瞬时受体电位粘脂素1介导细胞自噬在远端缺血后处理保护大鼠脑缺血-再灌注损伤中的作用研究

doi:10.3877/cma.j.issn.1674-6880.2024.03.002

目的

研究远端缺血后处理（RIPostC）对脑卒中大鼠脑缺血半暗带的保护作用及最佳干预时间，初步探讨RIPostC脑保护的相关机制。

方法

构建大鼠大脑中动脉栓塞（MCAO）模型，栓塞2 h后再灌注，依据RIPostC开始干预时间将36只Sprague Dawley大鼠分为假手术组（Sham组）、缺血-再灌注（I/R）组、缺血后立即干预组（RIPostC-0 h组）、2 h后干预组（RIPostC-2 h组）、6 h后干预组（RIPostC-6 h组）和12 h后干预组（RIPostC-12 h组）。RIPostC进行4个循环，每个循环5 min。再灌注24 h后采用改良大鼠神经功能缺损（mNSS）评分进行神经功能评估，2,3,5-氯化三苯基四氮唑（TTC）染色评估脑梗死体积，干-湿重法测定脑含水量，荧光定量PCR检测瞬时受体电位粘脂素1（TRPML1）信使RNA（mRNA）表达水平，western-blotting法检测TRPML1和自噬相关蛋白Beclin1、微管相关蛋白1轻链3（LC3）、p62的蛋白表达水平。

结果

6组大鼠mNSS评分、脑梗死体积、脑含水量、LC3-Ⅱ/LC3-Ⅰ比值、Beclin1、p62以及TRPML1 mRNA和蛋白表达水平比较，差异均具有统计学意义（F = 4.640、9.968、10.211、83.414、32.074、8.234、172.232、27.462，P均< 0.05）。与I/R组比较，各RIPostC组mNSS评分、脑梗死体积、脑含水量、p62蛋白均显著降低（P均< 0.05）；而LC3-Ⅱ/LC3-Ⅰ比值、Beclin1以及TRPML1 mRNA和蛋白表达水平均显著升高，且RIPostC-0 h组TRPML1 mRNA和蛋白表达水平升高更明显（P均< 0.05）。此外，RIPostC-0 h组、RIPostC-2 h组、RIPostC-6 h组脑梗死体积均较RIPostC-12 h组减少更明显（P均< 0.05）。

结论

RIPostC可显著减少大鼠脑梗死体积、减轻脑水肿、改善神经功能缺损症状，且在I/R之后立即实施效果最佳，RIPostC的脑保护作用可能是通过上调TRPML1提高脑I/R区的自噬水平实现的。

关键词: 瞬时受体电位粘脂素1, 细胞自噬, 远端缺血后处理, 缺血-再灌注损伤

Objective

To investigate the protective effect and optimal intervention time of remote ischemic post-conditioning (RIPostC) on ischemic penumbra of stroke rats, and preliminarily explore the brain protective mechanisms of RIPostC.

Methods

A rat middle cerebral artery occlusion (MCAO) model was constructed and reperfusion was performed after 2 hours of embolization. Thirty-six Sprague Dawley rats were divided into a sham operated group (Sham group), an ischemic/reperfusion group (I/R group), an immediate intervention after ischemia group (RIPostC-0 h group), an intervention after ischemia for 2 h group (RIPostC-2 h group), an intervention after ischemia for 6 h group (RIPostC-6 h group), and an intervention after ischemia for 12 h group (RIPostC-12 h group). RIPostC was performed 5 minutes each time with four cycles. After 24 hours of reperfusion, the neurological function was evaluated using the modified neurological severity score (mNSS), the cerebral infarction volume was evaluated by staining with 2,3,5-triphenyltetrazolium chloride (TTC), the brain water content was measured by a dry-wet weight method, the transient receptor potential mucolipin 1 (TRPML1) messenger RNA (mRNA) was detected by fluorescence quantitative PCR, and the protein expression of TRPML1, autophagy related protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), and p62 was detected by western-blotting.

Results

Significant statistical differences were observed in the mNSS score, cerebral infarction volume, brain water content, LC3-II/LC3-I ratio, Beclin1, p62, and TRPML1 mRNA and protein of the six groups (F = 4.640, 9.968, 10.211, 83.414, 32.074, 8.234, 172.232, 27.462; all P < 0.05). Compared with the I/R group, the mNSS score, cerebral infarction volume, brain water content, and p62 protein were significantly reduced, and the LC3-II/LC3-I ratio, Beclin1, and TRPML1 mRNA and protein were significantly increased in all RIPostC groups (all P < 0.05). The RIPostC-0 h group also had a more significant increase in TRPML1 mRNA and protein than other RIPostC groups (all P < 0.05). In addition, the cerebral infarction volume in the RIPostC-0 h group, RIPostC-2 h group, and RIPostC-6 h group was decreased more significantly than that in the RIPostC-12 h group (all P < 0.05).

