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中华妇幼临床医学杂志(电子版)

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2023, Vol. 19 ›› Issue (05): 540 -549. doi: 10.3877/cma.j.issn.1673-5250.2023.05.007

Original Article

The protective effect and mechanism of overexpression of transcription factor 12 on the cerebral cortex of rats with sepsis-associated encephalopathy

Xiaoyan Zhang, Dongqiong Xiao, Hu Gao, Lin Chen, Fajuan Tang, Xihong Li†()   

  1. Department of Emergency Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
  • Received:2023-07-01 Revised:2023-09-08 Published:2023-10-01
  • Corresponding author: Xihong Li
  • Supported by:
    National Natural Science Foundation of China(82071353); Key R&D Project of Sichuan Provincial Department of Science and Technology(2021YFS0029, 2023YFS0025)
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Objective

To investigate protective effects and mechanism of overexpressed transcription factor 12 (Tcf12) on the cortex of rats with sepsis-associated encephalopathy (SAE).

Methods

A total of 144 male Sprague Dawley (SD) rats at 30 days of age were selected into this study. They were randomly divided into four groups according to the processing method: cecal ligation and perforation (CLP) model group (CLP group), Tcf12 negative control group (Veh+ CLP group), Tcf12 overexpression group (AD-Tcf12+ CLP group), and sham surgery group (Sham group). The Sham group and CLP group received 5 μL physiological saline injected into each lateral ventricle. The AD-Tcf12+ CLP group received 5 μL AD-Tcf12, and the Veh+ CLP group received 5 μL Veh. After 48 hours of lateral ventricle injection, CLP surgery was performed. The Sham group only underwent laparotomy and examination of the cecum. The mean arterial pressure (MAP), heart rate, and neurobehavioral scores were monitored at 3, 6, 12, 24, and 48 hours after modeling. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the cerebral cortex of each group at 24 hours after modeling. Western blotting was used to detect the protein expression of Tcf12, cleaved caspase-3, Bax, Bcl2, P38, and p-P38 in the cerebral cortex of rats. Immunofluorescence staining was used to observe the expression of Tcf12 in neurons of the cerebral cortex. This experiment was approved by the Animal Protection Research Committee of West China Second University Hospital, Sichuan University [Approval No. 2020(022)], and animal experiments were carried out in accordance with corresponding guidelines and regulations.

Results

① Compared with Sham group, the MAP and neurobehavioral scores of CLP group and Veh+ CLP group showed a decreasing trend at different time points, while heart rate showed an increasing trend. The most significant changes occurred at 24 hours after modeling, and the differences were statistically significant (P<0.05). In addition, the neurobehavioral scores of CLP group and Veh+ CLP group were less than 6 points at 24 hours, consistent with the diagnosis of SAE. ② Compared with Veh+ CLP group, AD-Tcf12+ CLP group showed increased MAP and neurobehavioral scores and decreased heart rate, with the most significant changes occurring 24 hours after modeling, and the differences were statistically significant (P<0.05). ② Western blotting detection results showed that compared with Sham group, there were statistically significant differences in the relative expression levels of Tcf12 at 12, 24, and 48 h after modeling in CLP group (P=0.045, 0.019, 0.030), reaching a low point at 24 hours. ③ Immunofluorescence staining at 24 h after modeling showed that Tcf12 expression was mainly located in the cytoplasm of neurons. At 24 h after modeling, the fluorescence signal of Tcf12 (75.11±6.39) in CLP group SD rats was significantly lower than that in Sham group (101.79±3.89), and the difference was statistically significant (t=8.73, P<0.001). ④ Compared with Sham group, the expression levels of cleaved caspase-3 and Bax, as well as p-P38/P38, were significantly increased in CLP group and Veh+ CLP group rats, while the expression level of Bcl2 protein was significantly decreased, with statistically significant differences (P<0.05). ⑤ HE staining showed that compared with Sham group, there were significant pathological changes in the cerebral cortex tissue of Veh+ CLP group and CLP group rats, including nuclear condensation, cell edema, decreased cell number, and disordered arrangement. However, the pathological changes in the cerebral cortex tissue of AD-Tcf12+ CLP group rats were significantly improved.

