NUFIP1-engineered exosomes derived from hUMSCs regulate apoptosis and neurological injury induced by propofol in newborn rats.

BACKGROUND: Propofol can increase neurotoxicity in infants but the precise mechanism is still unknown. Our previous study revealed that nuclear FMR1 interacting protein 1 (NUFIP1), a specific ribophagy receptor, can alleviate T cell apoptosis in sepsis. Yet, the effect of NUFIP1-engineered exosomes elicited from human umbilical cord blood mesenchymal stem cells (hUMSCs) on nerve injury induced by propofol remains unclear. This study intended to investigate the effect of NUFIP1-engineered exosomes on propofol-induced nerve damage in neonatal rats. METHODS: Firstly, NUFIP1-engineered exosomes were extracted from hUMSCs serum and their identification was conducted using transmission electron microscopy (TEM), Flow NanoAnalyzer, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB). Subsequently, the optimal exposure duration and concentration of propofol induced apoptosis were determined in SH-SY5Y cell line using WB. Following this, we co-cultured the NUFIP1-engineered exosomes in the knockdown group (NUFIP1-KD) and overexpression group (NUFIP1-OE) with SH-SY5Y cells and assessed their effects on the apoptosis of SH-SY5Y cells using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay, Hoechst 33258 staining, WB, and flow cytometry, respectively. Finally, NUFIP1-engineered exosomes were intraperitoneally injected into neonatal rats, and their effects on the learning and memory ability of neonatal rats were observed through the righting reflex and Morris water maze (MWM) test. Hippocampi were extracted from different groups for hematoxylin-eosin (HE) staining, immunohistochemistry, immunofluorescence, and WB to observe their effects on apoptosis in neonatal rats. RESULTS: TEM, Flow NanoAnalyzer, qRT-PCR, and WB analyses confirmed that the exosomes extracted from hUMSCs serum exhibited the expected morphology, diameter, surface markers, and expression of target genes. This confirmed the successful construction of NUFIP1-KD and NUFIP1-OE-engineered exosomes. Optimal exposure duration and concentration of propofol were determined to be 24 hours and 100 µg/ml, respectively. Co-culture of NUFIP1 engineered exosomes and SH-SY5Y cells resulted in significant up-regulation of pro-apoptotic proteins Bax and c-Caspase-3 in the KD group, while anti-apoptotic protein Bcl-2 was significantly decreased. The OE group showed the opposite trend. TUNEL apoptosis assay, Hoechst 33258 staining, and flow cytometry yielded consistent results. Animal experiments demonstrated that intraperitoneal injection of NUFIP1-KD engineered exosomes prolonged the righting reflex recovery time of newborn rats, and MWM tests revealed a significant diminution in the time and number of newborn rats entering the platform. HE staining, immunohistochemistry, immunofluorescence, and WB results also indicated a significant enhancement in apoptosis in this group. Conversely, the experimental results of neonatal rats in the OE group revealed a certain degree of anti-apoptotic effect. CONCLUSIONS: NUFIP1-engineered exosomes from hUMSCs have the potential to regulate nerve cell apoptosis and mitigate neurological injury induced by propofol in neonatal rats. Targeting NUFIP1 may hold great significance in ameliorating propofol-induced nerve injury.

Apoptotic mechanism of propofol-induced developmental toxicity in zebrafish embryos.

General anesthetics can cause neurological damage and long-term behavioral/cognitive impairment during fetal and early postnatal life. However, the adverse influence on embryo development induced by propofol is unclear. We used embryonic zebrafish to explore the effects of propofol on embryonic and larval growth and development, and the related apoptotic mechanism. Zebrafish embryos were immersed in propofol (1, 2, 3, 4, and 5 µg/ml) dissolved in E3 medium from 6 to 48 hours post fertilization (hpf). The survival rate, locomotion, heart rate, hatchability, deformity rate, and body length were analyzed at defined stages. Terminal deoxynucleotidyl transferase nick-end-labeling was used to detect zebrafish embryo apoptosis, and the expression levels of apoptosis-related genes were determined using quantitative real-time reverse transcription PCR and whole-mount in situ hybridization. Larvae at 48 hpf were anesthetized by immersion in E3 culture medium containing 2 µg/ml propofol, the reasonable anesthetic concentration for zebrafish embryos, which caused significant caudal fin dysplasia, light pigmentation, edema, hemorrhage, and spinal deformity, and decreased the hatchability, body length, and heart rate. The numbers of apoptotic cells in propofol-treated 12, 48 and 72 hpf embryos increased significantly, and the mRNA expression levels of intrinsic apoptosis pathway-related casp3a, casp3b, casp9, and baxb genes were upregulated, mainly in the head and tail. Propofol decreased apoptosis in the head and back of 24 hpf zebrafish, which was consistent with the mRNA expression analysis. Our findings demonstrated that zebrafish embryos and larvae exposed to propofol experienced developmental toxicity, which correlated with the intrinsic apoptosis pathway with casp3a, casp3b, casp9, and baxb as the key genes.

