in different temperature,obvious autolysis occurred. On the9th day for stored at4°C, the3rd day forstored at17°C and25°C respectively, the varying degrees of fuzzy renal cellstructure, missed nucleus, dissolved cytoplasm and homogenization in the redwere all observed. But it is impossible to make a distinction between controlgroup and stressful injury group about the renal pathologic changes.
2The effect of4°C and17°C storage on GRP78and CHOP proteinstability
In the kidneys stored at4°C for1-9days and17°C for1-3days, thelevels of the GRP78and CHOP proteins gradually reduced in their distribution and intensity (SI group), but were still higher than that in the control group(p<0.05).
3The effect of25°C storage on GRP78and CHOP protein stability
In the kidneys stored at25°C for1-3days, the levels of the GRP78protein gradually reduced in their distribution and intensity (SI group), butwere still higher than that in the control group (p<0.05).
In the kidneys stored at25°C for1day, the levels of the CHOP proteingradually reduced in their distribution and intensity (SI group), but were stillhigher than that in the control group (p<0.05).
Summary:
1GRP78and CHOP had good stabilities as antigens in rat kidney, andboth of their expressions could be tested for fixed within a week after ratstressful injury.
2GRP78and CHOP had good stabilities as antigens. After rat stressfulinjury, with the kidneys stored in4°C for1-9days,17°C for1-3days and25°C for1-3days, the expression of GRP78could still be tested. While CHOPhad a weaker stability as antigens than GRP78, it still could be tested with thekidneys stored in4°C for1-9days,17°C for1-3days and25°C for1day.
In summary, a rat stressful injury model consisting of restraint stress andcrush injury was successful established. Pathologic changes in kidney wereobserved after injury; ERS, apoptosis and inflammation were tested in ratrenal injury; and the effects of the fixed time and environmental temperatureon the antigen stability of GRP78and CHOP were tested in rat kidneys. Thediagnosis indicator system of renal stressful injury was successful established:
(1) The clear surface sign of mechanical trauma (traffic accident injuries,earth-quake, mine disaster, confessions obtained by torture and so on) wasobserved.
(2) Pathologic changes: swelling and congestive glomeruli, dropped epithelialcell of renal tubule, lots of inflammatory cell infiltration and interstitialhyperemia were observed in the kidney.
(3) Localization of apoptosis by the TUNEL assay was observed and ultrastructure changes in rat kidney were evaluated by TEM, includingwidened nuclear-week gap, nuclear chromatin located mainly in the nuclearmembrane and so on, which suggesting apoptosis.
