Cerebral ischemia-reperfusion aggravated cerebral infarction injury and possible differential genes identified by RNA-Seq in rats

doi:10.1016/j.brainresbull.2019.12.014

Brain Research Bulletin

Volume 156, March 2020, Pages 33-42

https://doi.org/10.1016/j.brainresbull.2019.12.014 Get rights and content

Highlights

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Cerebral I/R aggravates neurological function injury and neuroinflammation.
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TLR4/MyD88 inflammatory signaling pathway plays an important role after cerebral I/R.
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HRAS/RAF1 neurotrophic signaling pathway plays an important roles after cerebral I/R.

Abstract

Numerous studies have shown that local excessive inflammatory response in brain tissue was an important pathogenesis of secondary injury following cerebral ischemia-reperfusion (I/R). However, the inflammatory-related targets and pathways after cerebral I/R injury are still unclear. This study was to investigate possible targets and mechanisms after cerebral I/R injury. Rats were subjected to transient or permanent middle cerebral artery occlusion (MCAO). Neurological deficit scores test was used to evaluate neurological function. Cerebral infarction was evaluated by MRI, TTC staining and Nissl staining. Microglia activation was detected by immunofluorescence using Iba-1 antibody. Inflammatory factors were detected by ELISA assay. RNA-sequencing transcriptome analysis was processed and the differential genes were verified by real-time quantitative PCR (qPCR) and western blotting. The results showed that neurological function of rats in I/R group was more severe than that in I group on the 7^th after cerebral I/R. Therefore, the differences between cerebral ischemia and cerebral I/R for 7 days were studied in further study. The results showed that the levels of pro-inflammatory factors in I/R group were higher and the levels of anti-inflammatory factors were lower than those in I group. KEGG pathway and gene network enrichment analysis revealed that some common differential up- and down-regulated genes were involved in most of significant pathways. These common differential up-regulated genes belonged to TLR4/MYD88 inflammatory signaling pathway and common differential down-regulated genes belonged to HRAS/RAF1 neurotrophic signaling pathway. Interestingly, according to the genetic interaction analysis of string database, these up-regulated differential genes might promote the development of inflammation, while the down-regulated differential genes might inhibit the development of inflammation. Furthermore, qPCR and WB results verified that these pro-inflammatory genes in the I/R group were higher than those in the I group, while possible anti-inflammatory genes in the I/R group were lower than those in the I group. It is concluded that TLR4/MYD88 inflammatory signaling pathway and HRAS/RAF1 neurotrophic signaling pathway may play different roles after cerebral I or I/R and may be therapeutic targets for stroke recovery.

Introduction

Cerebral ischemia-reperfusion (I/R) injury refers to the recanalization of blood flow after cerebral ischemia, leading to further tissue damage and dysfunction, which is a complex pathophysiological process involving multiple factors (Feigin et al., 2009; Patel et al., 2013; Huang et al., 2006). Cerebral I/R can cause a wide range of microvascular dysfunction and changes in tissue barrier function. The inflammatory response caused by cerebral I/R injury can cause systemic inflammatory response or multiple organ dysfunction syndrome, accounting for 30–40 % of severe mortality (Perego et al., 2011; Eltzschig and Eckle, 2011; Zhang et al., 2018a, b). Therefore, it is urgent to prevent and treat cerebral I/R by exploring the mechanisms of cerebral I/R injury.

Numerous studies have shown that inflammatory immune response is widely involved in cerebral I/R injury, and local excessive inflammatory response in brain tissue is an important pathogenesis of secondary injury following cerebral I/R (Gelderblom et al., 2009; Zhou et al., 2013). Neuroinflammation is like a double-edged sword, which on the one hand aggravates neuronal damage and on the other hand promotes tissue repair and reincarnation (Patel et al., 2013). Therefore, exploring the mechanism of neuroinflammation and finding new targets will be of great significance in the prevention and treatment of cerebral I/R injury.

