Remote ischaemic perconditioning reduces the infarct volume and improves the neurological function of acute ischaemic stroke partially through the miR-153-5p/TLR4/p65/IkBa signalling pathway

Introduction

Cerebrovascular disease, which is characterized by high disability, morbidity, and mortality, is currently one of the focuses of the prevention and treatment of diseases of the elderly [14]. Stroke is currently the most important cause of disability and death in China, among which ischaemic stroke accounts for about 87% of all strokes [9]. The only effective treatment for ischaemic stroke is recombinant tissue plasminogen activator (rt-PA) thrombolytic therapy. However, it is restricted by its strict time window, indications, and contraindications. Currently, less than 1% of patients in China qualify for rt-PA thrombolytic therapy [3,17]. Therefore, it is urgent to actively explore new, effective, and safe treatment methods to improve the prognosis of ischaemic stroke.
The American Stroke Association (ASA) has published research showing that before the occurrence of cerebral infarction, patients with repeated “small strokes” (i.e. transient ischemic attacks [TIA]) have lower infarct volume and better prognosis than patients without “small strokes” [20]. This may indicate that repeated and short-term ischaemia and hypoxia of brain tissue can enhance the ability of brain tissue to resist ischaemia. Remote ischaemic conditioning (RIC) is a new method of ischaemic adaptation, which protects target organs from continuous ischaemic damage through transient ischaemia-reperfusion of remote organs [10,26]. According to the implementation time of RIC, it can be divided into remote ischaemic preconditioning (RIPC), remote ischaemic perconditioning (RIPerC), and remote ischaemic postconditioning (RIPostC). RIPerC refers to the implementation of RIC after cerebral ischaemia and before reperfusion. It is worth noting that there have been many studies on the application of RIPerC to ischaemic animal models, and it was initially found that RIPerC can significantly reduce the infarct volume of the ischaemic animal model organs [5,27]. However, the mechanism by which RIPerC interferes with ischaemic stroke to reduce cerebral infarction volume and improve neurological function has not yet been reported.
MicroRNA (miRNA) is an endogenous gene that encodes a non-coding single-stranded RNA molecule with a length of approximately 20 nucleotides [6]. After being combined with the target mRNA, it can effectively inhibit the translation of target mRNA and induce the degradation process. With the development of whole-genome sequencing technology, miRNAs are receiving increasing attention. Studies have shown that miRNAs are involved in various biological functions, such as brain neurodevelopment [2], nerve cell apoptosis [16], and changes in synaptic plasticity [22]. Interestingly, when the brain is in a hypoxic and ischaemic environment, neurological impairment and neurodegenerative diseases will also have a close relationship with miRNA [4]. Several studies have reported that miR-153-5p has significant anti-tumour effects [7,23]. However, there is no report about the expression characteristic and potential value of miR-153-5p after ischaemic stroke.
In this study, a middle cerebral artery occlusion (MCAO) mouse model and oxygen-glucose deprivation (OGD) cell model were constructed in vivo and in vitro. In clinical terms, we further explored the relationship between the plasma miR-153-5p level within 6 hours of the onset of acute ischaemic stroke and the clinical characteristics and prognosis. The purpose is to clarify the mechanism of RIPerC in reducing the infarct volume and neurological damage of acute ischaemic stroke.

Material and methods

Collection of plasma samples from acute ischaemic stroke patients and healthy controls, and 3-month follow-up of patients

The study group comprised 68 patients with acute ischaemic stroke treated in our hospital from October 2018 to August 2019, including 42 males and 26 females. The National Institutes of Health Stroke Scale (NIHSS) score was assessed on admission. We collected plasma from patients with acute ischaemic stroke within 6 hours of onset and stored it at –80° for future use. Demographic and clinical information, including gender, age, hypertension, hypercholesterolaemia, diabetes mellitus, body mass index (BMI), alcoholism, and smoking were also collected. The cerebral infarct volume of patients was calculated by MRI (Vision Plus1.5T, Siemens, Germany). According to the time when the patients were included in the study, they were followed up for 3 months. During the same period, 68 healthy controls who underwent physical examinations from the Health Examination Centre were collected. Plasma was acquired from the subject with written informed consent. Our research plans were approved by the Human Ethics Committee of our hospital, Kunming, China (no. 20180811H), according to the Declaration of Helsinki (as revised in 2013).