(4) ERS markers, GRP78and CHOP were significantly increased in thekidney.
But the diagnosis indicator system still needs to be validated in actual forensiccases.
损伤作为躯体应激原可引起机体应激反应；此外当事人在遭受损伤后往往会产生焦虑、紧张或抑郁等不良情绪，作为心理社会因素常引起机体超常应激反应，这种情况常被忽视。应激时，机体通过神经内分泌系统的协调作用对应激原作出整体反应，如蓝斑-交感-肾上腺髓质系统和下丘脑-垂体-肾上腺皮质轴的强烈兴奋，并伴有多种内分泌激素的改变。本室前期研究证明，束缚应激可加重挤压伤大鼠肾脏损伤，表明应激在肾损伤发生中可能发挥重要作用，但机制尚不清楚。
内质网是细胞内蛋白质折叠和成熟的场所。大量的研究表明应激激素及其代谢性产物，细胞因子，氧自由基等均可干扰蛋白质加工运输，引发内质网应激(endoplamic reticulum stress, ERS)。ERS是应激时重要的细胞反应之一，其本质上对于增强细胞对损伤的抵抗及适应能力具有重要意义，但过度强烈或持续时间过长的ERS可通过诱导凋亡和炎症反应造成组织、细胞损伤。大量研究表明，ERS可能是多种肾损伤发生的重要因素之一，但非致命性机械性损伤导致肾损伤的机制是否与ERS有关尚不明确。
挤压伤模型是公认的复制肢体软组织挫伤的动物模型，束缚应激模型是公认的复制心理社会应激的动物模型。因此，为模拟实际案情，明确应激性损伤机制，本实验将采用束缚应激加挤压伤的复合模型即应激性损伤模型，探讨束缚应激加重挤压伤大鼠肾损伤的机制是否与ERS有关，为揭示非致命性机械性损伤导致肾损伤的确切机制提供实验依据。此外，实际法医检案中，免疫组织化学(Immunohisto chemistry，IHC)染色技术的应用受到组织固定时间和死后变化等因素的影响。故本实验拟在明确ERS在应激性肾损伤中的作用机制后进一步探讨死后组织固定时间和环境温度对内质网应激相关蛋白抗原稳定性的影响，以期为非致命性机械性损伤及应激参与的死亡案件的死因鉴定提供理论依据，为建立诊断应激性损伤的指标体系提供实验技术方法。
第一部分大鼠应激性损伤模型的建立以及肾脏内质网应激的激活
目的：建立应激性损伤模型，观察大鼠肾损伤的组织形态学变化和超微结构的变化，并从整体水平探讨大鼠应激性肾损伤时，ERS是否参与束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
实验分为正常对照组，禁食水组，束缚应激组，挤压伤组，应激性损伤(即束缚应激+挤压伤复合模型)组，溶剂DMSO对照组，Salubrinal (ERS抑制剂)组，Salubrinal+应激性损伤组，每组5只大鼠。于造模成功后处死大鼠，取材。每天实验后分别称取正常组、禁食水组和束缚应激组大鼠体重，以评估是否建立束缚应激模型。采用高效液相色谱-电化学检测法(highperformance liquid chromatography coupled to electrochemical detection,HPLC-ECD)，检测血浆中NE、E的含量变化；应用Western blot方法检测应激性肾损伤时GRP78蛋白表达水平；采用透射电子显微镜(transmissionelectron microscope, TEM)技术观察大鼠肾脏的超微结构变化；采用苏木素-伊红(Hematoxylin and Eosin, HE)染色法观察大鼠后肢肌肉组织、肾脏损伤的形态学变化；采用苦味酸法检测血浆中肌酐(creatinine, Cre)的含量变化；采用二乙酰肟法检测血尿素氮(blood urea nitrogen, BUN)的含量变化。
结果：
1大鼠后肢肌肉组织病理学观察
与正常对照组相比，挤压伤组和应激性损伤组大鼠后肢肌肉组织均可见横纹肌溶解，间质出血水肿伴炎细胞浸润，该结果提示大鼠存在软组织损伤，故本实验成功的建立了大鼠挤压伤模型。
2大鼠体重的变化
正常对照组大鼠体重逐渐增加。而与正常对照组和禁食水组相比，束缚应激大鼠体重增长明显减缓(p<0.05)，该结果提示本实验成功的建立了束缚应激模型。
3大鼠血浆中NE、E浓度变化
与正常对照组相比，束缚应激组和挤压伤组大鼠血浆中NE、E的浓度明显升高(p<0.05)。该结果提示束缚应激和挤压伤均能引发应激反应。而与挤压伤组相比，应激性损伤组大鼠血浆中NE、E的浓度明显升高(p<0.05)。与正常对照组相比，应激性损伤组大鼠血浆中NE、E的浓度分别增高了2.29倍和3.30倍。这一结果提示，应用束缚应激后挤压的方式成功建立了应激性损伤模型，双重应激原共同刺激引发了机体超常应激反应。
4大鼠肾脏组织超微结构观察
禁食水组大鼠肾小管细胞核周间隙增大，内质网高度扩张，线粒体嵴和膜融合。束缚应激组大鼠肾小管细胞核固缩，核周间隙增大，内质网扩张，线粒体嵴和膜融合，质膜内褶排列紊乱。挤压伤组大鼠肾小管扩张，线粒体嵴和膜模糊融合，粗面内质网空泡化，细胞核轻度固缩。与挤压伤组相比，应激性损伤组大鼠肾脏组织超微结构损伤最明显，肾小管细胞核周间隙增大，染色质轻度边移，核周细胞器减少，线粒体外膜基本消失，内质网扩张，质膜内褶排列紊乱，肾小管细胞膜破裂。该结果提示束缚应激加重挤压伤大鼠肾脏超微结构损伤。
5Western blot检测GRP78蛋白的表达
与挤压伤组相比，应激性损伤组大鼠肾脏GRP78的表达明显增高(p<0.05)，而给予ERS抑制剂Sal可明显抑制应激性损伤诱导的GRP78表达升高(p<0.05)。该结果提示Sal可明显抑制ERS的启动，故本实验进一步观察了应用Sal后应激性肾损伤的形态学变化。
6大鼠肾脏组织病理学观察
与正常对照组相比，挤压伤组大鼠可见肾小球肿胀充血，少量炎细胞浸润及间质充血，而束缚应激组大鼠除可见上述病理改变外，尚可见炎细胞浸润。与挤压伤组大鼠相比，应激性损伤组大鼠肾脏组织病理学变化最明显，可见肾小球肿胀淤血，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。该结果表明束缚应激可加重挤压伤大鼠肾损伤。而应用ERS抑制剂Sal可明显减轻应激性损伤组大鼠肾损伤程度。而DMSO组和Salubrinal组大鼠肾脏仅可见少量的炎细胞浸润。该结果提示ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
7大鼠血浆Cre和BUN含量的变化
