TRPV1在应激性急性胃粘膜损伤中的作用及其与舒芬太尼关系的研究
发布时间：2018-09-01 09:23
【摘要】：研究背景 急性胃粘膜损伤(Acute Gastric Mucosal Lesion, AGML)是临床上重大手术应激、严重烧创伤或者危重症患者的常见并发症,AGML的发病机制与机体神经体液失调、胃粘膜的防御功能减弱以及损伤因素增强等因素密切相关。虽然影响AGML的发生、发展因素非常复杂,但持续性、过度伤害应激是诱发急性胃粘膜损伤发生、发展的最直接、最重要的因素。在严重创伤条件下,各种应激因素作用于胃肠道和中枢神经系统,通过内分泌、神经、免疫等系统与消化道(消化道神经、内分泌网络调控)相互作用而产生胃粘膜病变。大量的文献已证实,在严重的应激状态下,由于胃粘膜能量代谢障碍、酸碱平衡紊乱、胃粘膜屏障功能破坏以及自由基产生增多,最终导致胃粘膜局部出现严重的血液循环障碍,主要表现为胃粘膜的微循环障碍以及胃血流量减少。已有研究表明,胃的脊髓传入神经通路内以及胃肠道分布有丰富的TRPV1受体及辣椒素敏感传入神经元,它们在维持胃肠黏膜的正常功能、对抗多种伤害性侵袭因素中发挥重要的作用。我们前期研究结果表明,TRPV1在应激性急性胃粘膜损伤的发生中起重要作用,本研究旨在从舒芬太尼预先给药以及浸水束缚应激不同时间的角度探讨TRPV1在应激性AGML中的作用及机制。 舒芬太尼(Sufentail)是一种以激活μ受体为主的阿片类麻醉镇痛药,其脂溶性高,能够迅速通过血-脑屏障进入脑内与阿片类受体结合发挥作用。我们前期动物实验证实,TRPV1和μ-阿片受体在胃的脊髓控制区的脊髓背根神经节(DRG)上共存表达,且二者功能上具有相关性；另有文献指出,向脑室内注射选择性μ-阿片受体激动剂可以减少酒精或吲哚美辛等诱导的急性胃粘膜损伤。因此本实验第一部分拟探索舒芬太尼预处理在WIRS诱导的AGML中的作用机制及其与TRPV1的关系。 由于急性胃粘膜损伤是临床上急危重患者的常见并发症,且已有文献证实TRPV1在胃脊髓传入神经通路以及胃粘膜都高表达,且我们前期研究表明TRPV1、ASIC3在胃脊髓控制区DRG神经元上共存表达,它们在应激性急性AGML的发生中起重要作用。本课题采用实时荧光定量PCR、免疫组化等方法,分别从下丘脑和胃粘膜二个层面探讨TRPV1在AGML发生中的作用及可能机制,并从应激控制的角度采用舒芬太尼预先给药进行反证,旨在获得TRPV1参与和调控应激性AGML形成的可靠证据。本实验亦从WIRS不同时间的角度探索胃粘膜TRPV1、ASIC3mRNA的动态变化过程,并计数胃粘膜损伤溃疡指数(UI),同时检测应激不同时间时血清SOD、MDA的活力水平,观察大鼠应激状态下机体氧化和抗氧化能力。结果表明应激性溃疡发生过程中SOD、MDA可作为一个早期敏感指标进行检测,能够为临床上选择合适的时间对应激性溃疡进行干预提供依据。通过初步探索TRPV1、ASIC3在AGML发生中的调控作用及其机制,为确立TRPV1作为防治应激性AGML的新靶点提供更充分的科学依据,也为围术期应激控制防治AGML的发生奠定坚实的基础。通过WIRS溃疡模型的复制,我们更好地了解AGML的发病机理,为临床上更好防治严重烧创伤、手术和急危重患者高AGML提供新思路。 研究目的 通过复制经典浸水束缚应激模型,初步探索：1.TRPV1在应激性急性胃粘膜损伤中的作用,并以舒芬太尼从中枢和外周两个层面进行验证,明确TRPV1mRNA与μ-阿片受体激动剂之间的关系,同时检测大鼠应激性AGML过程中胃粘膜及血清中SOD和MDA的水平,观察机体在应激过程中的抗氧化能力；2.从浸水束缚应激不同时间的角度探讨胃粘膜层面TRPV1mRNA和ASIC3mRNA之间的关系以及机体的氧化和抗氧化能力变化。 实验方法 (1)成年雄性SPF级Wistar大鼠30只,随机分为3组,分别为：正常组(NC组)、浸水束缚应激组(WIRS组)和舒芬太尼预先给药组(SF组)。舒芬太尼预先给药组在大鼠束缚浸水5min前腹腔注射25μg/kg舒芬太尼,WIRS组以等容积生理盐水进行腹腔注射；浸水束缚应激(WIRS)模型制作参照以往研究方法。实验内容包括：1各组大鼠麻醉后刮取胃粘膜、取下丘脑液氮冻存,提取组织总RNA,采用实时荧光定量PCR的方法检测胃粘膜、下丘脑TRPV1mRNA的表达变化；2留取胃液后冲洗净胃内容物,显微镜下观察胃粘膜损伤情况,计数溃疡指数(UI),检测胃液pH,观察舒芬太尼对大鼠应激性急性胃粘膜病变的作用；3各组胃组织损伤明显处用组织剪剪取0.2×0.1cm大小组织块,中性福尔马林溶液固定,采用HE染色方法观察舒芬太尼预先给药对WIRS大鼠胃粘膜组织形态的影响；4各组大鼠麻醉后取其腹主动脉血,离心并留取血清；测定胃粘膜MDA蛋白浓度,以检测各组大鼠组织和血清中SOD、MDA含量； (2)成年雄性SPF级Wistar大鼠12只,随机分为3组,分别为：正常组(NC组)、浸水束缚应激组(WIRS组)和舒芬太尼预先给药组(SF组),采用免疫组化技术观察TRPV1在胃组织的定位及表达变化。 (3)成年雄性SPF级Wistar大鼠40只,随机分为4组,分别为：正常组(NC组)、浸水束缚应激2h组(WIRS2h组)、浸水束缚应激4h组(WIRS4h组)和浸水束缚应激6h组(WIRS6h组),内容包括：1各组大鼠麻醉后刮取胃粘膜液氮冻存,提取组织总RNA,采用实时荧光定量PCR的方法检测浸水束缚应激不同时间胃粘膜TRPV1mRNA和ASIC3mRNA的表达变化；2冲洗净胃内容物,显微镜下观察胃粘膜损伤情况,计数溃疡指数(UI)；3各组胃组织损伤明显处用组织剪剪取0.2×0.1cm大小组织块,中性福尔马林溶液固定,采用HE染色方法观察浸水束缚应激不同时间大鼠胃组织形态的变化；4各组大鼠麻醉后取其腹主动脉血,离心并留取血清,以检测不同时间点大鼠血清SOD、MDA含量。 实验结果 (1)NC组大鼠胃腔内见少量棕灰色食物残渣,胃液呈透明淡黄色,冲洗后见胃粘膜光滑完整,呈淡红色,未见水肿及出血。WIRS组大鼠胃腔内见大量红褐色内容物及血性积液,冲洗后见胃粘膜明显水肿,伴随点状、条索状出血、糜烂甚至溃疡。SF预处理组大鼠胃腔内容物呈棕色,胃粘膜粘液增厚,偶见点状及线状出血点,损伤较WIRS组明显减轻。光镜下观察NC组大鼠腺体排列整齐,结构完整,未见细胞结构改变；WIRS组大鼠胃粘膜细胞排列紊乱,黏膜上皮黏膜脱落、坏死,细胞间隙增宽,无法辨认腺体及具体结构；舒芬太尼预先给药组则明显减轻胃粘膜层损伤程度,但仍有腺体结构排列紊乱。浸水束缚应激6h可引起大鼠胃粘膜损伤的溃疡指数显著升高(P0.05),舒芬太尼预先给药可显著降低UI(P0.05),但仍高于正常组。WIRS组胃液pH值显著低于正常组(P0.05),舒芬太尼预先给药组与正常组相比大鼠胃液pH值变化不明显,显著高于WIRS组。舒芬太尼预先给药可明显减少浸水束缚应激引起的脂质过氧化产物含量和胃粘膜组织局部SOD活力(P0.05),但血清SOD活力水平在各组间变化不明显。荧光定量PCR结果显示,应激组胃粘膜TRPV1mRNA的相对表达量是正常组的(0.2±0.3)倍,而舒芬太尼预先给药组TRPV1mRNA的表达则是正常组的(4.2+0.5)倍,两组差异均有显著性(P0.05)；下丘脑内,舒芬太尼预先给药组TRPV1mRNA的相对表达量是正常组的(3.2±0.8)倍,差异亦具有统计学意义(P0.05),正常组与应激组相比TRPV1mRNA则变化不明显。 (2)免疫组化检测结果显示棕色颗粒为TRPV1受体的阳性表达,结构显示,浸水束缚应激模型组大鼠胃组织TRPV1受体的表达量略高于正常组大鼠,而舒芬太尼预先给药组大鼠TRPV1受体的表达量显著高于应激组和正常组(P0.05)。 (3)荧光定量PCR结果显示,胃粘膜TRPV1mRNA在WIRS2h和WIRS4h时变化不明显,但在WIRS6h时显著减低(P0.05)；与NC组相比,ASIC3mRNA相对表达量在应激初期即出现明显升高(P0.05)；与NC组、WIRS2h、 WIRS4h和WIRS6h相比,ASIC3mRNA在WIRS6h时明显降低(P0.05)。各组大鼠胃沿大弯剪开冲洗后,见正常组大鼠胃粘膜光滑完整,呈淡红色,无充血及水肿。WIRS2h及WIRS4h组大鼠胃内容物呈褐色,冲洗时偶见出血点及糜烂,且WIRS2h组程度不及WIRS4h组,偶见出血点及条索状溃疡；WIRS6h组大鼠胃腔内见大量红褐色内容物,冲洗时见红色血痂,胃粘膜充血水肿,呈点状、片状糜烂坏死,多与胃纵轴平行。WIRS2h、4h、6h引起溃疡指数升高(13.00±2.24vs0.00±0.00、16.40±3.05vs0.00±0.00、30.40±2.41vs0.00±0.00,P0.05),明显高于NC组。胃粘膜损伤指数倾向于随着WIRS时间的延长而逐渐增加。血清SOD活力在应激过程中先增高后降低,WIRS4h时最高,同时检测到的MDA水平在WIRS4h时最低。WIRS2h、 WIRS4h、WIRS6h组胃粘膜内TRPV1mRNA的相对表达量分别是正常组胃粘膜TRPV1mRNA的(1.43±0.19)、(1.20±0.13)和(0.19±0.03)倍。与NC组比较,胃粘膜WIRS6h组的TRPV1mRNA表达减少,其差别具有显著统计学意义(P0.05)。 实验结论 (1)舒芬太尼预先给药可有效减轻WIRS诱导的急性胃粘膜损伤,其机制可能与控制机体氧化应激反应、减少胃酸分泌以及调控中枢和外周TRPV1mRNA的表达有关。 (2)应激性溃疡发生过程中,机体存在一个代偿过程,血清SOD、MDA水平可作为一个早期指标进行检测,为选择合适的时间对应激性溃疡进行干预提供依据。 (3)TRPV1和ASIC3在应激性急性胃粘膜病变的过程中发挥一定的作用,二者的变化趋势具有一致性。对于WIRS诱导的急性胃粘膜损伤,ASIC3比TRPV1更为敏感。
[Abstract]:Research background