Conclusions

RIPostC can significantly reduce infarction volumes, alleviate brain edema, and improve neurological deficits, which should be implemented immediately after reperfusion. The neuroprotective effect of RIPostC may be achieved by upregulating TRPML1 to increase autophagy levels in the cerebral I/R area.

Key words: Transient receptor potential mucolipin 1, Autophagy, Remote ischemic post-conditioning, Cerebral ischemic/reperfusion injury

表1 各组大鼠mNSS评分比较

组别	只数	评分（分， ± s）
Sham组	6	0
I/R组	6	14.5 ± 1.5^a
RIPostC-0 h组	6	11.3 ± 1.4^ab
RIPostC-2 h组	6	11.3 ± 1.5^ab
RIPostC-6 h组	6	12.2 ± 1.3^ab
RIPostC-12 h组	6	12.3 ± 1.6^ab
F值		4.640
P值		0.006

表1 各组大鼠mNSS评分比较

组别	只数	评分（分， ± s）
Sham组	6	0
I/R组	6	14.5 ± 1.5^a
RIPostC-0 h组	6	11.3 ± 1.4^ab
RIPostC-2 h组	6	11.3 ± 1.5^ab
RIPostC-6 h组	6	12.2 ± 1.3^ab
RIPostC-12 h组	6	12.3 ± 1.6^ab
F值		4.640
P值		0.006

图1 各组大鼠脑梗死体积和脑含水量比较注：I/R.缺血-再灌注；RIPostC.远端肢体缺血后处理；TTC. 2,3,5-氯化三苯基四氮唑；Sham组为假手术组；RIPostC-0 h组、RIPostC-2 h组、RIPostC-6 h组、RIPostC-12 h组分别为RIPostC立即干预组、2 h后干预组、6 h后干预组和12 h后干预组；a图为TTC染色下各组大鼠脑组织梗死情况，红色区域为正常脑组织区域，白色区域为脑梗死区域；b图与c图分别为各组大鼠脑梗死体积百分比及脑含水量比较，与I/R组相比，^aP < 0.05；与RIPostC-12 h组比较，^bP < 0.05；与Sham组比较，^cP < 0.05

图2 RIPostC对大鼠脑缺血半暗带区细胞自噬相关蛋白表达的影响注：RIPostC.远端肢体缺血后处理；LC3.微管相关蛋白1轻链3；I/R.缺血-再灌注；Sham组为假手术组；RIPostC-0 h组、RIPostC-2 h组、RIPostC-6 h组、RIPostC-12 h组分别为RIPostC立即干预组、2 h后干预组、6 h后干预组和12 h后干预组；与Sham组比较，^aP < 0.05；与I/R组比较，^bP < 0.05

图3 各组大鼠脑缺血半暗带区TRPML1 mRNA及蛋白表达水平的比较注：TRPML1.瞬时受体电位粘脂素1；mRNA.信使RNA；I/R.缺血-再灌注；Sham组为假手术组；RIPostC.远端肢体缺血后处理；RIPostC-0 h组、RIPostC-2 h组、RIPostC-6 h组、RIPostC-12 h组分别为RIPostC立即干预组、2 h后干预组、6 h后干预组和12 h后干预组；与Sham组比较，^aP < 0.05；与I/R组比较，^bP < 0.05；与RIPostC-0 h组比较，^cP < 0.05