Neuroinflammation caused by activated microglia and astrocytes is a key determinant in the short-term and long-term prognosis after cerebral I/R (Kyritsis et al., 2012; Macrez et al., 2011; Tobin et al., 2014). Microglia, as a resident macrophage in the brain, directly participates in neuroinflammation after polarization activation (Peruzzotti-Jametti et al., 2014; Nowicka et al., 2008). There are two major types of macrophage/microglia phenotypes: classical activation (Ml) and alternative activation (M2). Although M1/M2 does not fully reflect the activation of macrophage/microglia diversity, it represents two extreme phenotypes of microglia activation and its mediated inflammatory response (Morioka et al., 1993; Kettenmann et al., 2011). Therefore, it has important value to regulate M1/M2 microglial activation to minimize the detrimental effect and/or maximize the protective role. In this study, RNA-sequencing was used to explore the key inflammatory targets which affecting reperfusion injury by comparing the reperfusion and non-reperfusion after cerebral ischemia in rats.

Section snippets

Animals

Male Sprague-Dawley rats (240−280 g) were purchased from Beijing Sibefu Biotechnology Co., Ltd., license number: SCXK (Beijing 2012-0001). All animals were housed in a room with controlled temperature of 23 ± 2 °C and humidity of 55 ± 5 % with a regular 12 h light-dark cycle and allowed to have water and food ad libitum. After acclimatization for 3 days, rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 min or permanent MCAO. The MCAO rats were inserted a suture

Differences of survival rate and neurobehavioral function between cerebral I/R and I in rats

As shown in Fig. 2A and B, there was no animal death in the sham group, but the mortality rate of the I/R group (23.3 %) was higher than that of the I group (13.3 %). These indicated that cerebral I/R had severe secondary damage. In addition, the body weight of normal rats increased steadily with time, and the body weight of rats in cerebral I/R group was slightly lower than that in cerebral I group (Fig. 2C).

The neurological behavior of the rats in the I/R group and the I group was both

Discussion

The pathophysiological process of cerebral I/R injury was complex and highly interactive (Lansberg et al., 2012; Gauberti et al., 2013, 2014). Inflammatory response played a key role in cerebral I/R injury (Smith et al., 2012; Li et al., 2017; Nagy and Nardai, 2017). The inflammatory response mediated microvascular dysfunction was an important reason why cerebral ischemia reperfusion caused secondary brain damage (Gulyas et al., 2012; Fernandez-Lizarbe et al., 2009).

The inflammatory response

Author statement

Xiao Cheng, Ying-Lin Yang, Wei-Han Li and Man Liu performed the animal experiments; Xiao Cheng and Yue-Hua Wang analyzed the data and wrote the manuscript; Yue-Hua Wang and Guan-Hua Du designed the experiments; and all authors read and approved the final manuscript.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by the National Key Research & Development Plan (2018YFC0311005); the National Natural Science Foundation of China (81473383), the Drug Innovation Major Projects (2018ZX09711001-003-019), the Medical and Health Innovation Project of Chinese Academy of Medical Sciences (2016-I2M-3-007), and Innovation Fund for Graduate of Beijing Union Medical College (2017-1007-02 & 2018-1007-04).