Acute Gastric Mucosal Lesion (AGML) is a common complication of severe surgical stress, severe burns or critical illnesses. The pathogenesis of AGML is closely related to neurohumoral disorders, impaired gastric mucosal defense and increased injury factors. The factors are very complicated, but persistent, and excessive injury stress is the most direct and important factor that induces the occurrence and development of acute gastric mucosal injury. A large number of literatures have confirmed that under severe stress, gastric mucosal microcirculation disorder is the main manifestation of gastric mucosal hemodynamic disturbance due to disturbance of energy metabolism, disorder of acid-base balance, destruction of gastric mucosal barrier function and increase of free radicals. Previous studies have shown that there are abundant TRPV1 receptors and capsaicin-sensitive afferent neurons in the spinal afferent pathway and gastrointestinal tract of the stomach, which play an important role in maintaining the normal function of gastrointestinal mucosa and resisting multiple noxious invasion factors. The purpose of this study was to investigate the role and mechanism of TRPV1 in stress-induced acute gastric mucosal injury (SAGML) from the perspective of sufentanil pretreatment and immersion restraint stress at different times.
Sufentail is an opioid anesthetic analgesic drug that activates mu receptors. It is highly liposoluble and can quickly enter the brain through the blood-brain barrier to bind to opioid receptors. Our previous animal experiments confirmed that TRPV1 and mu-opioid receptors coexist in the dorsal root ganglion (DRG) of the spinal cord in the spinal cord control region of the stomach. The first part of this study is to explore the mechanism of sufentanil preconditioning in WIRS-induced AGML and its relationship with TRPV1.
