高压氧对永久性大脑中动脉栓塞大鼠心肾组织自噬活性的影响

The effect of hyperbaric oxygen treatment on autophagy activity in heart and kidney of permanent middle cerebral artery occlusion rats

目的 :观察永久性大脑中动脉栓塞(permanent middle cerebral artery occlusion,p MCAO)模型大鼠心、肾组织内自噬标志物微管相关蛋白轻链3(LC3)和P62的变化情况,探讨高压氧对心、肾组织自噬的影响及意义。方法:雄性Sprague-Dawley大鼠共75只,随机分为对照组、p MCAO组和高压氧组(p MCAO后给予高压氧处理);每组按造模后6 h和5 d 2个时间点处死大鼠并分成2个亚组。通过改良神经功能缺陷评分(m NSS)评估大鼠神经功能损伤情况,利用2,3,5-氯化三苯基四氮唑(TTC)法测定大鼠脑梗死面积,蛋白质印迹法(Western blotting)检测心、肾组织蛋白表达情况,实时荧光定量(real time)PCR检测组织内基因水平。结果:高压氧干预可有效减少脑缺血损伤后梗死面积,有助于神经功能恢复。除了高压氧6 h亚组的肾组织外,各组心、肾组织LC3和P62的蛋白表达水平变化基本与基因水平相符,且LC3与P62蛋白表达变化趋势基本一致。在大鼠心、肾组织中,脑缺血6 h后LC3-Ⅱ/LC3-Ⅰ和P62表达升高,高压氧处理后下降(P<0.05);缺血后5 d结果基本与6 h相似。结论:脑缺血损伤后心、肾组织内LC3-Ⅱ/LC3-Ⅰ和P62蛋白表达水平上调,高压氧干预后可下调,高压氧可能通过抑制自噬活性减少脑缺血后继发的心、肾损伤。

Objective To observe the changes of autophagy marker light chain 3（LC3） and P62 in heart and kidney of permanent middle cerebral artery occlusion（p MCAO） rats and analyze the effect and significance of hyperbaric oxygen（HBO） treatment on autophagy of these tissues. Methods Seventy-five male Sprague-Dawley rats were randomly divided into control group, p MCAO model group and HBO group. And each group was made up of two subgroups which were sacrificed at 6 h and 5 d, respectively. Modified neurological severity score（m NSS） was used to evaluate the extent of neurologic injury, and the 2, 3, 5-triphenyl tetrazolium chloride（TTC） method was used to determine the infarcted area in brain. Western blotting was used to detect the protein expression and real time PCR was used to detect m RNA level of LC3 and P62 in heart and kidney. Results HBO could effectively reduce the infarction area after cerebral ischemic injury,which could promote functional recovery of nerve. Except in kidney of 6 h HBO group, changes in m RNA level of LC3 and P62 were in accordance with their protein expression. The trends of changes of expression of these two autophagic proteins were fundamentally the same. At 6 h after p MCAO, both LC3 and P62 were elevated（P〈0.05）, but declined after treated with HBO（P〈0.05）. Results at 5 d after ischemia were basically similar to that of 6 h after ischemia. Conclusions Levels of LC3-Ⅱ/LC3-Ⅰ and P62 protein were up-regulated after cerebral ischemia both in heart and kidney, and could be down-regulated by HBO treatment. HBO may alleviate the secondary heart and kidney damage after cerebral ischemia by inhibiting the autophagy activity.