Conclusions

The expression of Tcf12 is downregulated in the cortical tissue of rats with SAE, especially in cortical neurons. Overexpression of Tcf12 could have neuroprotective effects on rats with SAE, and its mechanism might be related to the neuronal apoptosis inhibited in the cerebral cortex.

Key words: Transcription factor 12, Apoptosis, Sepsis related encephalopathy, Neurons, Mechanism, Rats
表1 4组30 d龄雄性SD大鼠不同时间点平均动脉压比较(mmHg，±s)
组别 鼠数(只) 3 h 6 h 12 h 24 h 48 h
①CLP组 6 96.5±3.6 95.7±3.1 81.0±2.4 63.5±4.4 78.0±1.8
②AD-Tcf12＋CLP组 6 98.8±2.0 97.0±2.4 95.2±3.4 91.5±2.7 94.2±3.7
③Veh＋CLP组 6 97.0±4.3 96.0±4.7 79.8±1.3 64.5±3.1 76.2±3.1
④Sham组 6 100.0±2.1 100.0±1.7 100.3±2.6 102.5±1.6 103.8±4.0
总体比较            
F值   1.58 2.30 99.05 237.11 100.47
P值   0.225 0.109 <0.001 <0.001 <0.001
多重比较的P值            
① vs ④   0.419 0.173 <0.001 <0.001 <0.001
③ vs ④   0.698 0.252 <0.001 <0.001 <0.001
③ vs ①   >0.999 >0.999 >0.999 >0.999 >0.999
② vs ③   >0.999 >0.999 <0.001 <0.001 <0.001
表2 4组30 d龄雄性SD大鼠不同时间点心率比较(次/min，±s)
组别 鼠数(只) 3 h 6 h 12 h 24 h 48 h
①CLP组 6 334.7±8.4 336.0±7.2 375.0±4.7 415.8±9.6 392.8±8.7
②AD-Tcf12＋CLP组 6 335.8±5.3 335.8±5.1 352.7±5.0 379.5±5.2 361.0±4.5
③Veh＋CLP组 6 335.2±6.0 336.8±7.9 376.2±7.6 417.8±9.8 396.7±8.6
④Sham组 6 330.3±2.1 330.3±3.5 332.5±8.1 330.8±7.8 333.5±6.7
总体比较            
F值   1.08 1.40 60.30 144.40 98.97
P值   0.382 0.273 <0.001 <0.001 <0.001
多重比较的P值            
① vs ④   >0.999 0.764 <0.001 <0.001 <0.001
③ vs ④   >0.999 0.498 <0.001 <0.001 <0.001
③ vs ①   >0.999 >0.999 >0.999 >0.999 >0.999
② vs ③   >0.999 >0.999 <0.001 <0.001 <0.001
表3 4组30 d龄雄性SD大鼠不同时间点神经行为学评分(分，±s)
组别 鼠数(只) 3 h 6 h 12 h 24 h 48 h
①CLP组 6 9.17±0.75 8.00±1.10 6.33±0.82 4.17±0.75 5.00±0.63
②AD-Tcf12＋CLP组 6 9.17±0.75 8.83±1.17 7.67±1.21 6.83±0.75 7.50±1.05
③Veh＋CLP组 6 9.00±0.89 8.17±1.17 6.17±0.75 4.17±0.75 5.33±1.37
④Sham组 6 10.00±0.00 10.00±0.00 10.00±0.00 10.00±0.00 10.00±0.00
总体比较            
F值   2.53 5.03 27.88 108.60 38.07
P值   0.086 0.009 <0.001 <0.001 <0.001
多重比较的P值            
① vs ④   0.306 0.014 <0.001 <0.001 <0.001
③ vs ④   0.130 0.027 <0.001 <0.001 <0.001
③ vs ①   >0.999 >0.999 >0.999 >0.999 >0.999
② vs ③   >0.999 >0.999 0.029 <0.001 0.003
图1 Western blotting检测Sham组和CLP组SD大鼠不同时间点大脑皮质神经细胞中Tcf12的相对表达水平注：CLP为盲肠结扎穿孔术
表4 Sham组与CLP组SD大鼠不同时间点大脑皮质神经细胞中Tcf12的相对表达水平比较(±s)
组别 鼠数(只) Tcf12
CLP组    
①术后3 h 6 1.05±0.26
②术后6 h 6 1.01±0.19
③术后12 h 6 0.81±0.20
④术后24 h 6 0.77±0.16
⑤术后48 h 6 0.79±0.16
⑥Sham组 6 1.17±0.19