Acute gastric mucosal injury is a common complication in critically ill patients, and the expression of TRPV1 in gastro-spinal afferent pathway and gastric mucosa has been proved to be high. Our previous studies showed that TRPV1 and ASIC3 coexist in DRG neurons in the gastro-spinal control region, and they play an important role in the pathogenesis of stress-induced acute AGML. In this study, real-time fluorescence quantitative PCR and immunohistochemistry were used to investigate the role and possible mechanism of TRPV1 in the development of AGML from hypothalamus and gastric mucosa, and sufentanil was used to pre-administer sufentanil in order to obtain reliable evidence that TRPV1 participated in and regulated the formation of stress-induced AGML. The dynamic changes of TRPV1 and ASIC3 mRNA in gastric mucosa were investigated by WIRS at different time points. The ulcer index (UI) of gastric mucosa injury was counted. The activities of SOD and MDA in serum were measured at different time points of stress. The oxidative and antioxidative abilities of rats were observed under stress conditions. As an early sensitive index, it can provide basis for choosing appropriate time to intervene stress ulcer in clinic. Through preliminary exploration of the regulatory role and mechanism of TRPV1 and ASIC3 in the occurrence of AGML, it can provide sufficient scientific basis for establishing TRPV1 as a new target for prevention and treatment of stress AGML, and also provide perioperative stress response. Through the duplication of WIRS ulcer model, we can better understand the pathogenesis of AGML, and provide new ideas for clinical prevention and treatment of severe burns, surgery and high AGML in critically ill patients.
research objective
The role of TRPV1 in stress-induced acute gastric mucosal injury was investigated by replicating the classical immersion restraint stress model. Sufentanil was used to verify the relationship between TRPV1 mRNA and mu-opioid receptor agonists in central and peripheral layers. SOD and MD in gastric mucosa and serum during stress-induced AGML in rats were detected. To investigate the relationship between TRPV1 mRNA and ASIC3 mRNA in gastric mucosa and the changes of oxidative and antioxidant capacity of the organism during water immersion restraint stress.