Role of adenosine A2A receptors in the loss of consciousness induced by propofol anesthesia.

The mechanism of propofol-anesthesia-induced loss of consciousness (LOC) remains largely unknown. We speculated that the adenosine A2A receptor serves as a vital molecular target in regulating LOC states under propofol anesthesia. c-Fos staining helped observe the changes in the neuronal activity in the nucleus accumbens (NAc). Initially, the adenosine signals in the NAc were measured under propofol anesthesia using fiber photometry recordings. Then, behavior tests and electrophysiological recordings were used to verify the effect of systemic A2A R agonist or antagonist treatment on propofol anesthesia. Next, the microinjection technique was used to clarify the role of the NAc A2A R under propofol anesthesia. Fiber photometry recordings were applied to assess the effect of A2A R agonist or antagonist systemic treatment on adenosine signal alterations in the NAc during propofol anesthesia. Then, as the GABAergic neurons are the main neurons in the NAc, we further measured the neuronal activity of GABAergic neurons. In our study, propofol anesthesia enhanced the neuronal activity in the NAc, and the adenosine signals were increased in the NAc. SCH58261 reduced the LOC time and sedative depth, while CGS21680 increased those via intraperitoneal injection. Additionally, CGS21680 increased the changes in delta, theta, alpha, beta, and low-gamma oscillations in the NAc. Moreover, microinjection of SCH58261 significantly shortened the LOC time, whereas microinjection of CGS21680 into the NAc significantly prolonged the LOC duration. The results illustrated that after A2A R agonist administration, the level of extracellular adenosine signals in the NAc was decreased and the neuronal activity of GABAergic neurons was enhanced, whereas after A2A R antagonist administration via intraperitoneal injection, the opposite occurred. This study reveals the vital role of the A2A R in propofol-induced LOC and that the A2A R could affect the maintenance of propofol anesthesia.

Neonatal Exposure to Propofol Interferes with the Proliferation and Differentiation of Hippocampal Neural Stem Cells and the Neurocognitive Function of Rats in Adulthood via the Akt/p27 Signaling Pathway.

Objective: Neonatal exposure to propofol has been reported to cause neurotoxicity and neurocognitive decline in adulthood; however, the underlying mechanism has not been established. Methods: SD rats were exposed to propofol on postnatal day 7 (PND-7). Double-immunofluorescence staining was used to assess neurogenesis in the hippocampal dentate gyrus (DG). The expression of p-Akt and p27 were measured by western blotting. The Morris water maze, novel object recognition test, and object location test were used to evaluate neurocognitive function 2-month-old rats. Results: Phosphorylation of Akt was inhibited, while p27 expression was enhanced after neonatal exposure to propofol. Propofol also inhibited proliferation of neural stem cells (NSCs) and decreased differentiation to neurons and astroglia. Moreover, the neurocognitive function in 2-month-old rats was weakened. Of significance, intra-hippocampal injection of the Akt activator, SC79, attenuated the inhibition of p-AKT and increase of p27 expression. SC79 also rescued the propofol-induced inhibition of NSC proliferation and differentiation. The propofol-induced neurocognition deficit was also partially reversed by SC79. Conclusion: Taken together, these results suggest that neurogenesis is hindered by neonatal propofol exposure. Specifically, neonatal propofol exposure was shown to suppress the proliferation and differentiation of NSCs by inhibiting Akt/p27 signaling pathway.

Edaravone alleviated propofol-induced neural injury in developing rats by BDNF/TrkB pathway.

