Effect of LncRNA OIP5-AS1/microRNA-186-5p on isoflurane-induced cognitive dysfunction in aged rats

Abstract

Background

Post-operative recognition dysfunction (POCD) is a kind of central nervous system complication that appears after operative anesthesia. Recent studies on the mechanism of long non-coding RNA (lncRNA) in neurodegenerative diseases are abundant.

Aims

The study aimed to explore the expression pattern and role of lncRNA OIP5-AS1 in POCD and to investigate its underlying mechanism in old rats.

Methods

The old rats were exposed to isoflurane to mimic the POCD in the elderly, and their cognitive function was tested via Morris water maze (MWM) test. Enzyme linked immunosorbent assay was applied for the concentration detection of inflammation and oxidative stress-related factors. Luciferase reporter assay was done for the target gene analysis.

Results

Downregulation of OIP5-AS1 was accompanied by isoflurane treatment in rats, overexpression of OIP5-AS1 induced the rats to spend more time in the target quadrant, and shorten escape latency time. OIP5-AS1 inhibited the release of TNF-α, IL-6 and IL-1β, GSH and superoxide dismutase, decreased the activation of caspase-3, but promoted malondialdehyde release. miR-186-5p was a target miRNA of OIP5-AS1, and exhibited high expression in rats after isoflurane exposure. miR-186-5p can abolish the beneficial role of OIP5-AS1 against cognitive impairment, inflammatory response, oxidative stress and neuron apoptosis.

Conclusion

OIP5-AS1 plays a neuroprotective role in elderly POCD rats through sponging miR-186-5p, and it is related to OIP5-AS1/miR-186-5p mediated inflammatory response, oxidative stress and neuron apoptosis.

Keywords

LncRNA Isoflurane Rats Postoperative recognition dysfunction Neuroprotection Mechanism

Introduction

Postoperative cognitive dysfunction (POCD) is a kind of central nervous system complication that often occurs after operative anesthesia.¹ The occurrence of POCD show close association with the age of patients.² In elderly patients who underwent general anesthesia, about 41.4% of cases can develop POCD.³ It seriously affects the quality of life of patients and increases the burden of social medical care. Although the pathogenesis of POCD is still unclear, general anesthesia has been identified to be one of the main inducements of POCD.⁴ Clinical reports suggest that POCD is more common in elderly patients under isoflurane inhalation.⁵ The results of animal studies also showed that isoflurane could induce memory impairment in older rats.⁶ Therefore, the pathogenesis elucidation of POCD may help to reduce and prevent the occurrence of complications after anesthesia and operation.

Long non-coding RNAs (lncRNAs) are non-coding RNAs over 200 nt in length. LncRNAs have been reported to participate in the onset of tumors, cardiovascular disease, as well as neurodegenerative disease.⁷ As a regulator of excessive inflammation, lncRNA can mediate neuronal apoptosis.⁸ Studies on the mechanism of lncRNA in neurodegenerative diseases are abundant, such as Alzheimer’s Disease (AD), and the pathogenesis and symptoms of AD are similar to POCD.⁹ LncRNA OIP5-AS1 is considered to be a neuroprotective factor against nerve injuries. For example, OIP5-AS1 is detected to be lowly expressed in rats with ischemic stroke, and OIP5-AS1 upregulation can protect against impaired cognitive function caused by cerebral ischemia.¹⁰ In addition, a recent study also demonstrated the downregulation of OIP5-AS1 in diabetic mice, which is accompanied by neurological deficits.¹¹ Thereby, we presented that the role of OIP5-AS1 in POCD may be worth studying.

It was speculated that OIP5-AS1 might be a protective factor against isoflurane-induced neuron injury, that might be related to its regulatory role in neuroinflammation and oxidative stress. In the current study, the old rats were exposed to isoflurane to mimic the POCD in the elderly, and the expression pattern and role of OIP5-AS1 in POCD were explored. Furthermore, the underlying mechanism was also preliminary explained.

Materials and methods

Laboratory animal

The full-term male Sprague-Dawley (SD) rats of clean grade were selected, aged 20 months with body weight of 350–400 g. All rats were kept at the temperature of 22°C, relative humidity of 60.5%, alternating light and dark for 12 h, ventilated and dry environment, providing an adequate standardized diet. This study was approved by the Animal Ethical and Welfare Committee of the Chinese PLA General Hospital (Beijing, China) (Ethics No. SCXK-2017-0013).