Experimental method
(1) 30 adult male SPF Wistar rats were randomly divided into three groups: normal group (NC group), immersion restraint stress group (WIRS group) and sufentanil pre-administration group (SF group). The experimental contents include: 1. After anesthesia, the gastric mucosa was scraped, the hypothalamus liquid nitrogen was frozen, the total RNA was extracted, and the expression of TRPV1 mRNA in gastric mucosa and hypothalamus was detected by real-time fluorescence quantitative PCR; 2. After gastric juice was removed, the gastric contents were washed out and observed under microscope. Gastric mucosal injury was observed, ulcer index (UI) was counted, gastric juice pH was measured, and the effect of sufentanil on stress-induced acute gastric mucosal lesion in rats was observed. Fourth, after anesthesia, abdominal aorta blood of rats in each group was taken, centrifuged and serum was taken. The concentration of MDA protein in gastric mucosa was determined to detect the content of SOD and MDA in tissues and serum of rats in each group.
(2) Twelve adult male SPF Wistar rats were randomly divided into three groups: normal group (NC group), immersion restraint stress group (WIRS group) and sufentanil pretreatment group (SF group). The location and expression of TRPV1 in gastric tissue were observed by immunohistochemistry.
(3) Forty adult male SPF Wistar rats were randomly divided into four groups: normal group (NC group), immersion restraint stress 2 h group (WIRS 2 h group), immersion restraint stress 4 h group (WIRS 4 h group) and immersion restraint stress 6 h group (WIRS 6 h group). The contents included: 1. After anesthesia, the rats in each group were scraped out for gastric mucosa nitrogen cryopreservation, and the total RNA was extracted by real-time fluorescence assay. The expression of TRPV1 mRNA and ASIC3 mRNA in gastric mucosa was detected by quantitative PCR at different time of immersion restraint stress; 2 gastric contents were washed and gastric mucosal lesions were observed under microscope and ulcer index (UI) was counted; 3 gastric tissue lesions in each group were markedly cut off with 0.2 *0.1 cm tissue mass, fixed with neutral formalin solution, and used. HE staining method was used to observe the changes of gastric tissue morphology in rats under water immersion restraint stress at different time points.
experimental result
(1) In NC group, a small amount of brown-gray food residue was found in the gastric cavity, and the gastric juice was transparent and yellowish. After washing, the gastric mucosa was smooth and intact, pale red, edema and bleeding were not found. In WIRS group, a large number of reddish-brown contents and hematogenous effusion were found in the gastric cavity. After washing, the gastric mucosa was evidently edematous, accompanied by punctate, corded bleeding, erosion and even ulcer. The contents of gastric cavity in pretreatment group were brown, the mucus of gastric mucosa thickened, occasional dot-like and linear bleeding spots were found, and the injury was significantly less than that in WIRS group. The ulcer index of gastric mucosal injury in rats was significantly increased after 6 hours of immersion restraint stress (P 0.05), and the UI was significantly decreased by sufentanil (P 0.05), but it was still higher than that in normal group. The pH value of gastric juice in RS group was significantly lower than that in normal group (P The results of fluorescence quantitative PCR showed that the relative expression of TRPV1 mRNA in gastric mucosa of stress group was (0.2 6550 The relative expression of TRPV1 mRNA in normal group was 3.2 (+ 0.8) times as much as that in normal group, and the difference was statistically significant (P 0.05).
(2) The results of immunohistochemistry showed that the expression of TRPV1 receptor was positive in brown granules. The structure showed that the expression of TRPV1 receptor in gastric tissue of immersion restraint stress group was slightly higher than that of normal group, while the expression of TRPV1 receptor in sufentanil pretreatment group was significantly higher than that of stress group and normal group (P 0.05).