Preparation of MCAO model and RIPerC

We purchased 50 C57BL/6J mice (male, 25 ±2 g) from the Experimental Animal Centre of Tianjin Medical University [SCXK (E) 2019-0115]. Experiments were performed under a project license (No. 20190408-3) granted by the Experimental Animal Centre of our hospital. Humane care was given during the experimental animal breeding and experimental procedures following the 3R principle of experimental animals. The mice were housed at 22°C, with humidity of 50%, and provided with an adequate diet. According to body weight, C57BL/6J mice were randomly divided into 5 groups (10 mice/group): Sham group, MCAO 3.0 h group, MCAO 4.5 h group, MCAO 3.0 h + RIPerC group, and MCAO 4.5 h + RIPerC group.
The MCAO was built based on a previous study [24]. The mice were anaesthetized with sodium pentobarbital at a dose of 30 mg/kg. A 6-0 surgical thread was inserted from the left external carotid artery of the mouse and extended into the internal carotid artery to block the middle cerebral artery and induce embolism. The blood flow of arterial embolism was detected by laser Doppler blood flow meter (Moor Instruments, UK). Ischaemia was defined when the blood flow dropped to 80% (compared to the baseline). Clinically, the best and latest time points for intravenous thrombolysis for patients with acute ischaemic stroke are within 3.0 h and 4.5 h of onset, respectively. Therefore, we chose the 2 time points of 3.0 h and 4.5 h.
RIPerC treatment of MCAO mice: After the successful establishment of the MCAO model, the mice were subjected to cerebral ischaemia for 2.0 h and 3.5 h, respectively, without removing the threaded plug. Then, a non-invasive tourniquet was used to tighten the right hind limb of the mouse for the first time for 10 min and loosen it for 10 min. For the second time, the right hind limb of the mouse was tightened for 10 min and loosened for 10 min. For the third time, the right hind limb of the mouse was tightened for 10 min and loosened for 10 min. After the above 3 cycles, the threaded plug was removed. Then, the blood supply was restored and blood flow after reperfusion was maintained at higher than 70%. Finally, the neurological function (Menzies score, Belayev score, and Garcia score) of each group of mice was scored. All the mice were sacrificed 24 hours after reperfusion.

Cell and model establishment

Mouse Neuro-2A neuroblastoma (Neuro-2a) cells were established by R. J. Klebe and F. H. Ruddle with spontaneous tumours of strain A mice, and most of them were neuron-like with axon-like structures. Therefore, we purchased Neuro-2a cells (CL-0168) from Wuhan Procell for in vitro research. Neuro-2a cells were inoculated in MEM complete medium (PM170410B, Procell, China) mixed with foetal bovine serum. When the serum-free medium was needed in subsequent experiments, the medium was replaced with a pure MEM medium (PM170409B, Procell, China). The conventional cell culture was uniformly completed in a Heracell™ VIOS 250i CO2 Incubator (Thermo Scientific™) at 37°C with 5% CO2.
An OGD cell model was constructed as described before [19]. Neuro-2a cells were adjusted to 1 × 105cells/ml and then uniformly inoculated in a pure MEM medium. Neuro-2a cells in the control group were inoculated in MEM complete medium. The cells in the OGD group were transferred to a 37°C Heal Force 3-gas incubator (HF100) containing 1% O2, 5% CO2, and 94% N2. The subsequent OGD treatment was conducted with the continuous filling of an anoxic mixture for 6 hours. During this period, the cells in the control group were cultured routinely.

Cell transfection

miR-153-5p mimic, small interfering RNA (siRNA)-miR-153-5p, and Toll-like receptor 4 (TLR4) overexpression plasmid, as well as their negative controls (NC), were synthesized by Shanghai HANBIO Company. The sequence of siRNAs was provided in Table I. After completing the plasmid preparation, we used the Lipofectamine 3000 liposome transfection reagent (L3000009, ThermoFisher) to transfect the overexpression plasmid into the Neuro-2a cells. The transfection efficiency was assessed 48 hours after transfection by quantitative real-time polymerase chain reaction (qRT-PCR).

