氯胺酮致大鼠肾脏损害机制及褪黑素的治疗作用研究

发布时间：2018-02-13 02:05

本文关键词： 氯胺酮肾损伤褪黑素氧化应激凋亡　出处：《广西医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景目前研究发现,氯胺酮对膀胱的损伤机制可能与氯胺酮的直接毒性作用、炎症损伤、血管损伤以及氧化应激的有关。但其对肾脏的损害作用机制尚不明确。本研究通过建立氯胺酮致SD大鼠肾脏损伤动物模型,观察SD大鼠行为学及体重变化、取肾脏行HE染色、马松染色观察病理学变化；测定肾组织匀浆中丙二醛(MDA)的含量、超氧化物气化酶(SOD)和谷胱甘肽过氧化物酶(GSH-P x)的活力,以及抗氧化剂褪黑素(Melatonni,MT)干预后是否对肾脏有保护作用。研究目的：探讨氯胺酮所致肾脏损伤的可能机制以及MT对氯胺酮染毒大鼠肾脏损伤的保护作用。研究方法：选择成年雄性SD大鼠30只,随机分为三组：(1)生理盐水组(对照组)：每日注射生理盐水lml；(2)氯胺酮组(K实验组)：氯胺酮按100mg/kg用生理盐水稀释至1m1每天腹腔注射。(3)MT+氯胺酮联合给药组(MT保护组)：氯胺酮按10Omg/kg+褪黑素10mg/kg用生理盐水稀释至1m1每天腹腔注射。三组大鼠饲养时间为12周。12周后经腹主动脉采血,测量血清中肌酐、尿素氮评估大鼠的肾脏功能。然后处死大鼠,取肾脏制作组织匀浆,测量丙二醛(MDA)的含量、超氧化物气化酶(SOD)和微量还原型谷胱甘肽(GSH)的活力。另取肾脏组织行HE、Masson染色和TUNEL凋亡检测。研究结果：1.大鼠行为学和体重变化对照组大鼠在注射等量生理盐水后,大鼠行为未见明显异常。而氯胺酮组和MT保护组大鼠在腹腔注射药物后,大鼠先出现兴奋增加,走路不稳、后肢瘫痪和共济失调等表现。其后均在lmin内出现深度麻醉,表现为嗜睡、对刺激无反应等。从第2周至12周,对照组大鼠的平均体重均高于K实验组和MT保护组,三组大鼠的体重变化差异有统计学意义(P0.05)。并且MT保护组大鼠体重平均值也高于K实验组,二者比较差异有统计学意义(P0.05)。2.大鼠肾功能检测结果大鼠血清Scr、BUN在各组之间差异无统计学意义(P0.05)。但MT保护组和K实验组的Scr和BUN值仍然分别有14只(14/20)、12只(12/20)高于对照组的均值,提示氯胺酮可能对大鼠的肾脏滤过功能有一定的损害作用。3.肾组织匀浆氧化指标测定结果三组肾组织匀浆MDA含量差异有统计学意义(P0.001),K实验组MDA含量明显高于对照组(P0.001)；而MT保护组MT明显抑制了氯胺酮引起的大鼠肾脏组织MDA的升高趋势,与K组比较明显降低(K0.001)。三组大鼠肾组织匀浆SOD活力差异有统计学意义(P0.001),K实验组明显低于对照组(P0.001),MT组增加了肾组织匀浆SOD活力,与K实验组比较差异有统计学意义(P0.05)。三组大鼠肾组织匀浆GSH-Px活力差异有统计学意义(P0.001),K实验组明显低于对照组(P0.001),MT组增加了肾组织匀浆GSH-Px活力,与K实验组比较差异有统计学意义(P0.05)。4.大鼠肾脏HE染色结果对照组大鼠的肾脏结构组织形态学正常,无肾小管水肿、淤血或管型形成。K实验组肾小管、肾小球周围能够见到大量炎症细胞浸润,肾间质有纤维化条索形成。肾小管大量水肿,正常结构消失,出现大量空泡样改变；肾小球变性萎缩,甚至出现闭锁；MT保护组肾小管、肾小球炎症细胞浸润、肾间质纤维化较K实验组减轻,部分肾小球出现萎缩。5.大鼠肾脏Masson染色结果与对照组比较,K实验组、MT保护组肾间质胶原纤维染色明显加深,可见纤维化条索形成,提示在长期腹腔注射氯胺酮后,肾间质胶原沉积增加、纤维化加重。但MT保护组较K实验组肾间质胶原沉积减少,纤维化程度减轻。半定量分析发现,三组间比较差异有统计学意义(F=178.83,P0.001)。对照组肾间质平均胶原面积比是(8.97%±0.02)；K实验组平均胶原面积比是(41.49%±0.03)；MT保护组平均胶原面积比是(28.54%±0.04)。K实验组和对照组比较,差异有统计学意义(P0.001),提示氯胺酮导致肾间质发生纤维化;MT保护组与K实验组比较,差异有统计学意义(P0.001),提示褪黑素在一定程度上能够减轻肾间质的纤维化程度。6.大鼠肾脏肾小球TUNE凋亡细胞检测对照组偶见凋亡阳性细胞。K实验组和MT保护组,凋亡细胞呈现明显增多。半定量分析发现,肾小管平均每高倍镜下阳性细胞K实验组为171.76±14.67个,MT保护组为149.92±27.87个,NS对照组为64.56±17.90个。三者比较,差异有统计学意义(F=73.33,P0.001)。K实验组和对照组比较,差异有统计学意义(P0.001),MT保护组和对照组比较,差异有统计学意义(P0.001)。MT保护组比K实验组凋亡细胞计数明显减少,差异有统计学意义(P0.05)。研究结论：1.长期腹腔注射氯胺酮是制造大鼠氯胺酮性肾脏损伤模型的可靠方法。2.氯胺酮导致大鼠肾组织匀浆中MDA的水平升高,SOD和GSH-Px的活力下降,并且在应用MT治疗后,可明显降低大鼠肾组织匀浆中MDA的含量,增加SOD和GSH-Px的活力。提示大鼠体内存在氧化损伤和氧化-抗氧化失衡,可能为氯胺酮致肾脏损伤的机制之一,褪黑素对肾脏具有一定的保护作用。3.氯胺酮导致肾脏炎症细胞浸润和肾脏细胞凋亡增加,而MT能够减轻肾脏的细胞凋亡,MT的保护机制可能和增强体内抗氧化应激能力有关系。
[Abstract]:On the background of the present study found a direct toxic effect, the possible mechanism of ketamine and ketamine on injury of bladder inflammation injury, vascular injury and oxidative stress damage to the kidneys. But its mechanism is still not clear. In this study we established a model of renal injury induced by ketamine animal SD rats, and to observe the change of body weight SD the behavior in the kidneys, HE staining was performed to observe the pathological changes of Masson staining; Determination of malondialdehyde in kidney homogenates (MDA) content, superoxide enzyme gasification (SOD) and glutathione peroxidase (GSH-P x) activity and melatonin (Melatonni, MT) after the intervention have protective effects on kidney research. Objective: To investigate the possible mechanism of renal injury induced by ketamine and the protective effect of MT on renal injury in rats exposed to ketamine. Methods: 30 male