总体比较    
F值   4.37
P值   0.004
多重比较的P值    
① vs ⑥   >0.999
② vs ⑥   >0.999
③ vs ⑥   0.045
④ vs ⑥   0.019
⑤ vs ⑥   0.030
图2 Sham组及CLP组SD大鼠造模后24 h时大脑皮质神经细胞中Tcf12表达及定位[图2A～2D分别为CLP组DAPI(蓝色)、Tcf12(绿色)、NeuN(红色)、Merge；图2E～2H分别为Sham组DAPI(蓝色)、Tcf12(绿色)、NeuN(红色)、Merge](免疫荧光染色，高倍)注：CLP为盲肠结肠穿孔术
图3 Western blotting检测4组SD大鼠造模后24 h时大脑皮质神经细胞中cleaved caspase-3、Bax、Bcl2、p-P38和P38相对表达水平
表5 4组30 d龄SD大鼠造模后24 h时cleaved caspase-3、Bax、Bcl2、p-P38/P38相对表达水平比较(±s)
组别 鼠数(只) cleaved caspase-3 Bax Bcl2 p-P38/P38
①CLP组 6 1.47±0.05 1.67±0.04 0.48±0.04 1.09±0.04
②AD-Tcf12＋CLP组 6 0.71±0.05 0.88±0.04 0.81±0.04 0.53±0.04
③Veh＋CLP组 6 1.49±0.04 1.68±0.04 0.48±0.03 1.07±0.06
④Sham组 6 0.47±0.06 0.71±0.05 1.00±0.06 0.40±0.02
总体比较          
F值   642.68 883.33 228.77 437.75
P值   <0.001 <0.001 <0.001 <0.001
多重比较的P值          
① vs ④   <0.001 <0.001 <0.001 <0.001
③ vs ①   >0.999 >0.999 >0.999 >0.999
② vs ③   <0.001 <0.001 <0.001 <0.001
图4 4组30 d龄SD大鼠造模后24 h时大脑皮质病理改变(图4A：CLP组与Veh＋CLP组大鼠大脑皮质组织病理改变相似，可见细胞核固缩，细胞水肿，细胞数目减少，排列紊乱、空泡改变；图4B：AD-Tcf12＋CLP组大鼠大脑皮质组织病理改变较CLP组与Veh＋CLP组明显好转，细胞水肿有所改善，形态结构及排列较规整；图4C：Veh＋CLP组；图4D：Sham组大鼠大脑皮质细胞排列有序、结构规整)(HE染色，中倍)注：CLP为盲肠结肠穿孔术
[1]
Evans L, Rhodes A, Alhazzani W, et al. Surviving sepsis campaign: international guidelines for management of sepsis and septic shock 2021 [J]. Crit Care Med, 2021, 49(11): e1063-e1143. DOI: 10.1097/CCM.0000000000005337.
[2]
Fleischmann-Struzek C, Mellhammar L, Rose N, et al. Incidence and mortality of hospital- and ICU-treated sepsis: results from an updated and expanded systematic review and Meta-analysis [J]. Intensive Care Med, 2020, 46(8)：1552-1562. DOI: 10.1007/s00134-020-06151-x.
[3]
Mei B, Li J, Zuo Z. Dexmedetomidine attenuates sepsis-associated inflammation and encephalopathy via central α2A adrenoceptor [J]. Brain Behav Immun, 2021, 91: 296-314. DOI: 10.1016/j.bbi.2020.10.008.
[4]
Ren C, Yao RQ, Zhang H, et al. Sepsis-associated encephalopathy: a vicious cycle of immunosuppression [J]. J Neuroinflammation, 2020, 17(1): 14. DOI: 10.1186/s12974-020-1701-3.
[5]
Liu D, Huang SY, Sun JH, et al. Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options [J]. Mil Med Res, 2022, 9(1): 56. DOI: 10.1186/s40779-022-00422-y.
[6]
Yang J, Zhang LI, Jiang ZY, et al. TCF12 promotes the tumorigenesis and metastasis of hepatocellular carcinoma via upregulation of CXCR4 expression [J]. Theranostics, 2019, 9(20): 5810-5827. DOI: 10.7150/thno.34973.
[7]