与正常对照组相比，束缚应激组和挤压伤组大鼠血浆中Cre和BUN的浓度明显升高(p<0.05)。该结果提示束缚应激和挤压伤均能引发肾损伤。而与挤压伤组相比，应激性损伤组大鼠血浆中Cre和BUN的浓度明显升高(p<0.05)。该结果表明束缚应激可加重挤压伤大鼠肾损伤。而应用ERS抑制剂Sal可明显减轻应激性损伤组大鼠肾损伤程度。该结果提示ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
小结：本部分实验成功建立大鼠应激性损伤模型，ERS抑制剂可减轻应激性肾损伤，初步证实ERS可能参与了束缚应激加重挤压伤大鼠肾损伤的过程。
第二部分ERS介导的束缚应激加重挤压伤大鼠肾损伤的机制研究
实验一、ERS介导的束缚应激加重挤压伤大鼠肾损伤的凋亡机制
目的：建立应激性损伤模型，从整体水平研究ERS诱导凋亡是否参与了束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
分组同第一部分实验。应用Western blot方法检测应激性肾损伤时ERS诱导凋亡的特异性蛋白CHOP、caspase-12和凋亡终末执行分子caspase-3蛋白表达水平；采用原位末端标记技术(TdT-mediated dUTP nickend labeling, TUNEL)观察大鼠肾脏的细胞凋亡情况。
结果：
1Western blot检测CHOP、caspase-12和caspase-3蛋白的表达
与正常对照组相比，束缚应激组和挤压伤组大鼠肾脏CHOP、caspase-12和caspase-3表达均明显增高(p<0.05)。与挤压伤组相比，应激性损伤组大鼠肾脏CHOP、caspase-12和caspase-3的表达均更加显著增高(p<0.05)。而应用ERS抑制剂Sal均可明显抑制上述蛋白的变化(p<0.05)。该结果提示束缚应激加重挤压伤大鼠肾损伤的机制与ERS诱导的细胞凋亡有关。
2TUNEL法检测肾脏组织细胞凋亡情况
与对照组相比，束缚应激组和挤压伤组大鼠肾脏组织阳性细胞数目均明显增多(p<0.05)；与挤压组相比，应激性损伤组大鼠肾脏组织的凋亡细胞数更加明显增加(p<0.05)，而应用Sal可使凋亡细胞数明显减少(p<0.05)。该结果证实束缚应激加重挤压伤大鼠肾损伤的机制确实与ERS诱导的细胞凋亡有关。
实验二、ERS介导的束缚应激加重挤压伤大鼠肾损伤的炎症机制
目的：建立应激性损伤模型，从整体水平研究ERS诱导的炎症反应是否参与了束缚应激加重挤压伤大鼠肾损伤的过程。
方法：
分组同第一部分实验。应用Western blot方法检测应激性肾损伤时单核细胞趋化蛋白-1(Monocyte chemoattractant protein-1, MCP-1)的蛋白表达水平；肾组织固定石蜡包埋后，连续切片，采用IHC法观察大鼠肾脏的GRP78和MCP-1的表达情况。
结果：
1Western blot检测大鼠肾脏MCP-1蛋白的表达
与正常对照组相比，束缚应激组和挤压伤组大鼠肾脏MCP-1表达均明显增高(p<0.05)。与挤压伤组相比，应激性损伤组大鼠肾脏MCP-1的表达更加明显增高(p<0.05)，同时，应用Sal可明显抑制MCP-1的表达(p<0.05)。该结果提示束缚应激加重挤压伤大鼠肾损伤的机制与ERS诱导的MCP-1相关的炎症反应有关。
2IHC法检测大鼠肾脏GRP78和MCP-1的表达
与正常对照组相比，连续切片染色可见应激性损伤组大鼠肾脏组织相同部位同时存在明显增高是GRP78和MCP-1的阳性表达。该结果为ERS诱导与MCP-1相关的炎症反应提供了形态学上的证据。
小结：束缚应激加重挤压伤大鼠肾损伤与ERS有关，ERS一方面通过激活CHOP和caspase-12凋亡通路诱导肾脏细胞凋亡，另一方面通过促进MCP-1的表达加重肾脏炎症反应。
第三部分固定时间和环境温度对ERS标志性蛋白GRP78和CHOP蛋白抗原稳定性的影响
实验一、GRP78和CHOP在大鼠应激性肾损伤中的持续表达
目的：由于当事人常经历非致死性创伤后一段时间内才发生死亡，故建立应激性损伤模型，从整体水平研究应激性肾损伤后一段时间内GRP78和CHOP的表达水平变化。
方法：
实验分为正常对照组和应激性损伤后0h,1d,3d,5d及7d组，每组5只大鼠。分别于造模后相应时间点处死大鼠，取材。采用IHC法观察大鼠肾脏的GRP78和CHOP的表达情况。
结果：
IHC半定量分析GRP78和CHOP在大鼠应激性肾损伤的持续表达
应激性损伤后0h-7d大鼠肾脏GRP78和CHOP的表达水平均随时间的延长逐渐升高，在第3天达峰值，之后有所下降，但与对照组相比，均有统计学意义。该结果表明应激性损伤后一段时间内应用IHC技术仍能检测到GRP78和CHOP的表达。实验二、固定时间对GRP78和CHOP蛋白抗原稳定性的影响
目的：研究固定时间对大鼠应激性损伤的肾组织中GRP78和CHOP抗原稳定性的影响。
方法：
实验分为正常对照组和应激性损伤组。每组5只大鼠，于造模后3d处死大鼠，取肾脏分别甲醛固定1d、3d、5d及7d后脱水，石蜡包埋。应用HE染色法观察大鼠肾脏组织形态学的变化；采用IHC发观察固定时间对大鼠肾脏的GRP78和CHOP抗原稳定性的影响。
结果：
1固定不同时间对肾脏HE染色结果的影响
固定时间对两组肾脏组织的HE染色结果没有明显影响。与正常对照组相比，应激性损伤组大鼠肾损伤明显加重，均可见肾小球肿胀淤血，肾小囊闭塞，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。
2固定时间对肾脏GRP78和CHOP抗原稳定性的影响
固定不同时间(3~7d)对肾脏GRP78和CHOP的抗原稳定性未见明显影响。与正常对照组相比，应激性损伤组肾脏GRP78和CHOP表达均明显增高(p<0.05)。
实验三、环境温度对GRP78和CHOP蛋白抗原稳定性的影响
目的：研究环境温度对大鼠应激性损伤后离体肾组织中GRP78和CHOP抗原稳定性的影响。
方法：
实验分为正常对照组和应激性损伤组。每组5只大鼠，于造模后3d处死大鼠，取肾脏：①在4℃下分别放置1d、3d、5d、7d、9d及11d；②在17℃及25℃下分别放置1d、3d、5d及7d后进行取材、固定、包埋。应用HE染色法观察不同环境温度下大鼠肾脏组织形态学的变化；采用IHC法观察环境稳定对大鼠肾脏GRP78和CHOP抗原稳定性的影响。
结果：
1不同环境温度下放置不同时间对肾脏组织结构的影响