(3) Fluorescence quantitative PCR showed that TRPV1 mRNA in gastric mucosa did not change significantly at WIRS 2H and WIRS 4h, but decreased significantly at WIRS 6h (P 0.05); ASIC3 mRNA relative expression increased significantly at the initial stage of stress (P 0.05) compared with NC group, WIRS 2h, WIRS 4H and WIRS 6h, ASIC3 mRNA decreased significantly at WIRS 6h (P 0.05). The gastric contents of rats in WIRS2h and WIRS4h groups were brown and occasionally bleeding spots and erosion were observed. The degree of bleeding spots and stripe ulcers in WIRS2h group was less than that in WIRS4h group. The ulcer index increased at 2 hours, 4 hours and 6 hours (13.00.24 vs 0.00.00, 16.40.05 vs 0.00.00, 30.40.41 vs 0.00.00, 30.40.41 vs 0.00.00.00, P 0.05), significantly higher than that of NC group. The activity of SOD in serum increased first and then decreased during stress. The highest level of MDA was detected at 4 hours of WIRS, and the lowest level was detected at 4 hours of WIRS. The relative expression of TRPV1 mRNA in gastric mucosa of WIRS 2h, WIRS 4H and WIRS 6h groups was (1.43 (0.19)), (1.20 (0.13) and (0.19 (0.03) times higher than that of NC group, respectively. The expression of TRPV1mRNA in group RS6h decreased, and the difference was statistically significant (P0.05).
empirical conclusion
(1) Sufentanil can effectively alleviate WIRS-induced acute gastric mucosal injury. The mechanism may be related to the control of oxidative stress, the decrease of gastric acid secretion and the regulation of TRPV1 mRNA expression in the central and peripheral regions.
(2) There is a compensatory process in the process of stress ulcer. The levels of serum SOD and MDA can be used as an early indicator to detect stress ulcer, and provide the basis for choosing the appropriate time to intervene in stress ulcer.
(3) TRPV1 and ASIC3 play a certain role in the process of stress-induced acute gastric mucosal lesion, and the change trend of them is consistent. ASIC3 is more sensitive to WIRS-induced acute gastric mucosal lesion than TRPV1.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614
本文编号：2216761
[Abstract]:Research background
Acute Gastric Mucosal Lesion (AGML) is a common complication of severe surgical stress, severe burns or critical illnesses. The pathogenesis of AGML is closely related to neurohumoral disorders, impaired gastric mucosal defense and increased injury factors. The factors are very complicated, but persistent, and excessive injury stress is the most direct and important factor that induces the occurrence and development of acute gastric mucosal injury. A large number of literatures have confirmed that under severe stress, gastric mucosal microcirculation disorder is the main manifestation of gastric mucosal hemodynamic disturbance due to disturbance of energy metabolism, disorder of acid-base balance, destruction of gastric mucosal barrier function and increase of free radicals. Previous studies have shown that there are abundant TRPV1 receptors and capsaicin-sensitive afferent neurons in the spinal afferent pathway and gastrointestinal tract of the stomach, which play an important role in maintaining the normal function of gastrointestinal mucosa and resisting multiple noxious invasion factors. The purpose of this study was to investigate the role and mechanism of TRPV1 in stress-induced acute gastric mucosal injury (SAGML) from the perspective of sufentanil pretreatment and immersion restraint stress at different times.
Sufentail is an opioid anesthetic analgesic drug that activates mu receptors. It is highly liposoluble and can quickly enter the brain through the blood-brain barrier to bind to opioid receptors. Our previous animal experiments confirmed that TRPV1 and mu-opioid receptors coexist in the dorsal root ganglion (DRG) of the spinal cord in the spinal cord control region of the stomach. The first part of this study is to explore the mechanism of sufentanil preconditioning in WIRS-induced AGML and its relationship with TRPV1.