Experimental grouping and administration method

SD rats were randomly divided into 6 groups (n = 12/group) by the random number table method. Control group (Ctrl): normal breeding; isoflurane group (Iso): rats were given 1.4% isoflurane for 2 h as previously reported,¹² in brief, the rats were placed in an anesthesia box, which was connected to the anesthesia machine in a constant temperature box at 25°C and 30%–70% N₂ mixture gas was used as the carrier gas, and the ventilation flow was 2 L·min⁻¹; isoflurane + negative control group (Iso+NC): rats were injected with the OIP5-AS1-NC, then given 1.4% isoflurane for 2 h; isoflurane + OIP5-AS1 group (Iso+OIP5-AS1): rats were injected with the OIP5-AS1, then given 1.4% isoflurane for 2 h; OIP5-AS1 + negative control of miR-186-5p mimic group (OIP5-AS1+mimic-NC): rats were injected with the OIP5-AS1 and miR-186-5p mimic-NC by lateral cerebroventricular injection, then given 1.4% isoflurane for 2 h; OIP5-AS1 + miR-186-5p mimic group (OIP5-AS1+miR-mimic): rats were injected with the OIP5-AS1 and miR-186-5p mimic by lateral cerebroventricular injection, then given 1.4% isoflurane for 2 h. Sequences of OIP5-AS1, miR-186-5p mimic, miR-NC were synthesized and provided by sequences were synthesized and provided by TIANGEN (Beijing, China).

Measurement of cognitive function

Morris water maze (MWM) test was applied for the evaluation of rats’ cognitive function as previously reported.¹³ Briefly, the rats were put into the pool, and the time from entering the water to climbing onto the platform was recorded as the escape latency time. The training was done for 4 consecutive days, 4 times a day. On the fifth day, the platform was removed and the rats were put into the water from the same entry point, and their percentage of time in the targeted quadrant within 120 s was recorded.

RNA extraction and reverse transcription-quantitative PCR

Total RNA molecules were isolated and extracted by adding TRIzol, chloroform, isopropanol, and 75% ethanol. After its purity and quality were detected, 500 ng isolated total RNA were retro-transcriptional to generate their complementary single-stranded cDNA through the FastKing gDNA Dispelling RT SuperMix (TIANGEN, China) or miRNA cDNA synthesis Kit (Takara, Japan). Finally, the SuperReal PreMix Plus (SYBR Green) kit (TIANGEN, China) or miRNA qPCR Assay kit (Takara, Japan), specific primers, and template cDNA (2 mug) were mixed to perform PCR amplification on ABI-Prism 7900 sequence detection system. The levels of OIP5-AS1 and miR-186-5p were calculated as 2^−ΔΔCt, and GAPDH or U6 snRNA expression was used for normalization.

Enzyme linked immunosorbent assay

After homogenization of rat hippocampal tissue samples, the lysate was added to fully lysate the cells. After centrifugation at 12,500 g for 3–5 min, the supernatant was collected. Then the concentration of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF-α), glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) was detected via Enzyme linked immunosorbent assay (ELISA) kit (Elisa biotech, Shanghai, China) based on the instruction book.¹⁴ The OD value was detected by the microplate reader, the sample concentration was calculated based on the standard curve.

Caspase-3 activity

The caspase-3 assay kit (Abcam, Cambridge, MA, USA) was applied for the measurement of the Caspase-3 level based on the manufacturer’s protocol. The hippocampal tissue was lysed using a specific lysis buffer, then the lysate was centrifuged at 15,000 r/min for 5 min at 4°C. The protein concentration in the supernatants was measured using the Bradford dye method.¹⁵ Caspase-3 activity was assessed by measuring the absorbance at 405 nm.

Luciferase reporter assay

Bioinformatics was used to predict the targeting relationship between lncRNA OIP5-AS1 and miR-186-5p.¹⁶ The wild-type (WT) or mutant (MUT) fragment of OIP5-AS1 containing the binding site was cloned into the luciferase vector of pGL3 and named WT- OIP5-AS1 or MUT- OIP5-AS1, respectively. Then WT- OIP5-AS1 or MUT- OIP5-AS1 were transfected with miR-186-5p mimic or inhibitor into 293T cells. After 48 h incubation, luciferase activity was detected using a dual-luciferase reporter gene assay system with a microplate reader (Molecular Devices, LLC). Renilla luciferase was used for normalization. Each sample was repeated 3 times.

Statistical analysis

SPSS 20.0 software was used for statistical analysis. Comparison between multiple groups was done by one-way ANOVA. The cell experiment was repeated 3 times with 3 multiple holes in each group. The p value less than .05 was considered to be statistically significant.