不同环境温度下，随着放置时间的延长，肾脏组织自溶变化逐渐明显加重。4℃放置至第9d，17℃和25℃分别放置至第3d，均可见不同程度的肾细胞结构模糊，部分细胞核缺失，胞浆溶解，胞浆呈均质化伊红色。但正常对照组和应激组大鼠肾脏形态学变化已无法区分。
24℃和17℃保存对GRP78和CHOP抗原稳定性的影响
4℃放置1~9d，17℃放置1~3d，应激性损伤组肾脏的GRP78和CHOP的表达强度与范围均逐渐减弱，但与对照组相比，仍有一定差异(p<0.05)。
325℃保存对GRP78和CHOP抗原稳定性的影响
放置1~3d，应激性损伤组肾脏GRP78的表达强度与范围均逐渐减弱，但与对照组相比仍明显增高(p<0.05)。
放置1d，与对照组相比，应激性损伤组肾脏CHOP的表达强度仍明显增强(p<0.05)，但随放置时间延长，其肾脏CHOP的表达强度与范围均逐渐减弱，放置3d~7d与对照组相比无明显差异。
小结：
1GRP78和CHOP抗原稳定性较好，在大鼠应激性损伤后肾脏组织固定7d以内，均可检测到GRP78、CHOP的表达。
2GRP78和CHOP抗原稳定性较好，大鼠应激性损伤后肾脏组织在4℃放置1-9d、17℃和25℃放置1-3d内均可检测到GRP78的表达。CHOP抗原稳定性稍弱于GRP78，大鼠应激性损伤后肾脏组织在4℃放置1-9d、17℃和25℃放置1d内仍能检测到CHOP的表达。
综上，本实验成功建立了大鼠束缚应激和挤压伤复合模型即应激性损伤模型，观察了损伤后大鼠肾脏的病理学变化；检测了肾脏中内质网应激、细胞凋亡及炎症因子表达情况；以及组织固定时间和环境变化对肾脏中GRP78和CHOP抗原稳定的影响，成功地建立了诊断应激性肾损伤的指标体系：
(1)明确的机械性损伤(交通伤、地震、矿难、刑讯逼供等)的体表征象。
(2)肾脏的组织学变化为肾小球肿胀淤血，肾小管上皮细胞脱落，大量炎细胞浸润，间质淤血。
(3)通过TUNEL可证实肾脏存在细胞凋亡，电镜下也可见肾小管细胞核周间隙增大，染色质轻度边移等凋亡现象。
(4)肾脏ERS特异性蛋白GRP78和CHOP的表达明显增高。但该指标体系尚有待于在实际检案中进行验证。
Objective: Clinical data and forensic autopsy reports confirm thatnon-lethal mechanical trauma, such as soft tissue injuries, traffic accidentinjuries, and confessions obtained by torture, often lead to kidney injury, renalfailure, and even death. Because this kind of injury itself is not sufficient tocause death, it is not yet clear how this kind of damage causes renal injury,which challenges clinicians and confuses clinicians and legal medical experts.Hence, there is an urgent need in forensic pathology to determine themechanism(s) underlying kidney injury following non-fatal mechanicaltrauma.
The damage as a physical stressor caused a stress response; and after thedamage the injured organism would have a bad mood, such as anxiety,helplessness, or depression, which can cause an extraordinary psychologicalstress response, that was often ignored. In response to stress, through thecoordinating role of neuroendocrine system the body made a overall reaction.For example, the hypothalamo-pituitary-adrenal axis and the sympatheticnervous system (SNS) are excited strongly, which results in significantchanges in kinds of endocrine hormones. Our previous studies havedemonstrated that restraint stress can aggravate the kidney damage in crushinjury rats, suggested that stress played an important role in happening ofkidney injury, but the mechanism(s) is not clear.
The endoplasmic reticulum (ER) is the site of protein synthesis and foldingin cell. Lots of studies have confirmed that processing and transport ofproteins can be disturbed by stress hormones, toxic metabolites, cytokines,oxygen radicals and so on, then ER stress (ERS) is activated. ERS is one ofthe importante cell response in stress, that is necessary for cells resisting injuryand adaptive capacity. But excessive ERS not only induces apoptosis, but also activates inflammatory signaling pathways, which can cause tissue and celldamage. Numerous studies have showed that ERS may be one of theimportant factors in a variety of kidney injury, but whether or not ERS isrelated with kidney injury caused by non-lethal mechanical trauma isunclear.