Acute gastric mucosal injury is a common complication in critically ill patients, and the expression of TRPV1 in gastro-spinal afferent pathway and gastric mucosa has been proved to be high. Our previous studies showed that TRPV1 and ASIC3 coexist in DRG neurons in the gastro-spinal control region, and they play an important role in the pathogenesis of stress-induced acute AGML. In this study, real-time fluorescence quantitative PCR and immunohistochemistry were used to investigate the role and possible mechanism of TRPV1 in the development of AGML from hypothalamus and gastric mucosa, and sufentanil was used to pre-administer sufentanil in order to obtain reliable evidence that TRPV1 participated in and regulated the formation of stress-induced AGML. The dynamic changes of TRPV1 and ASIC3 mRNA in gastric mucosa were investigated by WIRS at different time points. The ulcer index (UI) of gastric mucosa injury was counted. The activities of SOD and MDA in serum were measured at different time points of stress. The oxidative and antioxidative abilities of rats were observed under stress conditions. As an early sensitive index, it can provide basis for choosing appropriate time to intervene stress ulcer in clinic. Through preliminary exploration of the regulatory role and mechanism of TRPV1 and ASIC3 in the occurrence of AGML, it can provide sufficient scientific basis for establishing TRPV1 as a new target for prevention and treatment of stress AGML, and also provide perioperative stress response. Through the duplication of WIRS ulcer model, we can better understand the pathogenesis of AGML, and provide new ideas for clinical prevention and treatment of severe burns, surgery and high AGML in critically ill patients.
research objective
The role of TRPV1 in stress-induced acute gastric mucosal injury was investigated by replicating the classical immersion restraint stress model. Sufentanil was used to verify the relationship between TRPV1 mRNA and mu-opioid receptor agonists in central and peripheral layers. SOD and MD in gastric mucosa and serum during stress-induced AGML in rats were detected. To investigate the relationship between TRPV1 mRNA and ASIC3 mRNA in gastric mucosa and the changes of oxidative and antioxidant capacity of the organism during water immersion restraint stress.
Experimental method
(1) 30 adult male SPF Wistar rats were randomly divided into three groups: normal group (NC group), immersion restraint stress group (WIRS group) and sufentanil pre-administration group (SF group). The experimental contents include: 1. After anesthesia, the gastric mucosa was scraped, the hypothalamus liquid nitrogen was frozen, the total RNA was extracted, and the expression of TRPV1 mRNA in gastric mucosa and hypothalamus was detected by real-time fluorescence quantitative PCR; 2. After gastric juice was removed, the gastric contents were washed out and observed under microscope. Gastric mucosal injury was observed, ulcer index (UI) was counted, gastric juice pH was measured, and the effect of sufentanil on stress-induced acute gastric mucosal lesion in rats was observed. Fourth, after anesthesia, abdominal aorta blood of rats in each group was taken, centrifuged and serum was taken. The concentration of MDA protein in gastric mucosa was determined to detect the content of SOD and MDA in tissues and serum of rats in each group.
(2) Twelve adult male SPF Wistar rats were randomly divided into three groups: normal group (NC group), immersion restraint stress group (WIRS group) and sufentanil pretreatment group (SF group). The location and expression of TRPV1 in gastric tissue were observed by immunohistochemistry.
(3) Forty adult male SPF Wistar rats were randomly divided into four groups: normal group (NC group), immersion restraint stress 2 h group (WIRS 2 h group), immersion restraint stress 4 h group (WIRS 4 h group) and immersion restraint stress 6 h group (WIRS 6 h group). The contents included: 1. After anesthesia, the rats in each group were scraped out for gastric mucosa nitrogen cryopreservation, and the total RNA was extracted by real-time fluorescence assay. The expression of TRPV1 mRNA and ASIC3 mRNA in gastric mucosa was detected by quantitative PCR at different time of immersion restraint stress; 2 gastric contents were washed and gastric mucosal lesions were observed under microscope and ulcer index (UI) was counted; 3 gastric tissue lesions in each group were markedly cut off with 0.2 *0.1 cm tissue mass, fixed with neutral formalin solution, and used. HE staining method was used to observe the changes of gastric tissue morphology in rats under water immersion restraint stress at different time points.