Gao S, Bian T, Zhang Y, et al. TCF12 overexpression as apoor prognostic factor in ovarian cancer [J]. Pathol Res Pract, 2019, 215(9): 152527. DOI: 10.1016/j.prp.2019.152527.
[8]
Zhang Y, Yu RZ, Li QS, et al. SNHG1/miR-556-5p/TCF12 feedback loop enhances the tumorigenesis of meningioma through Wnt signaling pathway [J]. J Cell Biochem, 2020, 121(2): 1880-1889. DOI: 10.1002/jcb.29423.
[9]
Gu J, Ge X, You A, et al. miR-218-5p inhibits the malignant progression of glioma via targeting TCF12 [J]. Tumori, 2022, 108(4): 338-346. DOI: 10.1177/03008916211018263.
[10]
Rittirsch D, Huber-Lang MS, Flierl MA, et al. Immunodesign of experimental sepsis by cecal ligation and puncture[J]. Nat Protoc, 2009, 4(1): 31-36. DOI: 10.1038/nprot.2008.214.
[11]
Tang F, Chen L, Gao H, et al. Munc18-1 contributes to hippocampal injury in septic rats through regulation of syntanxin1A and synaptophysin and glutamate levels. Neurochem Res, 2023, 48(3): 791-803. DOI: 10.1007/s11064-022-03806-7.
[12]
Ilker MK, Murat U, Sinan B，et al. Sepsis induces apoptotic cell death in different regions of the brain in a rat model of sepsis [J]. Acta Neurobiol Exp (Wars). 2010；70(3)：246-60. DOI: 10.55782/ane-2010-1796.
[13]
Salomão R, Ferreira BL, Salomo MC, et al. Sepsis: evolving concepts and challenges [J]. Braz J Med Biol Res, 2019, 52(4): e8595. DOI: 10.1590/1414-431X20198595.
[14]
Mazeraud A, Righy C, Bouchereau E, et al. Septic-associated encephalopathy: a comprehensive review [J]. Neurotherapeutics, 2020, 17(2): 392-403. DOI: 10.1007/s13311-020-00862-1.
[15]
Gu M, Mei XL, Zhao YN. Sepsis and cerebral dysfunction: BBB damage, neuroinflammation, oxidative stress, apoptosis and autophagy as key mediators and the potential therapeutic approaches [J]. Neurotox Res, 2021, 39(2): 489-503. DOI: 10.1007/s12640-020-00270-5.
[16]
Gao J, Wang Y, Ma S, et al. Secukinumab alleviates cognitive impairment by attenuating oxidative stress and neuronal apoptosis via the IL-17RA/AKT/ERK1/2 pathway in a rat model of sepsis [J]. Exp Neurol, 2023, 359: 114263. DOI: 10.1016/j.expneurol.2022.114263.
[17]
Catarina AV, Branchini G, Bettoni L, et al. Sepsis-associated encephalopathy: from pathophysiology to progress in experimental studies [J]. Mol Neurobiol, 2021, 58(6): 2770-2779. DOI: 10.1007/s12035-021-02303-2.
[18]
Gao S, Bian T, Su M, et al. miR-26a inhibits ovarian cancer cell proliferation, migration and invasion by targeting TCF12 [J]. Oncol Rep, 2020, 43(1): 368-374. DOI: 10.3892/or.2019.7417.