Results

Improvements of OIP5-AS1 on spatial learning and memory in rats

In this experiment, spatial learning and memory were assessed using MWM test, and escape time was significantly reduced for all rats after completion of all training (Figure 1(B)). Compared with the control group, the escape latency time was prolonged and the time in the target quadrant decreased in the isoflurane group (Figure 1(B) and (C)). These data indicated that the POCD animal model has been successfully constructed. As shown in Figure 1(A), isoflurane treatment caused a decrease of OIP5-AS1 level, reflecting that OIP5-AS1 dysregulation might be involved in isoflurane-related POCD. The gain and loss function experiments revealed that overexpression of OIP5-AS1 contributed the rats to spend more time in the target quadrant, and shorten escape latency time. It reflected that OIP5-AS1 alleviated cognitive dysfunction induced by anesthesia.

Figure 1.

The effect of OIP5-AS1 on the improvements spatial learning and memory in rats. (a) Levels of lncRNA OIP5-AS1 in the hippocampus tissues of rats of different groups. (b) Escape latency of rats. (c) Time spent in the target quadrant of rats. The test was repeated 3 times in each rat. Comparison among different groups was performed via one-way ANOVA, and *** means p less than .05. Ctrl: rats with normal breeding; Iso group: rats were treated with isoflurane; Iso+NC: rats were injected with the OIP5-AS1-NC, then given isoflurane; Iso+OIP5-AS1: rats were injected with the OIP5-AS1, then given isoflurane.

Improvements of OIP5-AS1 on inflammatory response and oxidative stress in rats

Enzyme linked immunosorbent assay was performed for the detection of inflammatory factors in the hippocampal tissue of rats, TNF-α, IL-6 and IL-1β were selected as the indicators of inflammation. As shown in Figure 2(A), TNF-α, IL-6 and IL-1β showed the lowest levels in the control group, and the highest concentration was observed in the isoflurane or isoflurane+NC group, indicating that isoflurane treatment can promote inflammation. In comparison with isoflurane+NC group, rats in isoflurane+OIP5-AS1 owned the suppressed levels of inflammatory factors, indicating the anti-inflammatory role of OIP5-AS1 during neuron injury.

Figure 2.

Concentration of inflammation-related factors (a), oxidative stress-related factors (b), caspase-3 activity (c) in the hippocampus tissues of rats. The test was repeated 3 times in each rat. Comparison among different groups was performed via one-way ANOVA, and *** means p less than .05. Ctrl: rats with normal breeding; Iso group: rats were treated with isoflurane; Iso+NC: rats were injected with the OIP5-AS1-NC, then given isoflurane; Iso+OIP5-AS1: rats were injected with the OIP5-AS1, then given isoflurane.

In addition, a similar trend was also found in oxidative stress-related factors. As shown in Figure 2(B), compared with the control group, the concentration of oxidative stress-related cytokine MDA was significantly increased in the isoflurane group, and the concentrations of antioxidant factors, including GSH and SOD, were significantly decreased in the isoflurane group, reflecting isoflurane-induced oxidative stress in rats. But the changes induced by isoflurane were counteracted by OIP5-AS1 upregulation. The results suggested that OIP5-AS1 plays a role in inhibiting oxidative stress.

Caspase-3 activity in the hippocampus was measured to detect cell apoptosis. As shown in Figure 2(C), the caspase-3 activity was significantly amplified in the hippocampal tissue of rats exposed to isoflurane compared with the control group. But the caspase-3 activity was decreased via OIP5-AS1 overexpression (Figure 2(C)). It was concluded that OIP5-AS1 can inhibit neuron apoptosis.

Interaction between OIP5-AS1 and miR-186-5p

Through the TargetScan dataset, the binding sites between OIP5-AS1 and miR-186-5p were displayed in Figure 3(A). Then the Luciferase reporting assay pointed out the luciferase activity inhibition induced by miR-186-5p mimic transfection, and the promotion of luciferase activity induced by miR-186-5p inhibitor (Figure 3(B)). Furthermore, qRT-PCR results indicated the elevation of miR-186-5p levels in rats exposed to isoflurane, while OIP5-AS1 transfection downturned the increasing trend (Figure 3(C)). The findings indicated the interaction between OIP5-AS1 and miR-186-5p, OIP5-AS1 acted as a sponge of miR-186-5p.

Figure 3.