experimental result
(1) In NC group, a small amount of brown-gray food residue was found in the gastric cavity, and the gastric juice was transparent and yellowish. After washing, the gastric mucosa was smooth and intact, pale red, edema and bleeding were not found. In WIRS group, a large number of reddish-brown contents and hematogenous effusion were found in the gastric cavity. After washing, the gastric mucosa was evidently edematous, accompanied by punctate, corded bleeding, erosion and even ulcer. The contents of gastric cavity in pretreatment group were brown, the mucus of gastric mucosa thickened, occasional dot-like and linear bleeding spots were found, and the injury was significantly less than that in WIRS group. The ulcer index of gastric mucosal injury in rats was significantly increased after 6 hours of immersion restraint stress (P 0.05), and the UI was significantly decreased by sufentanil (P 0.05), but it was still higher than that in normal group. The pH value of gastric juice in RS group was significantly lower than that in normal group (P The results of fluorescence quantitative PCR showed that the relative expression of TRPV1 mRNA in gastric mucosa of stress group was (0.2 6550 The relative expression of TRPV1 mRNA in normal group was 3.2 (+ 0.8) times as much as that in normal group, and the difference was statistically significant (P 0.05).
(2) The results of immunohistochemistry showed that the expression of TRPV1 receptor was positive in brown granules. The structure showed that the expression of TRPV1 receptor in gastric tissue of immersion restraint stress group was slightly higher than that of normal group, while the expression of TRPV1 receptor in sufentanil pretreatment group was significantly higher than that of stress group and normal group (P 0.05).
(3) Fluorescence quantitative PCR showed that TRPV1 mRNA in gastric mucosa did not change significantly at WIRS 2H and WIRS 4h, but decreased significantly at WIRS 6h (P 0.05); ASIC3 mRNA relative expression increased significantly at the initial stage of stress (P 0.05) compared with NC group, WIRS 2h, WIRS 4H and WIRS 6h, ASIC3 mRNA decreased significantly at WIRS 6h (P 0.05). The gastric contents of rats in WIRS2h and WIRS4h groups were brown and occasionally bleeding spots and erosion were observed. The degree of bleeding spots and stripe ulcers in WIRS2h group was less than that in WIRS4h group. The ulcer index increased at 2 hours, 4 hours and 6 hours (13.00.24 vs 0.00.00, 16.40.05 vs 0.00.00, 30.40.41 vs 0.00.00, 30.40.41 vs 0.00.00.00, P 0.05), significantly higher than that of NC group. The activity of SOD in serum increased first and then decreased during stress. The highest level of MDA was detected at 4 hours of WIRS, and the lowest level was detected at 4 hours of WIRS. The relative expression of TRPV1 mRNA in gastric mucosa of WIRS 2h, WIRS 4H and WIRS 6h groups was (1.43 (0.19)), (1.20 (0.13) and (0.19 (0.03) times higher than that of NC group, respectively. The expression of TRPV1mRNA in group RS6h decreased, and the difference was statistically significant (P0.05).
empirical conclusion
(1) Sufentanil can effectively alleviate WIRS-induced acute gastric mucosal injury. The mechanism may be related to the control of oxidative stress, the decrease of gastric acid secretion and the regulation of TRPV1 mRNA expression in the central and peripheral regions.
(2) There is a compensatory process in the process of stress ulcer. The levels of serum SOD and MDA can be used as an early indicator to detect stress ulcer, and provide the basis for choosing the appropriate time to intervene in stress ulcer.
(3) TRPV1 and ASIC3 play a certain role in the process of stress-induced acute gastric mucosal lesion, and the change trend of them is consistent. ASIC3 is more sensitive to WIRS-induced acute gastric mucosal lesion than TRPV1.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614
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