右美托咪定对脓毒症小鼠急性肾损伤的影响

Effect of dexmedetomidine on acute kidney injury in septic mice

中文关键词: 右美托咪定  脓毒症  急性肾损伤  细胞焦亡

英文关键词: Dexmedetomidine  Sepsis  Acute kidney injury  Pyroptosis

中文摘要:

      
目的 探讨右美托咪定对脓毒症小鼠急性肾损伤的影响及与肾脏细胞焦亡的关系。
方法 健康清洁级ICR小鼠32只，雌雄各半，8~12周龄，体重20~25 g。采用随机数字表法将小鼠分为四组：对照组（C组）、脂多糖（LPS）组（L组）、LPS+右美托咪定组（LD组）和LPS+右美托咪定+阿替美唑组（LT组），每组8只。L组、LD组和LT组腹腔注射LPS 400 μg/kg，8 h后腹腔注射LPS 10 mg/kg建立脓毒症急性肾损伤模型。L组于建模即刻、建模后0.5、2、2.5、4、4.5 h腹腔注射生理盐水0.5 ml；LD组于建模即刻、建模后2、4 h腹腔注射生理盐水0.5 ml，于建模后0.5、2.5、4.5 h分别腹腔注射右美托咪定40 μg/kg；LT组于建模即刻、建模后2、4 h腹腔注射阿替美唑750 μg/kg，于建模后0.5、2.5、4.5 h分别腹腔注射右美托咪定40 μg/kg；C组在各时点腹腔注射等量生理盐水。所有小鼠于建模后24 h麻醉处死。采用全自动生化分析仪检测血清肌酐（Scr）和尿素氮（BUN）浓度，化学发光法测量肾皮质细胞三磷酸腺苷(ATP)和血清ATP浓度，ELISA法检测肾组织IL-1β和IL-18浓度，qRT-PCR法检测肾组织caspase-11、泛连接蛋白1（pannexin-1）、P2X7 mRNA表达量，Western blot法检测肾组织caspase-11、pannexin-1、P2X7蛋白含量，HE染色法观察肾组织病理结构，TUNEL染色记录肾小管上皮细胞凋亡细胞数并计算细胞凋亡率。
结果 与C组比较，L组、LD组和LT组血清BUN、Scr、ATP浓度均明显升高（P<0.05），肾皮质细胞ATP浓度明显降低（P<0.05），肾组织IL-1β和IL-18浓度、肾组织caspase-11、pannexin-1、P2X7 mRNA表达量及蛋白含量均明显升高（P<0.05），肾小管上皮细胞凋亡率明显升高（P<0.05）。与L组比较，LD组血清Scr、BUN、ATP浓度均明显降低（P<0.05），肾皮质细胞ATP浓度明显升高（P<0.05），肾组织IL-1β浓度、肾组织caspase-11、pannexin-1 mRNA表达量均明显降低（P<0.05），肾小管上皮细胞凋亡率明显降低（P<0.05）；LD组和LT组肾组织IL-18浓度、肾组织caspase-11、pannexin-1蛋白含量、肾组织P2X7 mRNA表达量及蛋白含量均明显降低（P<0.05）。与LD组比较，LT组血清BUN、Scr、ATP浓度均明显升高（P<0.05），肾皮质细胞ATP浓度明显降低（P<0.05），肾组织IL-1β和IL-18浓度、肾组织caspase-11、pannexin-1、P2X7 mRNA表达量及蛋白含量均明显升高（P<0.05），肾小管上皮细胞凋亡率明显升高（P<0.05）。
结论 右美托咪定减轻了LPS导致的脓毒症小鼠肾脏病理学损伤，降低肾组织IL-1β和IL-18浓度，降低肾小管上皮细胞凋亡率，可能通过非经典途径减轻了肾脏细胞焦亡。

英文摘要:

      
Objective To investigate the effect of dexmedetomidine on acute kidney injury and pyroptosis of renal cellse in septic mice.
Methods Thirty-two healthy ICR mice, half male and half female, aged 8-12 weeks, weighing 20-25 g, were divided into four groups using random number table method: control group (group C), lipopolysaccharide(LPS) group (group L), LPS + dexmedetomidine group (group LD), and LPS + dexmedetomidine + atimezole group (group LT), 8 mice in each group. The mice in groups L, LD and LT were injected intraperitoneally with LPS in dose of 400 μg/kg, and LPS was injected intraperitoneally in dose of 10 mg/kg after 8 hours to establish septic actue renal injury model. Group L was intraperitoneally injected with 0.5 ml of normal saline immediately after modeling and 0.5, 2, 2.5, 4, 4.5 hours after modeling. Group LD was intraperitoneally injected with 0.5 ml normal saline immediately after modeling and 2, 4 hours after modeling, and dexmedetomidine in dose of 40 μg/kg 0.5, 2.5, 4.5 hours after modeling, respectively. Group LT was intraperitoneally injected with atimezole in dose of 750 μg/kg immediately after modeling and 2, 4 hours after modeling, dexmedetomidine in dose of 40 μg/kg 0.5, 2.5 and 4.5 hours after modeling, respectively. Group C was intraperitoneally injected with the same amount of normal saline at each time point. Mice were anesthetized and killed 24 hours after the establishment of the model. The levels of blood serum creatinine (Scr) and blood urea nitrogen (BUN) were detected using automatic biochemical analyzer. The content of adenosine triphosphate (ATP) in renal cortex and serum was measured using chemiluminescence. The contents of IL-1β and IL-18 in renal tissue were determined by ELISA. qRT-PCR was used to detect the mRNA expression levels of caspase-11, pannexin-1 and P2X7. The relative protein expresions of caspase-11, pannexin-1 and P2XT in renal tissues were detected by Western blot. The pathological structure of renal tissue was observed by hematoxylin eosin staining. The number of apoptotic cells in renal cortex was tested by TUNEL staining and the apoptosis rate was calculated.
Results Compared with group C, serum BUN, Scr and ATP concentrations were significantly increased (P < 0.05), the concentration of ATP in renal cortex were decreased (P < 0.05), the concentrations of IL-1β and IL-18, mRNA expression and protein expressions of caspase-11, pannexin-1 and P2X7 in renal tissues were significantly increased (P < 0.05), the apoptosis rate of renal tubular epithelial cells were significantly increased in groups L, LD and LT (P < 0.05). Compared with group L, the serum concentrations of Scr, BUN and ATP were significantly decreased (P < 0.05), the concentration of ATP in renal cortex was increased, the concentration of IL-1β and the mRNA expressions of caspase-11 and pannexin-1 in renal tissues were significantly decreased (P < 0.05), the apoptosis rate of renal tubular epithelial cells was significantly decreased in group LD (P < 0.05). Compared with group L, the concentrations of IL-18, protein expressions of caspase-11 and pannexin-1 in renal tissues were significantly decreased (P < 0.05), mRNA expression and protein expression of P2X7 in renal tissues were significantly decreased in groups LD and LT (P < 0.05). Compared with group LD, serum BUN, Scr and ATP concentrations were significantly increased (P < 0.05), the concentration of ATP in renal cortex were decreased (P < 0.05), the concentrations of IL-1β and IL-18, mRNA expressions and protein expressions of caspase-11, pannexin-1 and P2X7 in renal tissues were significantly increased in group LT (P < 0.05), the apoptosis rate of renal tubular epithelial cells were significantly increased (P < 0.05).
Conclusion Dexmedetomidine alleviates LPS-induced renal pathological damage in mice with sepsis reduces the release of IL-1β and IL-18 in renal tissues, and reduces the apoptosis rate of renal tubular epithelial cells, possibly alleviating renal pyroptosis through a non-classical pathway.