As a variety of free radical scavenger, edaravone has shown its potential in producing antioxidant, anti-inflammatory and neuroprotective effects in various disease models. However, the underlying mechanism behind the neuroprotective effects of edaravone remained unclear. This study is aimed at determining the effects of edaravone on neuroprotection and anti-inflammatory through a propofol-induced neural injury rat model. Firstly, an observation was made of apoptosis and neuroinflammation in the hippocampus of developing under the influence of propofol. It was found out that propofol could produce inflammatory effects in the hippocampus by enhancing the astrogliosis (GFAP) activation and elevating the level of neuronal nitric oxide synthase (nNOS), pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). Meanwhile, the increase of apoptosis cells and the decrease of neurons (NeuN) were speculated to aggravate neural injury. Furthermore, it was demonstrated that edaravone intervention can reverse the neural apoptosis and inflammation. Additionally, the intraperitoneal injection of edaravone, the intraperitoneal injection of the brain-derived neurotrophic factor (BDNF)-mimicking small compound (7,8 dihydroxyflavone) and the intracranial injection of the exogenous BDNF were all respectively effective in alleviating the propofol-induced neural apoptosis and inflammation in the hippocampus. It was also found out that edaravone-activated downstream signalling through tyrosine kinase receptor B (TrkB) receptors in astrocyte, microglia and neuron. However, the neural injury of propofol had no impact on long-term learning and memory, except causing a short-term neurotoxicity. In conclusion, edaravone could alleviate the propofol-induced neural injury in developing rats through BDNF/TrkB pathway.

CircRNA 001372 Reduces Inflammation in Propofol-Induced Neuroinflammation and Neural Apoptosis through PIK3CA/Akt/NF-κB by miRNA-148b-3p.

OBJECTIVES: To investigate effects of circular RNA (circRNA) 001372 and its antagonist miRNAs-148b-3p on propofol-induced neurotoxicity and neuroinflammation in rat brain and pheochromocytoma cells. METHODS: Sprague Dawley rats in propofol model group (n = 6) were intraperitoneal injected with propofol (50 mg/kg) and in sham control group (n = 6) without any treatment. Twenty-four h later, brain tissues were acquired during pentobarbital anesthesia. PC-12 cells were transfected with or without circRNA001372 mimics, circRNA001372 inhibitor, negative mimics or miRNA-148b-3p for 48 h and then treated with propofol (100 µM) for 48 h. Quantitative reverse transcription PCR and gene chips were used for detecting levels of circRNA001372, Haemotoxylin and Eosin staining for cell morphology, MTT for cell viability, flow cytometry for apoptosis, enzyme-linked immunosorbent assay for lactate dehydrogenase (LDH), interleukin-1ß (IL-1ß), IL-6, IL17 and IL-18, and Western blots for phosphoinositide 3-kinase (PI3K), Akt, phosphorylated Akt, and nuclear factor (NF) κB, dual-light luminescent reporter gene assay for luciferase reporter. RESULTS: The propofol anesthesia in rats decreases levels of circRNA001372 and increases levels of cytokines including IL-1ß, IL-6, IL17 and IL-18, resulting in the neurocyte damage in brain. In propofol-treated PC-12 cells, the inhibition of circRNA001372 increases apoptosis and cell damage makers, including LDH, IL-1ß, IL-6, IL17, IL-18, resulting in the reduction of cell viability, which have been revised after over-expression of circRNA001372. MiRNA-148b-3p reduces circRNA001372-incresed PI3K and pAKt levels but enhances the circRNA001372-decreased NFκB level. CONCLUSIONS: CircRNA001372 suppresses propofol-induced neurotoxicity and neuroinflammation through PI3K/Akt/NF-κB signaling pathway in rat brain and neurocytes. MiRNA-148b-3p antagonizes the effects of circRNA001372.

Propofol induces the elevation of intracellular calcium via morphological changes in intracellular organelles, including the endoplasmic reticulum and mitochondria.

Propofol, most frequently used as a general anesthetic due to its versatility and short-acting characteristics, is thought to exert its anesthetic actions via GABAA receptors; however, the precise mechanisms of its adverse action including angialgia remain unclear. We examined the propofol-induced elevation of intracellular calcium and morphological changes in intracellular organelles using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, and HUVECs loaded with fluorescent dyes for live imaging. Although propofol (>50 µM) increased intracellular calcium in a dose-dependent manner in these cells, it was not influenced by the elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Studies using U-73122, xestospongin C, and dantrolene revealed that propofol-induced calcium elevation was not mediated by G-protein coupled receptors, IP3 receptors, or ryanodine receptors. We performed live imaging of the ER, mitochondria and Golgi apparatus during propofol stimulation using fluorescent dyes. Concomitant with the calcium elevation, the structure of the ER and mitochondria was fragmented and aggregated, and these changes were not reversed during the observation period, suggesting that propofol-induced calcium elevation occurs due to calcium leakage from these organelles. Although the concentration of propofol used in this experiment was greater than that used clinically (30 µM), it is possible that the concentration exceeds 30 µM at the site where propofol is injected, leading the idea that these phenomena might relate to the various propofol-induced adverse effects including angialgia.