The crush injury model is an accepted animal model to simulate softtissue injuries, while the restraint stress model is an accepted animal model toreproduce psychological stress. Thus, in order to simulate the actual case andclear injury mechanisms, the present study established a stress injury modelconsisting of restraint stress and crush injury to determine whether or notERS is related with the mechanism of that restraint stress can aggravatekidney damage caused by crush injury. Hoping to provide experimentbasis for kidney injury caused by non-lethal mechanical trauma.Additionally, in actual forensic cases, the use of immunohistochemical (IHC)indicators was affected by the tissue fixation time, postmortem changes and soon. So after clearing the mechanisms of ERS in stressful kidney injury, thisstudy further discussed the effects of tissue fixation time and environmentaltemperature on the stability of ERS related proteins, which would providetheoretical basis and experimental methods for setting up diagnosisindicator system of stressful injury.
Part1: The establishment of rat stressful injury model and the activationof renal ERS
Objective: To establish a rat stressful injury model, and to observe thepathologic, ultrastructure changes and to research whether or not restraintstress could aggravate kidney damage caused by crush injury through ERS.
Methods:
The rats were randomly divided into the following8groups (n=5pergroup): control (Con); food and water deprivation (Dep); restraint stress (RS);crush injury (CI); stressful injury (SI); Solvent (dimethyl sulfoxide) control(DMSO); and solute (salubrinal [ERS inhibitor]) control (Sal); Sal+SI. Therats were sacrificed after the end of experimental procedures. On the first day before stress and on the end of daily stress the body weights were recorded inCon, Dep and RS groups, to evaluate whether or not the RS model isestablished. Plasma levels of NE and E were evaluated by high performanceliquid chromatography coupled to electrochemical detection (HPLC-ECD);western blot was used to analysis the GRP78protein level in rat renal stressfulinjury; ultrastructure changes in rat kidney were evaluated by transmissionelectron microscope (TEM); pathologic findings in rat hind limb muscles andrenal injury were evaluated by Hematoxylin and Eosin staining (HE); plasmalevel of creatinine (Cre) was evaluated by picric acid method, and blood ureanitrogen (BUN) was evaluated by diacetyl monoxide's reagent colorimetricmethod.