[19]
Cui T, Liu P, Chen X, et al. Identification and functional characterization of caspases in turbot (scophthalmus maximus) in response to bacterial infection [J]. Fish Shellfish Immunol, 2023, 137: 108757. DOI: 10.1016/j.fsi.2023.108757.
[20]
刘硕儒，王功炜，张斌，等. 新型溶瘤病毒M1激活内质网应激致前列腺癌细胞凋亡的机制[J/OL]. 中华腔镜泌尿外科杂志(电子版)，2023，17(4)：388-393. DOI：10.3877/cma.j.issn.1674-3253.2023.04.015.
[21]
Zhang Q, Lei X, Wang F, et al. ERK1-mediated immunomodulation of mesenchymal stem cells ameliorates inflammatory disorders [J]. iScience, 2023, 26(10): 107868. DOI: 10.1016/j.isci.2023.107868.
[22]
Moon HR, Yun JM. Neuroprotective effects of hesperetin on H2O2-induced damage in neuroblastoma SH-SY5Y cells [J]. Nutr Res Pract, 2023, 17(5): 899-916. DOI: 10.4162/nrp.2023.17.5.899.
[23]
Kyriakou S, Potamiti L, Demosthenous N, et al. A naturally derived watercress flower-based phenethyl isothiocyanate-enriched extract induces the activation of intrinsic apoptosis via subcellular ultrastructural and Ca2＋ efflux alterations in an in vitro model of human malignant melanoma [J]. Nutrients, 2023, 15(18): 4044. DOI: 10.3390/nu15184044.
[24]
Li L, Han Q, Chen Y, et al. β-nicotinamide mononucleotide rescues the quality of aged oocyte and improves subsequent embryo development in pigs [J]. PLoS One, 2023, 18(10): e0291640. DOI: 10.1371/journal.pone.0291640.
[25]
Liao YJ, Lee CY, Twu YC, et al. Isolation and biological evaluation of alfa-mangostin as potential therapeutic agents against liver fibrosis [J]. Bioengineering (Basel), 2023, 10(9): 1075. DOI: 10.3390/bioengineering10091075.
[26]
Zhou RX, Ying J, Qiu X, et al. A new cell death program regulated by toll-like receptor 9 through p38 mitogen-activated protein kinase signaling pathway in a neonatal rat model with sepsis associated encephalopathy [J]. Chin Med J (Engl), 2022, 135(12): 1474-1485. DOI: 10.1097/CM9.0000000000002010.
[27]
Zhou RX, Yang X, Li XH, et al. Recombinant CC16 inhibits NLRP3/caspase-1-induced pyroptosis through p38 MAPK and ERK signaling pathways in the brain of a neonatal rat model with sepsis [J]. J Neuroinflammation, 2019, 16(1): 239. DOI: 10.1186/s12974-019-1651-9.
[28]
Zhou RX, Li YY, Qu Y, et al. Regulation of hippocampal neuronal apoptosis and autophagy in mice with sepsis-associated encephalopathy by immunity-related GTPase M1 [J]. CNS Neurosci Ther, 2020, 26(2): 177-188. DOI: 10.1111/cns.13229.
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