A computational analysis of the effect of sevoflurane in a human ventricular cell model of long QT syndrome: Importance of repolarization reserve in the QT-prolonging effect of sevoflurane.

The slowly and rapidly activating delayed rectifier K+ channels (IKs and IKr, respectively) contribute to the repolarization of ventricular action potential in human heart and thereby determine QT interval on an electrocardiogram. Loss-of-function mutations in genes encoding IKs and IKr cause type 1 and type 2 long QT syndrome (LQT1 and LQT2, respectively), accompanied by a high risk of malignant ventricular arrhythmias and sudden cardiac death. This study was designed to investigate which cardiac electrophysiological conditions exaggerate QT-prolonging and arrhythmogenic effects of sevoflurane. We used the O'Hara-Rudy dynamic model to reconstruct human ventricular action potential and a pseudo-electrocardiogram, and simulated LQT1 and LQT2 phenotypes by decreasing conductances of IKs and IKr, respectively. Sevoflurane, but not propofol, prolonged ventricular action potential duration and QT interval in wild-type, LQT1 and LQT2 models. The QT-prolonging effect of sevoflurane was more profound in LQT2 than in wild-type and LQT1 models. The potent inhibitory effect of sevoflurane on IKs was primarily responsible for its QT-prolonging effect. In LQT2 model, IKs was considerably enhanced during excessive prolongation of ventricular action potential duration by reduction of IKr and relative contribution of IKs to ventricular repolarization was markedly elevated, which appears to underlie more pronounced QT-prolonging effect of sevoflurane in LQT2 model, compared with wild-type and LQT1 models. This simulation study clearly elucidates the electrophysiological basis underlying the difference in QT-prolonging effect of sevoflurane among wild-type, LQT1 and LQT2 models, and may provide important information for developing anesthetic strategies for patients with long QT syndrome in clinical settings.

Developmental and cardiac toxicities of propofol in zebrafish larvae.

Propofol, a commonly used anesthetic, is convenient to use, induces quick effect, enables rapid recovery, and is widely accessible given its stable supply. However, its adverse effects are a concern. Reportedly, propofol exhibits a significant inhibitory effect on the respiratory and circulatory systems. Furthermore, intravenous administration of this drug results in hypotension, rapid heart rate, and respiratory failure. Because many pregnant women are administered propofol during childbirth, it may have a significant negative effect on the development of infants. Propofol can cause considerable developmental neurotoxicity and has known activity on the heart. However, the underling mechanisms of these toxicities remain unclear. In the present study, zebrafish embryos were exposed to propofol at different concentrations (0.05, 0.1, 0.5, 1, 5, 10, and 20 µg/ml) to determine its developmental and cardiac toxicities. Propofol exposure decreased the survival rate and hatchability of zebrafish embryos. Additionally, the embryo malformation rate increased in a concentration-dependent manner. Different types of malformations were observed following propofol administration. The proportion of pericardial cysts increased, whereas the heart rate and size decreased with an increase in propofol concentration. The quantitative reverse-transcription polymerase chain reaction revealed that propofol significantly altered the expression of genes related to cardiac development and functions in zebrafish. Collectively, our findings indicate that propofol exposure induces significant developmental and cardiac toxicities in zebrafish.

Inhibition of the electron transport chain in propofol induced neurotoxicity in zebrafish embryos.

Fetal and neonatal exposure to propofol can lead to neuronal death and long-term neurobehavioral deficiencies in both rodents and nonhuman primates. Zebrafish embryo, which is fertilized ex-utero, has provided us a new model species to study the effects of general anesthetics on developing brain. Inhibited electron transport chain leads to mitochondrial dysfunction and insufficient energy production. The aim of this study was to dissect the role of electron transport chain in propofol-induced neurotoxicity. 6 h post fertilization (hpf) zebrafish embryos were exposed to control or 1, 2 or 4 µg/ml propofol until 48hpf. Acridine orange staining was used to assess cell apoptosis in the brain of zebrafish embryos. The activity of mitochondrial electron transport chain complex was assessed using colorimetric method. Expression of key subunit of cytochrome c oxidase was assessed by western blot and transcription level of cox4i1 was assessed by quantitative real time-PCR. The mitochondrial membrane potential and ATP content were assessed. Exposure to 1, 2 and 4 µg/ml propofol induced significant increases in cell apoptosis in the brain of zebrafish embryos in a dose-dependent manner and led to significant decreases in electron transport chain complex IV activity from (0.161 ± 0.023)µmol/mg/min in blank control-treated group to (0.096 ± 0.015)µmol/mg/min, (0.083 ± 0.013)µmol/mg/min and (0.045 ± 0.014)µmol/mg/min respectively, accompanied by decreased expression of key regulatory subunit of cytochrome c oxidase-subunit IV and decreased transcription level of cox4i1. Propofol exposure also decreased the mitochondrial membrane potential and ATP content. Our findings demonstrate that inhibition of the electron transport chain is involved in the mechanisms by which propofol induces neurotoxicity in the developing brain.