Results:
1Pathologic findings in hind limb muscles
Rhabdomyolysis, interstitial hemorrhage, edema, and an abundance ofinflammatory cell were observed in the hind limb muscles of the crush andstressful injury groups, compared with the normal histologic structures of thecontrol group. These results suggested that the rats had a soft tissue injury.Therefore, the present experiment successfully established the rat crush injurymodel.
2The changes of rat body weights
Body weight of rats increased gradually in Con group. The weight gain inRS group was slowed down compaired with the control and Dep groups(p<0.05). Therefore, the present study successfully established the rat restraintstress model.
3Catecholamine changes in rat plasma samples
The mild increase in NE and E concentrations following RS and CIsuggested that a stress response was induced. The most significant changes inNE and E concentrations were noted in the SI group, compaired with the CIgroup. The NE and E concentrations in SI group were2.29and3.30timesrespectively higher than these of Con group, suggested that the rat stressfulinjury model was successful established though using restraint stress and crush injury, inducing extraordinary strong stress response caused by double attack.
4Ultrastructure changes in rat kidney
Dep group: the nuclear-week gap was widened, ERs were highlyenlarged, mitochondria crest and membrane were fused. RS group: the nucleiwere pyknotic, nuclear-week gap became widened, ERs were enlarged,mitochondria crest and membrane were fused, the plasma membraneinfoldings were arranged irregularly. CI group: tubules were dilated, ERs werevacuolation, mitochondria crest and membrane were fused. The nuclei weremild pyknotic. SI group, the pathological became more serious as comparedwith crush injury group. The nuclear-week gap was widened, nuclearchromatin located mainly in the nuclear membrane. Some mitochondria withruptured outer membrane, enlarged ER, disordered plasma membraneinfoldings, ruptured cell membrane and cast were observed. Those resultssuggested that restraint stress could aggravate kidney ultrastructure damagecaused by crush injury.
5Western blot analysis of the GRP78protein level
The GRP78protein levels were significantly increased in the SI group ascompared with the CI group (p<0.05). Moreover, the GRP78protein level wassignificantly decreased by an ERS inhibitor, Sal (p<0.05). Those resultssuggested that Sal could obviously suppress the activation of ERS. So thepresent study further observed the pathologic changes in the rat renal stressfulinjury with the Sal treatment.
6Pathologic changes in the kidney
Compared with the normal histologic structures of kidney in Con group,swelling and congestive glomeruli, minimal inflammatory cell infiltration andinterstitial hyperemia were observed in the CI group. In addition to the abovechanges, inflammatory cell infiltration was observed in the RS group. Thepathologic changes became more serious in SI group compared with the CIgroup. Swelling and congestive glomeruli, dropped epithelial cell of renaltubule, lots of inflammatory cell infiltration and interstitial hyperemia wereobserved in the SI group, suggesting that restraint stress could exacerbate rat kidney injury caused by a crush injury. Meanwhile, as an ERS inhibitor, Saltreatment alleviated the kidney injury obviously induced by stressful injury,and a few of inflammatory cells infiltration were observed in DMSO groupand Sal group, suggested that ERS might be one of mechanisms throughwhich restraint stress exacerbated rat kidney injury caused by crush injury.
7Cre and BUN changes in rat plasma samples
The mild increase in Cre and BUN concentrations following RS and CIsuggested that a kidney injury was induced. The most significant changes inCre and BUN concentrations were noted in the SI group, compaired with theCI group (p<0.05), suggested that restraint stress could exacerbate rat kidneyinjury caused by a crush injury. Meanwhile, Sal treatment alleviated thekidney injury obviously induced by stressful injury (p<0.05), suggested thatERS might be one of mechanisms through which restraint stress exacerbatedrat kidney injury caused by crush injury.
Summary: In the present part study, a rat stressful injury model wassuccessful established. The present study found that ERS inhibitor couldreduced the renal stressful injury, and preliminary confirmed that restraintstress could aggravate kidney damage caused by crush injury, and itsunderlying mechanism may be involved with ERS.