SD rats were randomly. Divided into three groups: (1) normal saline group (control group): daily injection of saline LML; (2) ketamine group (K group): Ketamine 100mg/kg diluted to 1m1 with normal saline by intraperitoneal injection every day. (3) MT+ ketamine combined drug group (MT group): according to the 10Omg/kg+ of melatonin 10mg/kg diluted with saline to 1m1 daily intraperitoneal injection of ketamine. Three rats were fed for 12 weeks.12 weeks after abdominal aortic blood, serum creatinine measurements, evaluation of rat kidney function of urea nitrogen. Then the rats were sacrificed and kidneys tissue homogenate, measurement of malondialdehyde (MDA) content, superoxide gasification enzyme (SOD) and trace glutathione (GSH) activity. The other kidney tissues were collected for detection of HE, Masson staining and TUNEL apoptosis. Results: 1. the behavior of rats and the change of body weight of rats in control group after the injection of normal saline, rats had no obvious differences Often. And ketamine group and MT group rats after intraperitoneal injection of drugs, rats first appeared excited increase, ataxia, ataxia and hind limb paralysis. Subsequently occurred within lmin depth of anesthesia, as lethargy, no response to stimulation. From second to 12 weeks, the average weight of control rats were higher than that of K group and MT group, there were statistically significant differences in body weight changes of the three groups of rats (P0.05) and MT protection group weight average value is higher than that of K group, there was significant difference between the two groups (P0.05) in serum of Scr rats renal function in rats with.2. detection results there was no significant difference of BUN between the groups (P0.05). But the MT protection group and K experimental group Scr and BUN were still 14 (14/20), 12 (12/20) higher than the control group, suggesting that ketamine may on rat glomerular filtration function have a certain role in.3. damage kidney group Oxidative index determination results between homogenate homogenate MDA content of renal tissue of three groups was statistically significant (P0.001), K MDA in the experimental group were significantly higher than the control group (P0.001); while the MT protection group MT obviously inhibited by ketamine in renal tissue of MDA rats increased, compared with the K group decreased significantly (K0.001). The three groups had statistical significance in kidney homogenates of rat SOD activity (P0.001), K group was significantly lower than the control group (P0.001), MT group increased SOD activity in renal tissue homogenate, and the difference had statistical significance K experimental group (P0.05). The three groups had statistical significance in kidney homogenates of rat GSH-Px activity the difference of K (P0.001), the experimental group was significantly lower than the control group (P0.001), MT group increased GSH-Px activity in renal tissue homogenate, and the difference had statistical significance in experimental group K (P0.05) HE in the kidney of.4. rat kidney tissue staining structure of rats in control group were morphological Often, no renal tubular edema, congestion or tube type.K experimental group of renal tubule formation can be seen around the