QRT-PCR

Total RNAs in Neuro-2a cells, mouse brain tissues, and stroke patients’ plasma were separated by TRIzol (15576425, Invitrogen, USA), and then cDNA was further synthesized by PrimeScriptTM RT reagent Kit (RR039A, TaKaRa, Japan). Pre-synthesized gene primers (Sangon, China), Roche SYBR Green Master (05629017212), and DEPC water were added to the cDNA and mixed and tested in the detection instrument (thermal cycler T100, Bio-Rad, USA), according to the following settings: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, and annealing at 58°C for 1 min, for a total of 40 cycles. For calculation of RNA levels, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as internal references. The gene RNA level was determined by the qRT-PCR detection and calculated by 2-DDCT. The amplification efficiencies of miR-153-5p, TLR4, HGF, GDAP1, TBX22, HNMT, FREM2, U6, and GAPDH were 98.7%, 101.5%, 99.4%, 96.5%, 104.7%, 99.6%, 97.2%, 103.3%, and 98.9%, respectively. The test was repeated 3 times for each sample. The detailed primer sequences are listed in Table I.

Apoptosis test

Neuro-2a cells were trypsinized and washed with phosphate-buffered saline (PBS). 1 × 105 Neuro-2a cells were resuspended in 100 µl of 1 × Binding Buffer (from Annexin V-FITC/PI Apoptosis Detection Kit; 39211ES60, YEASEN, China), then stained by Annexin V-FITC (5 µl) and PI Staining Solution (5 µl) in dark condition. Next, 400 µl of 1 × Binding Buffer was added to the Neuro-2a cell mixture, and the apoptosis changes were analysed by BD FACSVerse flow cytometer (USA).

Western blot

The RIPA (P0012K, Beyotime, China) and the BCA detection kit (P0010S, Beyotime, China) were applied in the extraction and quantification of proteins from Neuro-2a cells or brain tissues. The proteins were transferred to the NC membrane (N8633, Millipore, USA) with a pore size of 0.22 µm by the SDS-PAGE method. After treating the NC membrane with the blocking solution at room temperature for 2 hours, corresponding primary antibodies were used to fully cover the protein-laden NC membrane (at 4°C). After 24 hours, the primary antibody was washed off and replaced with the corresponding secondary antibody for further incubation. The NC membrane was developed with Beyotime ECL Luminescent Solution (P0018S) after 1 h. The fluorescent signal generated by the protein on the membrane was collected by the BIO-RAD Gel Doc™ XR+ instrument and processed into a corresponding greyscale band image. GAPDH was an internal reference. The primary and secondary antibodies used in the experiment were purchased from Abcam and CST in the United States as follows: Bcl-2 (1 : 2000, ab196495, 26 kDa, Abcam), Bax (1 : 2000, ab53154, 21 kDa, Abcam), cleaved Caspase-3 (1 : 5000, ab52072, 17 kDa, Abcam), phosphorylated-p65 (p-p65; 1 : 1000, ab76311, 60 kDa, Abcam), p65 (1 : 1000, ab19870, 65 kDa, Abcam), TLR4 (1 : 1000, ab22048, 96 kDa, Abcam), GAPDH (1 : 10000, ab8245, 36 kDa, Abcam), Rabbit Anti-Mouse antibody (1 : 5000, ab46540, Abcam), Goat Anti-Rabbit antibody (1 : 5000, ab97051, Abcam), p-inhibitor a of NF-kB (p-IkBa; 1 : 1000, #9246, 40 kDa, CST), and IkBa (1 : 1000, #4814, 39 kDa, CST).

Dual-luciferase reporter experiment

Based on the binding sequence provided by the starBase database, we constructed TLR4-wt and TLR4-mut reporter plasmid by pmirGLO vector (E1330, Promega, USA). The reporter plasmid and miR-153-5p mimic or mimic control were co-transfected into Neuro-2a cells (48 h). Neuro-2a cells treated with Dual-Luciferase® Reporter Assay kit (E1910, Promega, USA) were then transferred to the GloMax 20/20 Luminometer (Promega) to analyse luciferase activity.

RNA immunoprecipitation (RIP)

We collected Neuro-2a cells with transfection (TLR4 + miR-153-5p mimic or TLR4 + mimic control), and the Neuro-2a cells were lysed with Cell lysis buffer in the RIP kit (KT100-01, GZSCBio, China). Cells in the RNase-free EP tubes were labelled as the IgG + mimic-NC group, anti-Argonaute-2 (Ago2) + mimic-NC group, IgG + miR-153-5p group, Ago2 + miR-153-5p group, Input-mimic-NC group, and Input-miR-153-5p group, respectively. The antibodies used were the Ago2 antibody (1 : 50, ab156870, Abcam, USA) and the NC antibody IgG provided in the kit. In RNA binding protein immunoprecipitation, the magnetic bead-antibody mixture was mixed with cell lysate for incubation overnight (at 4°C). The Input-mimic-NC group and the Input-miR-153-5p group were not given the magnetic bead-antibody mixture. On the second day, the purified RNA was analysed for the expression of TLR4 by qRT-PCR, and the enrichment level was calculated.