Relationship between OIP5-AS1 and miR-186-5p. (a). The binding sites between OIP5-AS1 and miR-186-5p were revealed by TargetScan. (b). Luciferase activity of cells transfected with different gene fragments. (c). Levels of miR-186-5p in hippocampus tissues of rats of different groups. The test was repeated 3 times in each rat. Comparison among different groups was performed via one-way ANOVA, and *** means p less than .05. Ctrl: rats with normal breeding; Iso group: rats were treated with isoflurane; Iso+NC: rats were injected with the OIP5-AS1-NC, then given isoflurane; Iso+OIP5-AS1: rats were injected with the OIP5-AS1, then given isoflurane.

Interaction of OIP5-AS1 and miR-186-5p on spatial learning and memory in rats

Figure 4(A) displayed the elevation of miR-186-5p induced by miR-186-5p mimic transfection. Then the MWM test demonstrated the elevation of escape latency and the inhibition of time spent in the target quadrant induced by miR-186-5p overexpression (Figure 4(B) and (C)). It was concluded that miR-186-5p reversed the protective role of OIP5-AS1 against cognitive dysfunction.

Figure 4.

Interaction of OIP5-AS1 and miR-186-5p on spatial learning and memory in rats. (a) Levels of miR-186-5p in the hippocampus tissues of rats of different groups. (b) Escape latency of rats. (c) Time spent in the target quadrant of rats. The test was repeated 3 times in each rat. Comparison among different groups was performed via one-way ANOVA, and *** means p less than .05. Iso+NC: rats were injected with the OIP5-AS1-NC, then given isoflurane; OIP5-AS1: rats were injected with the OIP5-AS1, then given isoflurane; OIP5-AS1+mimic NC: rats were injected with the OIP5-AS1 and mimic NC, then given isoflurane; OIP5-AS1+miR-mimic: rats were injected with the OIP5-AS1 and miR-186-5p NC, then given isoflurane.

Interaction of OIP5-AS1 and miR-186-5p on inflammatory response and oxidative stress in rats

The influence of miR-186-5p on the indicators of oxidative stress and inflammation was further detected. As shown in Figure 5(A), miR-186-5p counteracted the restrained role of OIP5-AS1 on inflammatory cytokines. Figure 5(B) displayed the restraint of miR-186-5p on GSH and SOD, and its promotion of MDA concentration. Furthermore, the cell death marker, caspase-3 activation was detected among different groups. As shown in Figure 5(C), the inhibition of caspase-3 activation induced by OIP5-AS1 overexpression was reversed by miR-186-5p. We concluded that miR-186-5p can abolish the role of OIP5-AS1 in the inflammatory response, oxidative stress, and neuron apoptosis in rats.

Figure 5.

Interaction of OIP5-AS1 and miR-186-5p on inflammatory response and oxidative stress in rats. Concentration of inflammation-related factors (a), oxidative stress-related factors (b) and caspase-3 activity (c) in the hippocampus tissues of rats. The test was repeated 3 times in each rat. Comparison among different groups was performed via one-way ANOVA, and *** means p less than .05. Iso+NC: rats were injected with the OIP5-AS1-NC, then given isoflurane; OIP5-AS1: rats were injected with the OIP5-AS1, then given isoflurane; OIP5-AS1+mimic NC: rats were injected with the OIP5-AS1 and mimic NC, then given isoflurane; OIP5-AS1+miR-mimic: rats were injected with the OIP5-AS1 and miR-186-5p NC, then given isoflurane.

Discussion

As is known, the incidence of POCD can be seen in all patients at any age, especially in elderly cases.¹⁷ At present, advanced age is an internationally recognized risk factor for the onset of POCD.¹⁸ With the improvement of the medical treatment, more and more elderly patients will receive clinical surgery and anesthesia. The patients suffering POCD will also increase. Therefore, better understanding of POCD will be necessary for the its occurrence after surgery. In this study, elderly rats aged 18–20 months were selected, to simulate the elderly patients receiving anesthesia in clinical practice.

In recent years, the MWM test has become one of the most commonly used methods in basic research to test the learning and memory function of rats in the experimental process.¹⁹ MWM test has been widely recognized by domestic and foreign research fields, and is considered to be the most classical behavioral detection method to study and evaluate the spatial learning and memory ability of experimental animals.²⁰ The present MWM test results demonstrated that rats in the isoflurane group owned long escape latency in comparison with the control groups. In addition, the time spent in the target image was limited in rats exposed to isoflurane. It was concluded that isoflurane can induce the decline of learning and memory function in aged rats, which was consistent with the previous reports.