Part2: Restraint stress aggravates rat kidney injury caused by a crushinjury through ERS
Experiment1Restraint stress aggravates rat kidney injury caused by acrush injury through apoptosis induced by ERS
Objective: To establish rat stressful injury model, and to researchwhether or not restraint stress aggravates rat kidney injury caused by a crushinjury through apoptosis induced by ERS
Methods:
The rats were randomly divided into groups the same as in part1.Western blot were used to analysis the apoptosis specific proteins induced byERS (CHOP and caspase-12) and an executioner of apoptosis (caspase-3)protein levels in rat renal stressful injury. TdT-mediated dUTP nick end labeling (TUNEL) was used to observe apoptotic renal cells.
Results:
1Western blot analysis of the CHOP, caspase-12, and caspase-3proteinlevels
The CHOP, caspase-12and caspase-3protein levels were significantlyincreased in the RS and CI groups as compared with the control group(p<0.05).Moreover, the levels of the above proteins were most markedly increased in SIgroup, compared with the CI group. While the above changes weresignificantly inhibited by Sal (p<0.05), suggesting that the mechanism of thatrestraint stress aggravates rat kidney injury caused by a crush injury wasrelated with apoptosis induced by ERS.
2Localization of apoptosis by the TUNEL assay
Apoptosis was observed in renal tubules after RS and CI compared withthe control group (p<0.05). The greatest numbers of apoptotic renal tubularepithelial cells were found in SI group, meanwhile Sal decreased the numberof apoptotic cells (p<0.05), confirmed that restraint stress aggravates ratkidney injury caused by a crush injury was indeed related with apoptosisinduced by ERS.
Experiment2Restraint stress aggravates rat kidney injury caused by acrush injury through inflammation induced by ERS
Objective: To establish rat stressful injury model, and to researchwhether or not restraint stress aggravates rat kidney injury caused by a crushinjury through inflammation induced by ERS
Methods:
The rats were randomly divided into groups the same as in part1.Western blot were used to analysis the Monocyte chemoattractantprotein-1(MCP-1) protein levels in rat renal stressful injury. The kidneys werefixed, then embedded in paraffin. Serial sections of kidney were immunohisto-chemically stained for the expressions of GRP78and MCP-1.
Results:
1Western blot analysis of the MCP-1protein level
The MCP-1protein levels were increased significantly in the RS and CIgroups, compared with the control group (p<0.05). Moreover, the MCP-1protein level was most significantly increased in SI group compared with theCI group (p<0.05). And Sal treatment inhibited the MCP-1expression inducedby stressful injury (p<0.05), suggested that ERS induced MCP-1expressionincreased is one of mechanism by which restraint stress aggravates rat kidneyinjury caused by a crush injury.
2IHC analysis of GRP78and MCP-1distribution
GRP78and MCP-1were abundantly expressed in the SI kidneycompared with the control group. We also found that in serial sections, theMCP-1-positive cells also had positive expressions of GRP78. Those resultsprovided a morphological basis for the inflammation related with MCP-1caused by ERS.
Summary: Restraint stress can aggravate kidney damage caused by crushinjury, which was attributed to ERS. ERS not only leads to initiation of therenal apoptotic processes promoted by CHOP and a caspase-12-dependentpathway, but also increase expression of MCP-1to aggravate the renalinflammatory response.
Part3: The effects of the fixed time and environmental temperature onthe antigen stability of ERS markers, GRP78and CHOP
Experiment1The continuous expressions of GRP78and CHOP in ratrenal stressful injury
Objective: Because the injured organism often died within a period oftime after the nonfatal injuries occurred, the aim of the present study is toobserve the expressions of GRP78and CHOP within a period of time after ratstressful injury.
Methods:
Animals were randomly divided into six experimental groups forsampling at0h,1,3,5and7days after SI and a control group (n=5per group).The kidneys were quickly removed and fixed in10%formalin. IHC stainingwas used to observe the expressions of GRP78and CHOP after stressful injury in rat kidneys.
Results:
Semi-quantitative evaluation of IHC staining of GRP78and CHOPcontinuous expressions in the rat renal stressful injury
Within0h-7d after the stressful injury, with the extension of time, theexpressions of GRP78and CHOP all gradually increased (p<0.05), and on the3days it up to a peak. Those results suggested that within a week afterstressful injury, GRP78and CHOP still could be detected by IHC staining.
Experiment2The effects of the fixed time on the antigen stability ofGRP78and CHOP
Objective: To observe the effects of fixed time on the antigen stability ofGRP78and CHOP in rat kidney stressful injury.