MTT assay

According to the recommended inoculation requirements, the cell density of Neuro-2a was adjusted to 1 × 104 cells/ml and 100 µl of it was transferred to a sterilized 96-well plate (6 wells per treatment group). After 48 hours of routine culture, 10 µl of APE × BIO MTT solution (B7567) was added to Neuro-2a cells in the control group and other treatment groups. After 4 hours, the Neuro-2a cells were detected by the Molecular Devices microplate reader (SpectraMax iD5) for absorbance (OD) at 490 nm.

Triphenyl tetrazolium chloride staining

The brain tissues of the sacrificed mice were quickly removed and transferred to an environment of –20°C for freezing for 30 min. The frozen brain tissues were incised according to the coronal position and made into brain slices with a thickness of 2 mm. The brain tissues were then reacted with triphenyl tetrazolium chloride (TTC) Stain Kit (D025-1-1, Nanjing Jiancheng Bioengineering Institute, China) for 30 min. The brain tissues were fully stained in a water bath (37°C). Image information was collected immediately after the removed brain tissues were washed with PBS. The white part of the result was the infarcted tissues. The proportion of the infarct area in the total area was measured and calculated.

Immunohistochemical analysis

Immunohistochemical analysis was constructed as described before [21]. Primary antibody TLR4 (1 : 1000, ab22048, 96 kDa, Abcam) was added to the brain tissues, and the biotinylated Goat Anti-Rabbit secondary antibody (1 : 5000, ab97051, Abcam) was incubated at 4°C. Finally, the sections were incubated with DAB substrate for 5 min.

Statistical analysis

All experiments were performed in triplicate unless specified. Experiments were performed at least 3 times. The results are represented as the mean ± standard error of the mean (SEM) or median (interquartile range – IQR). The differences between normally distributed numeric variables were evaluated by Student’s t-test, whereas non-normally distributed variables were analysed by Mann-Whitney U-test. One-way ANOVA was used for the comparison among multiple groups if the variance was homogeneous, while non-normally distributed variables were evaluated by Kruskal-Wallis variance analysis. Multiple comparisons between the groups were performed using the S-N-K method. Correlations were analysed using the Spearmen method. The survival curve was analysed by the Kaplan-Meier method. P < 0.05 was considered significant.

Results

RIPerC treatment reduces cerebral infarction volume and neurological damage and promotes the expression of miR-153-5p in the MCAO animal models

In this study, 5 groups (10 mice/group) of mice were analysed, namely the Sham group, MCAO 3.0 h group, MCAO 4.5 h group, MCAO 3.0 h + RIPerC group, and MCAO 4.5 h + RIPerC group. As shown in Figure 1A, B, and Table II, compared with MCAO mice not treated with RIPerC, the proportion of infarcts in the entire brain tissues and neurological damage (Menzies score, Belayev score, and Garcia score) of MCAO mice treated with RIPerC were significantly reduced (p < 0.05). Moreover, compared with MCAO mice not treated with RIPerC, the relative miR-153-5p expression level in the brain of MCAO mice treated with RIPerC was significantly increased (Fig. 1C, p < 0.05).
Up-regulated miR-153-5p enhanced the viability of OGD cells and reduced apoptosis by regulating apoptosis-related proteins, while si-miR-153-5p had the opposite effect

The cell transfection experiment fully confirmed that the transfection study was successfully constructed. The miR-153-5p expression level was up-regulated in the miR-153-5p mimic group but down-regulated in the si-miR-153-5p group (Fig. 2A, p < 0.01). In vivo, as expected, the expression of miR-153-5p was suppressed by the OGD treatment (Fig. 2B, p < 0.01). In the following loss or gain cell function experiments, OGD treatment produced greater effects on inhibiting cell viability and increasing apoptosis of Neuro-2a cells (Fig. 2C, D, p < 0.05). More importantly, OGD treatment up-regulated Bax and cleaved caspase 3 proteins but prevented the activation of Bcl-2 (Fig. 2E, p < 0.05). Exogenous up-regulation or silencing of miR-153-5p also produced reversal or enhanced effects: miR-153-5p overexpression reduced cell damage and protein regulation caused by OGD treatment, whereas silenced miR-153-5p further increased OGD’s damage effects on Neuro 2a cells (Fig. 2C-E, p < 0.05).