glomerular infiltration of inflammatory cells, renal interstitial fibrosis cord formation. Renal tubular edema, normal structure disappeared, the emergence of a large number of vacuolar changes; glomerular atrophy and degeneration, even atresia; MT group of renal tubules glomerular, inflammatory cell infiltration, renal interstitial fibrosis than K experimental group, partial glomerular atrophy of renal Masson in rats with.5. staining results compared with the control group, K group, MT group of renal interstitial collagen fiber staining was deepened, fibrosis cord formation, suggesting that in the long term after intraperitoneal injection of ketamine, kidney collagen deposition and fibrosis. But the MT protection group compared with the K experimental group of renal interstitial collagen deposition decreased, reduce the degree of fibrosis. Semi quantitative analysis showed that there were significant differences between the three groups (F=178 .83, P0.001). The control group of renal interstitial collagen area ratio is the average (8.97% + 0.02); K group average collagen area ratio is (41.49% + 0.03); MT group average collagen area ratio is (28.54% + 0.04).K experimental group and the control group, the difference was statistically significant (P0.001). It indicates that ketamine lead to renal interstitial fibrosis; MT group and K experimental group, the difference was statistically significant (P0.001), suggesting that the degree of fibrosis in.6. rat kidney TUNE cell apoptosis of melatonin can reduce the renal interstitial in a certain extent, the control group occasionally positive apoptotic cells in experimental group.K and MT group. Apoptotic cells showed significantly increased. Semi quantitative analysis showed that the renal tubular average high magnification of K positive cells in experimental group was 171.76 + 14.67, 149.92 + 27.87 MT protection group, NS control group is 64.56 + 17.90. Three comparison, the difference was statistically significant (F=73. 33, P0.001).K experimental group and the control group, the difference was statistically significant (P0.001), MT group and control group, the difference was statistically significant (P0.001) in.MT group was obviously lower than that of K group the number of apoptotic cells, the difference was statistically significant (P0.05). Conclusions: 1. long term ketamine intraperitoneal injection ketamine is a reliable method for manufacturing renal injury in rats.2. ketamine resulted in elevated rat renal tissue homogenate levels of MDA, SOD and GSH-Px activity decreased, and the application of MT after treatment, can significantly reduce the content of rat kidney tissue homogenate in MDA, SOD and GSH-Px increased vitality. Suggesting the presence of oxidation damage and oxidant / antioxidant imbalance in rats, one of the possible mechanisms of renal damage induced by ketamine, melatonin has a protective effect on renal.3. ketamine must lead to renal inflammatory cell infiltration and apoptosis of kidney cells, MT can reduce the apoptosis of the kidneys, and the protective mechanism of MT may be associated with the enhancement of anti oxidative stress in the body.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R692

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