Methods:
Animals were randomly divided into a control group and four SI groups(n=5per group). The rats were sacrificed on the3rd day after the end of theexperimental procedures. The kidneys were quickly removed and fixed in10%formalin for different fixation times (1,3,5and7days) following by paraffinembedding. Pathologic changes in kidney were evaluated by HE staining; IHCwere used to observe the effects of fixed time on the antigen stability ofGRP78and CHOP in rat kidney.
Results:
1Pathologic changes in the kidney after stressful injury in samples fixedwithin a week
Fixation time had no significant effect on the kidneys in both of the twogroups. Swelling and congestive glomeruli, narrowed renal capsule,substantial inflammatory cells infiltration, narrowed renal capsule, droppedepithelial cell of renal tubule and interstitial hyperemia were observed in theSI group.
2The stabilities of GRP78and CHOP for fixed within a week
Kidney fixation over3–7days did not adversely affect the levels ofGRP78and CHOP. Compared with the control group, GRP78and CHOP levels were significantly higher in kidney samples from the S1group(p<0.05)。Experiment3The effects of environmental temperature on the antigenstability of GRP78and CHOP
Objective: To observe the effects of environmental temperature on theantigen stability of GRP78and CHOP in rat isolated kidney after stressfulinjury.
Methods:
Animals were randomly divided into two experimental groups (n=5pergroup): control group and stressful injury group. Rats were sacrificed on the3rd day after the end of the experimental procedures.①The kidneys werequickly removed and stored at4°C for sampling at1,3,5,7,9and11days;②The kidneys were quickly removed and stored at17°C and25°C forsampling at1,3,5, and7days respectively. And then the kidneys were fixedin10%formalin for paraffin embedding. Pathologic changes in kidney wereevaluated by HE staining; IHC were used to observe the effects ofenvironmental temperature on the antigen stability of GRP78and CHOP in ratkidney.
Results:
1Pathologic changes in the kidney after stressful injury in samples forstored at different environmental temperature
As kidney storage became more prolonged in different temperature,obvious autolysis occurred. On the9th day for stored at4°C, the3rd day forstored at17°C and25°C respectively, the varying degrees of fuzzy renal cellstructure, missed nucleus, dissolved cytoplasm and homogenization in the redwere all observed. But it is impossible to make a distinction between controlgroup and stressful injury group about the renal pathologic changes.
2The effect of4°C and17°C storage on GRP78and CHOP proteinstability
In the kidneys stored at4°C for1-9days and17°C for1-3days, thelevels of the GRP78and CHOP proteins gradually reduced in their distribution and intensity (SI group), but were still higher than that in the control group(p<0.05).
3The effect of25°C storage on GRP78and CHOP protein stability
In the kidneys stored at25°C for1-3days, the levels of the GRP78protein gradually reduced in their distribution and intensity (SI group), butwere still higher than that in the control group (p<0.05).
In the kidneys stored at25°C for1day, the levels of the CHOP proteingradually reduced in their distribution and intensity (SI group), but were stillhigher than that in the control group (p<0.05).
Summary:
1GRP78and CHOP had good stabilities as antigens in rat kidney, andboth of their expressions could be tested for fixed within a week after ratstressful injury.
2GRP78and CHOP had good stabilities as antigens. After rat stressfulinjury, with the kidneys stored in4°C for1-9days,17°C for1-3days and25°C for1-3days, the expression of GRP78could still be tested. While CHOPhad a weaker stability as antigens than GRP78, it still could be tested with thekidneys stored in4°C for1-9days,17°C for1-3days and25°C for1day.
In summary, a rat stressful injury model consisting of restraint stress andcrush injury was successful established. Pathologic changes in kidney wereobserved after injury; ERS, apoptosis and inflammation were tested in ratrenal injury; and the effects of the fixed time and environmental temperatureon the antigen stability of GRP78and CHOP were tested in rat kidneys. Thediagnosis indicator system of renal stressful injury was successful established:
(1) The clear surface sign of mechanical trauma (traffic accident injuries,earth-quake, mine disaster, confessions obtained by torture and so on) wasobserved.
(2) Pathologic changes: swelling and congestive glomeruli, dropped epithelialcell of renal tubule, lots of inflammatory cell infiltration and interstitialhyperemia were observed in the kidney.
(3) Localization of apoptosis by the TUNEL assay was observed and ultrastructure changes in rat kidney were evaluated by TEM, includingwidened nuclear-week gap, nuclear chromatin located mainly in the nuclearmembrane and so on, which suggesting apoptosis.
(4) ERS markers, GRP78and CHOP were significantly increased in thekidney.
But the diagnosis indicator system still needs to be validated in actual forensiccases.
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