Karagiannidis Ioannis, Kataki Agapi, Glustianou Georgia, Memos Nikolaos, Papalois Apostolos, Alexakis Nikolaos, Zografos George C, Konstadoulakis Manoussos M
*Laboratory of Surgical Research, 1st Department of Propaedeutic Surgery, University of Athens Hippocration Hospital †Histopathological Diagnosis, Soutsou, Athens ‡Experimental Research Center ELPEN Pharmaceuticals, Pikermi §1st Department of Propaedeutic Surgery, University of Athens Hippocration Hospital, Athens, Greece.
Shock. 2016 Feb;45(2):139-47. doi: 10.1097/SHK.0000000000000505.
The impact of a potential autophagy (LC3a/b) deregulation in hyper and in hypo stages during sepsis-induced kidney injury and the temporal profile of phosphorylated extracellular signal-related kinase, P38 (pP38), Akt (pAKT), and 13-3-3β protein were investigated in the current study, using a rat cecal ligation and puncture (CLP) model, by means of flow cytometry and immunohistochemistry. Cell viability was assessed by protein C zymogen concentrate (PC), 7-aminoactinomycin D (7-AAD) staining and inflammation by S100 protein immunostaining. The impact of reduced kidney inflammation in autophagy was assessed by PC administration, an anti-inflammatory and cytoprotective substance. Sepsis induction increased LC3a/b expression, which presented two peaks at 6 and 36 h after CLP, both in the percentage of positive cells (P = 0.024, P = 0.025, respectively) and in fluorescence intensity. At 6 h when inflammation was already apparent, LC3a/b increase was escorted by phosphorylated extracellular signal-related kinase stimulation and high cell viability (65%), designating autophagy as a cytoprotective mechanism against microbial infection. The phosphorylation of P38 was delayed to 12 h after CLP, when autophagy was reduced. pAkt and 14-3-3β expression was stimulated between 6 and 36 h after CLP, although a slight inhibition of pAkt within each cell was detected (lower MnIX value). During the second peak, inflammation was intensified, necrosis was significantly increased with LC3a/b+/7-AAD + cells to present a 1.5-fold increase. Protein C zymogen concentrate administration declined autophagy at 6 and 36 h after CLP and reduced necrosis, whereas double positive LC3a/b and 7-AAD cells were increased by 1.68 and 2.78-fold, respectively. These data open new prospectives in sepsis treatment, since they further support that autophagy represents a cytoprotective mechanism triggered by stress conditions, rather than an alternative cell death pathway.
在本研究中，我们使用大鼠盲肠结扎穿孔（CLP）模型，通过流式细胞术和免疫组织化学，研究了脓毒症诱导的肾损伤过程中高炎症和低炎症阶段潜在的自噬（LC3a/b）失调的影响，以及磷酸化细胞外信号调节激酶、P38（pP38）、Akt（pAKT）和13 - 3 - 3β蛋白的时间变化情况。通过蛋白C酶原浓缩物（PC）、7 - 氨基放线菌素D（7 - AAD）染色评估细胞活力，通过S100蛋白免疫染色评估炎症情况。通过给予PC（一种抗炎和细胞保护物质）来评估减轻肾脏炎症对自噬的影响。脓毒症诱导增加了LC3a/b的表达，在CLP后6小时和36小时出现两个峰值，无论是阳性细胞百分比（分别为P = 0.024，P = 0.025）还是荧光强度。在6小时炎症已经明显时，LC3a/b的增加伴随着磷酸化细胞外信号调节激酶的刺激和较高的细胞活力（65%），表明自噬是针对微生物感染的一种细胞保护机制。P38的磷酸化在CLP后延迟至12小时，此时自噬减少。pAkt和14 - 3 - 3β的表达在CLP后6至36小时受到刺激，尽管在每个细胞内检测到pAkt有轻微抑制（较低的MnIX值）。在第二个峰值期间，炎症加剧，坏死显著增加，LC3a/b + /7 - AAD + 细胞增加了1.