At present, the pathogenesis of POCD is still not completely clear. The importance of inflammatory response mechanisms in POCD has aroused widespread concern in recent years.²¹ Studies have shown that inflammation in the body, especially in nervous system, can affect the related functions of the central nervous system, thus causing changes in the body’s cognitive function.²² In recent years, the relationship between inflammation and the pathogenesis of POCD has been gradually revealed in various clinical studies and experimental studies.²³ Consistently, excessive release of inflammatory cytokines was detected in rats exposed to isoflurane in the present study, which was balanced out by lncRNA OIP5-AS1 overexpression. Consistent with the current results, the anti-inflammation role of OIP5-AS1 has been previously reported in many diseases. For example, in rats with middle cerebral artery occlusion/reperfusion (MCAO/R) injury, up-regulation of OIP5-AS1 can attenuate the activation of microglia/macrophage induced by MCAO/R and inhibit the release of inflammatory cytokines.¹⁰ Based on the current results, the involvement of lncRNA OIP5-AS1 in neuroinflammation caused by isoflurane was confirmed. Besides anti-inflammation, during the development of ischemic stroke, the neuroprotective role of OIP5-AS1 is also related to its anti-oxidative stress role.¹⁰ But the proinflammatory role of OIP5-AS1 is also reported during acute lung injury,²⁴ which suggests that OIP5-AS1 might play various roles in inflammatory responses induced by different diseases. Oxidative stress also plays a crucial role in the mechanism of POCD.²⁵ The present study also demonstrated the anti-oxidative stress role of OIP5-AS1 in isoflurane-treated rats. Furthermore, OIP5-AS1 also inhibited neuron cell apoptosis induced by MCAO in rats, which was reflected by the decrease of caspase-3 labeled apoptotic neurons by immunohistochemistry.¹⁰ The inhibition of caspase-3 activation induced by OIP5-AS1 was also observed in the hippocampus of rats based on the current results. All the findings led us to propose a hypothesis that, OIP5-AS1 is a powerful anti-inflammatory and anti-oxidative stress mediator, OIP5-AS1 might play a neuroprotective role in elderly POCD rats by inhibiting the inflammatory response, oxidative stress and neuron apoptosis.

LncRNAs act as competitive endogenous RNAs (ceRNAs) to regulate gene expression, they can regulate the mRNA expression through competition and binding microRNAs (miRNAs), thus forming a ceRNA regulatory mechanism (lncRNA-miRNA-mRNA) network.²⁶ miR-186-5p has been identified to be a target of OIP5-AS1 in previous research.^10,27 In the present study, up-regulated expression of miR-186-5p was detected in POCD rats, which was opposite to the level of OIP5-AS1. In addition, the luciferase activity assay verified the targeted relation between OIP5-AS1 and miR-186-5p. MiR-186-5p is a neuron-related microRNA and it has been demonstrated to promote neuron apoptosis, neuroinflammation and oxidative stress in several cell and animal experiments.^10,28 Besides, in mice with vascular cognitive impairment, downregulation of miR-186-5p is also suggested to participate in the beneficial role of aerobic exercise in cognitive function.²⁹ Currently, in POCD rats, miR-186-5p can abolish the beneficial role of OIP5-AS1 against cognitive impairment, inflammatory response, oxidative stress and neuron apoptosis. It was concluded that OIP5-AS1 exerts neuroprotective factor through sponging miR-186-5p.

Previous studies have demonstrated that a common start of neuroinflammation is microglial activation and the subsequent inflammatory cascade, which lead to micro-environment for central nervous system disorder and ultimately affect cognitive function.^30,31 Notably, the role of the NLRP3 inflammasome in neurodegenerative diseases has recently been investigated, and the functional NLRP3 inflammasome activation is limited to the microglial compartment in the mouse brain.³² The current study only suggested the role of OIP5-AS1/miR-186-5p in neuron inflammatory response, the detailed mechanism for its role in isoflurane-induced NLRP3 inflammasome activation needs to be investigated in the future studies.

In conclusion, the protective role of OIP5-AS1 in elderly POCD rats was firstly examined in rats in the present study. Its neuroprotective effect is related to OIP5-AS1/miR-186-5p mediated inflammatory response, oxidative stress and neuron apoptosis.

Footnotes

Nomenclature

Authors’ contributions

Yujing Sun carried out the design and coordinated the study, conducted the experiments on the animals and prepared the manuscript. Yawei Yuan provided assistance in design of the study and contribute to the result analysis and preparation of the manuscript. Long Wang contributed in the design of the experiments and performance of surgical procedures. Sen Sun contributed in the design and conduct of the experiments and undertook a lot of work in the process of revision, including the completion of supplementary experiments and language polishing. All authors read the manuscript and agreed to its publication.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Long Wang

Sen Sun

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