TLR4 was a downstream target gene of miR-153-5p

In this study, HGF, TLR4, GDAP1, TBX22, HNMT, and FREM2 with MCAO differences were screened (Fig. 3A). We further determined the expression changes of the above 6 target genes under miR-153-5p mimic intervention. The results showed that TLR4 was the most inhibited (Fig. 3B, p < 0.01). Therefore, TLR4 was determined as the downstream target gene of miR-153-5p for further analysis. We first verified the association between TLR4 and miR-153-5p based on the binding sequence predicted by the miRDB database (Fig. 3C). As shown in Fig. 3D, the co-transfection of TLR4-wt with miR-153-5p mimic significantly inhibited the luciferase activity of the cells (p < 0.01). Further RIP testing also confirmed the binding between miR-153-5p and TLR4 (Fig. 3E, p < 0.01).
Overexpressed TLR4 neutralized the anti-apoptosis and gene regulation effects of miR-153-5p mimic in OGD cells

The transfection of overexpressed TLR4 into Neuro-2a cells significantly upregulated the mRNA level of TLR4 (Fig. 4A, p < 0.05). In the following Western blot detection, miR-153-5p mimic further inhibited the TLR4 expression overexpressed by OGD, while overexpressed TLR4 neutralized the above regulatory effect of miR-153-5p mimic (Fig. 4B, p < 0.05). Overexpressed TLR4 also showed the same neutralization effect in the subsequent apoptosis test (Fig. 4C, p < 0.05). We also analysed the regulation of TLR4 on the p65/IkBa pathway. The results showed that TLR4 overexpression promoted the activation of OGD on the p65/IkBa pathway (and the ratio of p-p65/p65 and p-IkBa/IkBa) (Fig. 4D, p < 0.05). More importantly, overexpression of TLR4 neutralized the OGD attenuation effect of the miR-153-5p mimic (Fig. 4D, p < 0.05).

The MCAO animal model confirmed the results of the cell experiment

Furthermore, we validated the results of the cell experiment through in vivo study. The results of immunohistochemistry and Western blot detection showed that the expression of TLR4 protein in the brain tissue of MCAO mice treated with RIPerC was significantly lower than that of the group without RIPerC treatment (Fig. 5A-C, p < 0.05). Interestingly, the ratios of p-p65/p65 and p-IkBa/IkBa also decreased significantly after treatment with RIPerC (Fig. 5B, D, E, p < 0.05). The above results suggest that RIPerC treatment may inhibit the infarction and neurological damage of MCAO mice by regulating the miR-153-5p/TLR4/p65/IkBa signalling pathway.

Plasma miR-153-5p levels in ischaemic stroke patients are negatively correlated with infarct volume and NIHSS, and low levels of miR-153-5p are associated with poor prognosis

Plasma was collected within 6 hours after the onset of ischaemic stroke, and the plasma miR-153-5p level was detected by qRT-PCR. As shown in Figure 6A, plasma miR-153-5p levels in patients with ischaemic stroke were significantly lower than healthy controls. Spearman correlation analysis showed that plasma miR-153-5p levels in patients with ischaemic stroke were significantly negatively correlated with cerebral infarction volume (Fig. 6B, r = –0.596, p < 0.001) and NIHSS score (Fig. 6C, r = –0.571, p < 0.001). Then, the patients were divided into 2 groups based on the median of their plasma miR-153-5p levels. Survival analysis showed that, compared with the high miR-153-5p group, the 3-month overall survival rate of patients in the low miR-153-5p group was significantly lower (Fig. 6D, c2 = 5.095, p = 0.024). Cox regression analysis is shown in Table III. Hypertension and infarct volume are independent risk factors for poor prognosis in patients with ischaemic stroke, while miR-153-5p is an independent protective factor.