1	GBD 2019 Stroke Collaborators. Global, regional, and national burden of stroke and its risk factors, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019[J]. Lancet Neurol, 2021, 20 (10): 795-820.
2	中国中西医结合学会急救医学专业委员会.中国急性缺血性脑卒中中西医急诊诊治专家共识[J].中华危重病急救医学，2018，30（3）：193-197.
3	Jovin TG, Li C, Wu L, et al. Trial of thrombectomy 6 to 24 hours after stroke due to basilar-artery occlusion[J]. N Engl J Med, 2022, 387 (15): 1373-1384.
4	Roaldsen MB, Jusufovic M, Berge E, et al. Endovascular thrombectomy and intra-arterial interventions for acute ischaemic stroke[J]. Cochrane Database Syst Rev, 2021, 6 (6): Cd007574.
5	Saini V, Guada L, Yavagal DR. Global epidemiology of stroke and access to acute ischemic stroke interventions[J]. Neurology, 2021, 97 (20 Suppl 2): S6-S16.
6	卢志刚，卢青，李钊硕. Nrf2信号通路在脑缺血/再灌注损伤神经保护机制中的研究进展[J].中华危重病急救医学，2022，34（3）：325-328.
7	Kan X, Yan Z, Wang F, et al. Efficacy and safety of remote ischemic conditioning for acute ischemic stroke: a comprehensive meta-analysis from randomized controlled trials[J]. CNS Neurosci Ther, 2023, 29 (9): 2445-2456.
8	林羽佳，宋斌，林庆明，等.自噬在缓激肽后处理心肺复苏大鼠神经保护中的作用机制[J].中华危重病急救医学，2021，33（9）：1099-1104.
9	Denton D, Kumar S. Autophagy-dependent cell death[J]. Cell Death Differ, 2019, 26 (4): 605-616.
10	Wang J, Han D, Sun M, et al. Cerebral ischemic post-conditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model[J]. Mol Med Rep, 2016, 14 (5): 4162-4172.
11	Qi ZF, Luo YM, Liu XR, et al. AKT/GSK3β-dependent autophagy contributes to the neuroprotection of limb remote ischemic postconditioning in the transient cerebral ischemic rat model[J]. CNS Neurosci Ther, 2012, 18 (12): 965-973.
12	Medina DL. TRPML1 and TFEB, an intimate affair[J]. Handb Exp Pharmacol, 2023 (278): 109-126.
13	Wang Y, Jiang SW, Liu X, et al. Degradation of TRPML1 in neurons reduces neuron survival in transient global cerebral ischemia[J]. Oxid Med Cell Longev, 2018, (2018): 4612727.
14	Li J, Hu XS, Zhou FF, et al. Limb remote ischemic postconditioning protects integrity of the blood-brain barrier after stroke[J]. Neural Regen Res, 2018, 13 (9): 1585-1593.
15	Chen HS, Cui Y, Li XQ, et al. Effect of remote ischemic conditioning vs usual care on neurologic function in patients with acute moderate ischemic stroke: the RICAMIS randomized clinical trial[J]. Jama, 2022, 328 (7): 627-636.
16	McDonald MW, Dykes A, Jeffers MS, et al. Remote ischemic conditioning and stroke recovery[J]. Neurorehabil Neural Repair, 2021, 35 (6): 545-549.
17	Geng X, Wang Q, Lee H, et al. Remote ischemic postconditioning vs. physical exercise after stroke: an alternative rehabilitation strategy?[J]. Mol Neurobiol, 2021, 58 (7): 3141-3157.
18	Ren C, Wang P, Wang B, et al. Limb remote ischemic per-conditioning in combination with post-conditioning reduces brain damage and promotes neuroglobin expression in the rat brain after ischemic stroke[J]. Restor Neurol Neurosci, 2015, 33 (3): 369-379.
19	Gao X, Liu Y, Xie Y, et al. Remote ischemic postconditioning confers neuroprotective effects via inhibition of the BID-mediated mitochondrial apoptotic pathway[J]. Mol Med Rep, 2017, 16 (1): 515-522.
20	Yang J, Balkaya M, Beltran C, et al. Remote postischemic conditioning promotes stroke recovery by shifting circulating monocytes to CCR2 (+) proinflammatory subset[J]. J Neurosci, 2019, 39 (39): 7778-7789.
21	Chen GZ, Shan XY, Li XS, et al. Remote ischemic postconditioning protects the brain from focal ischemia/reperfusion injury by inhibiting autophagy through the mTOR/p70S6K pathway[J]. Neurol Res, 2018, 40 (3): 182-188.
22	Huang P, Xu M, Wu Y, et al. Multiple facets of TRPML1 in autophagy[J]. Cell Calcium, 2020 (88): 102196.
23	Li Z, Cui X, Lv H, et al. Remote ischemic postconditioning attenuates damage in rats with chronic cerebral ischemia by upregulating the autophagolysosome pathway via the activation of TFEB[J]. Exp Mol Pathol, 2020 (115): 104475.
24	Scotto Rosato A, Montefusco S, Soldati C, et al. TRPML1 links lysosomal calcium to autophagosome biogenesis through the activation of the CaMKKβ/VPS34 pathway[J]. Nat Commun, 2019, 10 (1): 5630.

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Role of transient receptor potential mucolipin 1 mediated autophagy in remote ischemic post-conditioning protecting against cerebral ischemic/reperfusion injury in rats

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Role of transient receptor potential mucolipin 1 mediated autophagy in remote ischemic post-conditioning protecting against cerebral ischemic/reperfusion injury in rats