高容量血液滤过对多器官功能障碍综合征犬内质网应激标志蛋白Caspase-12基因水平及表达的影响

Effect of high volume hemofiltration on the expression of caspase-12, the protein marker of endoplasmic reticulum stress, in dogs with multiple organ dysfunction syndrome

目的探讨高容量血液滤过(HVHF)治疗多脏器功能障碍综合征(multiple organ dysfunction syndrome,MODS)对内质网应激相关性细胞凋亡及应激标志蛋白Caspase-12基因水平及表达的影响,并探讨高容量血液滤过(high volume hemofiltration,HVHF)治疗MODS的机制。方法经二次打击建立犬MODS模型。分HVHF组和MODS组,于术前(T1)、内毒素注射完后0h(T2)、6h(T3)、12h(T4)及24h(T5)留血标本。体内实验:应用荧光定量PCR法检测肝、肾及肺组织Caspase-12m RNA基因表达水平。体外实验:2组血清体外诱导人脐静脉内皮细胞(HUVECs)建立内质网应激凋亡模型。测定si RNA干扰前后Caspase-12m RNA基因表达、蛋白表达及内皮细胞凋亡率。结果体内实验:与MODS组相比,HVHF组肾、肺组织Caspase-12m RNA基因表达水平明显降低,差异有统计学意义(P<0.05),而肝组织Caspase-12m RNA基因表达水平无明显差异(P>0.05)。体外实验:干扰前与MODS组相比,HVHF组在T2-T5HUVECs Caspase-12m RNA基因表达水平、Caspase-12蛋白表达水平及细胞凋亡率均明显降低,差异有统计学意义(P<0.01)。干扰后2组HUVECs Caspase-12m RNA基因表达水平、Caspase-12蛋白表达水平及细胞凋亡率均较干扰前显著降低,差异有统计学意义(P<0.01)。结论内质网应激凋亡信号通路在MODS演进过程中占重要地位,经过HVHF治疗后内质网应激(ERS)凋亡信号通路中的关键蛋白Caspase-12基因表达、Caspase-12蛋白表达水平及细胞凋亡率均明显下降,此作用有助于减轻机体细胞凋亡程度,有效地救治MODS。而RNA干扰(RNAi)技术有望成为治疗MODS新的治疗靶点。

Objective To investigate the effect of high volume hemofiltration （HVHF） on the gene expres- sion ofendoplasmic reticulum stress markers in multiple organ dysfunction syndrome （MODS） dogs. Meth- ods MODS model of Beagle dogs were established by a ＂two-hit＂ injury and then randomly divided into two groups, HVHF group （n=6） and MODS group （n=6）. Blood samples were drawn in the two groups at pre- operative （T1） and after injection of endotoxin for Oh （T2）, 6h （T3）, 12h （T4） and 24 h （T5）. Quantitative real- time PCR was used to measure caspase-12 mRNA levels in liver, kidney and hmg. In addition, dog serum sam- ples were applied to human umbilical venous endothelial cells （HUVECs） to induce endoplasmic reticulum stress, and the expression of caspase-12 and apoptosis in HUVECs were then assayed before and after down- regulation of caspase-12 expression by siRNA interference. Results Caspase-12 mRNA levels in kidney and lung were lower in HVHF group than in MODS group （P〈0.05）, but the mRNA levels in liver was similar be- tween the two groups （P〉0.05）. In HUVECs before siRNA interference, caspase-12 mRNA, caspase-12 pro- tein and apoptosis decrease more by T2-T5 samples from HVHF group than by those from MODS group （P〈 0.01）. In HUVECs induced with serum samples from HVHF group and MODS group, caspase-12 mRNA, cas- pase-12 protein and apoptosis decrease more in HUVECs after the siRNA interference than in those before the siRNA interference （P〈0.01）. Conclusion The endoplasmic reticulum stress pathway plays an important role in the evolution processes of MODS. RNA interference may become a new therapeutic modality for the treatment of MODS.