Discussion

Brain tissue ischaemia could cause local cerebral ischaemic injury. RIPerC refers to repetitive and intermittent blood flow blocking stimulation of the limbs, through nerve conduction, body fluids, and systemic inflammatory responses, to modulate the endogenous protective mechanism of motivation [5,10,26]. Thereby, it induces the tolerance of the heart, brain, liver, kidney, lung, and other organs to ischaemia and hypoxia, and improves the ability of remote vital organs to resist ischaemia and hypoxia damage. A study has shown that RIPerC cannot only protect ischaemic brain tissue, but also can further improve the effect of thrombolytic therapy for myocardial infarction [15]. To better improve the clinical treatment effect of RIPerC, the improvement effect of miR-153-5p on cerebral ischaemia injury was examined. The up-regulation of miR-153-5p effectively improved the cell damage caused by OGD and reduced the brain tissue infarction induced by MCAO in mice. At the molecular level, overexpression of miR-153-5p prevented the excessive activation of pro-apoptotic genes (Bax and cleaved caspase 3), and the p65/IkBa inflammatory pathway, while promoted the expression of apoptosis inhibitor Bcl-2. These data suggested that miR-153-5p is a potential marker of cerebral ischaemia injury. Thus, the pathway in which miR-153-5p exerted its effects was further analysed.
Further screening of mRNAs and verification demonstrated that among the mRNAs screened, TLR4 changed the most obviously after being regulated by miR-153-5p. The TLR4 gene is an important member of the interferon regulatory factor (IRF) family and a specific transcription factor that can bind to the A/T-rich DNA sequence to regulate the expressions of related genes [11]. Studies have shown that TLR4 is the core transcription factor for heart development [12,25]. Abnormal expression of TLR4 induces abnormal development of the heart and blood vessels, leading to the death of mice in the embryo [25]. TLR4 also has a wide range of regulatory effects on cell apoptosis and inflammation [1,13]. These reports confirmed the results of this study that TLR4 expression in OGD cells was up-regulated by MCAO but down-regulated by miR-153-5p. More importantly, TLR4 aggravated the cell damage caused by OGD and neutralized the regulatory effect of miR-153-5p. Our experimental results revealed that by regulating the miR-153-5p/TLR4 axis, RIPerC alleviated the damage to tissues and cells caused by cerebral ischaemia.
In addition, according to our results, miR-153-5p regulated the apoptosis and inflammation of tissues and cells through the miR-153-5p/TLR4 axis. Consistent with previous reports, up-regulation of Bcl-2, which inhibited apoptosis, blocked the expression of pro-apoptotic genes (Bax and Cleaved caspase 3), thereby reducing the apoptosis of Neuro-2a cells [18]. It also blocked the p65/IkBa pathway and alleviated the excessive activation of the inflammatory response. Similar results have also been reported in the research of Hsieh et al. [8].
Based on the MCAO mouse model and the OGD cell experiments, we further verified the relationship between the expression of plasma miR-153-5p level and cerebral infarction volume, neurological damage, and prognosis in clinical patients. We found that low levels of plasma miR-153-5p are closely related to poor prognosis of patients with ischaemic cerebral infarction, and the follow-up results further confirmed our conclusion. Combined with the above research results, it seems that RIPerC can inhibit the damage caused by cerebral ischaemia by regulating the miR-153-5p/TLR4/p65/IkBa pathway. However, this study has some shortcomings: 1. We only applied RIPerC to the MCAO mouse model, and the clinical application effect of RIPerC in patients with ischaemic stroke needs to be further verified. 2. The way through which RIPerC treatment affects the expression of miR-153-5p remains to be explored. 3. In this study, we found for the first time that RIPerC intervention can inhibit ischaemic brain damage by regulating the expression of miR-153-5p in patients with ischaemic stroke. Then, the expression regulation of miR-153-5p in the ischaemia of the myocardium, liver, kidney, and other important organs needs to be further explored.
In conclusion, this study confirmed that RIPerC intervention reduces apoptosis and inflammatory response in cerebral ischaemia injury by regulating the miR-153-5p/TLR4/p65/IkBa pathway, suggesting that RIPerC may be a potential therapeutic intervention to inhibit cerebral ischaemic injury. Our research only elucidated the protective effect of RIPerC/miR-153-5p at a basic level; therefore, in-depth research is also required to further confirm the current findings.

Acknowledgments

Thanks for the support of the Emergency Department of our hospital in this study.

Funding sources

This work was supported by the applied basic research foundation of Yunnan Province (202101AT070229, 2019FB090), the Innovation and Entrepreneurship Training Program for College Students in Yunnan Province (202110678069), and the in-hospital science and technology project of the Second Affiliated Hospital of Kunming Medical University (2020yk008).

Disclosure

The authors report no conflict of interest.

References