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Cerebral ischemia-reperfusion (I/R) injury is a pathophysiological disease involving multiple factors. It is after cerebral ischemia, the subsequent perfusion restores blood flow and recanalization, which further cause severe brain tissue damage and dysfunction (Cheng et al., 2020; Ding et al., 2020). Cerebral injury mechanisms remain unclear and complex, but existing studies believe that they involve the production of oxidative free radicals, neuroinflammation, Ca2+ overload, cell apoptosis, etc.(Guan and Chen, 2021; He et al., 2020; Shi et al., 2021).
Buyang Huanwu Decoction (BYHW) is a classic representative formula for treating qi deficiency and the blood stasis syndrome of stroke in the Qing Dynasty physician Wang Qingren's Correction on the Errors of Medical Works. However, the research on the mechanism of BYHW in the treatment of stroke is not systematic and comprehensive.
Combined with multi-omics analysis methods to explore the potential targets of BYHW in the treatment of cerebral ischemia-reperfusion (I/R).
The rat middle cerebral artery occlusion (MCAO) model was established to study the effect of BYHW on cerebral I/R injury in rats. Then, the potential targets and pathways of BYHW in the treatment of cerebral I/R injury were analyzed by proteomic, transcriptomic, and metabolomic methods. Finally, 4D-PRM was used to validate potential targets.
BYHW effectively improved the neurological function scores of MCAO rats and significantly reduced the rate of cerebral infarction in MCAO rats. Multi-omics analysis had identified 15 potential targets and 4 potential signaling pathways. The results of 4D-PRM targeted proteomics verification showed that Pde1b was reversed up-regulated, and Aprt, Gpd1, Glb1, HEXA, and HEXB were reversed down-regulated.
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Ovatodiolide (OVA), a bioactive substance extracted from the bioactive component of Anisomeles indica, is reported to be endowed with anti-inflammatory properties. Nonetheless, its function in ischemia-reperfusion (I/R)-induced neurological deficits and microglial inflammation remains unclear.
A middle cerebral artery occlusion (MCAO) model was set up in SD rats, which were then dealt with varying doses of OVA. The rats’ neurological functions were estimated at diverse periods postoperatively. The dry and wet method, triphenyl tetrazolium chloride (TTC) staining, and Nissl’s staining were conducted to measure brain edema, cerebral infarction area and neuronal damage, respectively. Immunohistochemistry (IHC) was performed to detect neuronal apoptosis and microglial activation, and the profiles of inflammatory factors in the cerebral tissues were estimated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In-vitro assays were implemented on HT22 neuronal cells and BV2 microglia to elaborate the effect of OVA against oxygen-glucose deprivation (OGD)-mediated effects.
OVA relieved HT22 cell apoptosis and eased inflammation in BV2 microglia, which were induced by OGD. OVA mitigated NF-κB phosphorylation in BV2 cells, whereas boosted SIRT1 expression. However, inhibiting SIRT1 abolished the anti-inflammatory effects of OVA in BV2 microglia under OGD stimulation. The condition medium (CM) of OGD-treated BV2 cells enhanced HT22 cell apoptosis and damage. OVA treatment in BV2 cells relieved BV2-mediated injury on HT22 cells, which was reversed by SIRT1 inhibitor. In-vivo results revealed that OVA dose-dependently attenuated I/R rats’ neurological deficits, reduced brain edema, cerebral infarction area, neuronal apoptosis and microglial overactivation. Additionally, OVA inactivated the NF-κB pathway and up-regulated SIRT1 in the I/R rat model.
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The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Cerebral ischemia-reperfusion aggravated cerebral infarction injury and possible differential genes identified by RNA-Seq in rats

Highlights

Abstract

Introduction

Section snippets

Animals

Differences of survival rate and neurobehavioral function between cerebral I/R and I in rats

Discussion

Author statement

Declaration of Competing Interest

Acknowledgements

Lancet Neurol.

J. Neurol. Sci.

Surg. Neurol.

Lancet Neurol.

Brain Res. Bull.

Neuroscience

Lancet Neurol.

Brain Res. Bull.

Neuroscience

Brain Res. Bull.

Neurosci. Lett.

Chin. J. Nat. Med.

Chin. J. Nat. Med.

Acute anti-inflammatory approaches to ischemic stroke

Ann. N. Y. Acad. Sci.

Ischemia and reperfusion-from mechanism to translation

Nat. Med.

Critical role of TLR4 response in the activation of microglia induced by ethanol

J. Immunol.

Effects of CD11b/18 monoclonal antibody on rats with permanent middle cerebral artery occlusion

Am. J. Pathol.

Ultra-sensitive molecular MRI of vascular cell adhesion molecule-1 reveals a dynamic inflammatory penumbra after strokes

Stroke

Molecular magnetic resonance imaging of brainimmune interactions

Front. Cell. Neurosci.

Temporal and spatial dynamics of cerebral immune cell accumulation in stroke

Stroke