Topiramate Reverses Physiological and Behavioral Alterations by Postoperative Cognitive Dysfunction in Rat Model Through Inhibiting TNF Signaling Pathway.

This study aimed to investigate the effects of topiramate (TPM) on rats with postoperative cognitive dysfunction (POCD) and elucidate the underlying mechanism. Differentially expressed genes in propofol-treated group and vehicle control group were filtered out and visualized in heatmap based on R program. POCD rat models were established for validation of TPM's anti-inflammatory action and Morris water maze (MWM) test was employed for assessment of spatial learning and memory ability of rats. Hematoxylin and eosin (HE) staining was applied to detect the neurodegeneration, and the apoptosis status was detected using TUNEL assay. In vitro, hippocampal microglia was treated with lipopolysaccharide or TPM to validate the TPM's anti-inflammatory action. Cell apoptosis was detected with flow cytometry. Inflammatory factors were detected by enzyme-linked immunosorbent assay, and factor-associated suicide (Fas), Fas-associated protein with death domain (FADD) expression were detected by western blot. As results, TPM administration improved the spatial learning and memory ability in POCD rat by decreasing the expression levels of Fas, FADD, and inflammatory factors (tumor necrosis factor-α, TNF-α; interleukin-1ß, IL-1ß; interleukin-6, IL-6) in POCD rats. In addition, TPM down-regulated cell apoptotic rate to suppress POCD by decreasing the expression of Caspase8, Bcl2-associated X (Bax), and poly ADP-ribose polymerase-1 (PARP1) yet enhancing B-cell lymphoma-2 (Bcl-2) expression. Besides, inhibition of Fas enhanced TPM-induced down-regulation of apoptosis of neuronal cell in hippocampus tissues of POCD rats. Our results revealed that treatment of POCD rats with TPM could suppress neuronal apoptosis in the hippocampus tissues, and the neuroprotective effects of TPM may relate with the regulation of tumor necrosis factor (TNF) signaling pathway.

Erythropoietin attenuates propofol-induced hippocampal neuronal cell injury in developing rats by inhibiting toll-like receptor 4 expression.

BACKGROUND: This study was to investigate the neuroprotective effect of erythropoietin (EPO) on hippocampal neuronal cell injury in developing rats. METHODS: The hippocampal neurons cells were obtained from SD rats aged 10 days and divided into control, propofol, EPO, and propofol + erythropoietin (E + P) groups. Cell proliferation and apoptosis were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Ki-67 immunofluorescence, and flow cytometry, respectively. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-4 and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA). Cellular immunohistochemistry was utilized to detect the expression of proliferating cell nuclear antigen (PCNA), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). Quantitative real time polymerase chain reaction (qRT-PCR) and western blot were used to detect the expression of Bax, Bcl-2, Caspase-3, toll-like receptor 4 (TLR4) and p65. Furthermore, TLR4 antagonist (TAK-242) and activator (LPS) were used to study the relationship between EPO and TLR4. RESULTS: Propofol treatment caused morphological and structural damage of hippocampal neurons. However, EPO significantly improved this damage, enhanced cell proliferation, decreased apoptosis and pro-inflammatory factor content, up-regulated the expression of Ki-67, PCNA, Bcl-2, NGF, BDNF and NT-3, as well as decreased the expression of Bax, Caspase-3, TLR4 and p65 (p < 0.05). After TAK-242 or LPS treatment, it showed similar results in propofol + TAK-242 (T + P) group and E + P group. CONCLUSION: Erythropoietin could attenuate propofol-induced hippocampal neuronal cell injury in developing rats, which may be related to inhibit TLR4 expression.

Lipid emulsion, but not propofol, induces skeletal muscle damage and lipid peroxidation.

PURPOSE: Prolonged propofol infusion induces skeletal muscle damage. However, it is well known that the lipid emulsion that is the solvent of propofol causes various types of tissue damage via lipid peroxidation, and that propofol, conversely, has an anti-lipid peroxidative effect. The purpose of this study was to determine whether propofol or the lipid emulsion is the cause of muscle damage following prolonged administration. METHODS: Rats were divided into four groups: NI group (no intervention), Cath group (venous catheter insertion only), Prop group (1% propofol (Maruishi) intravenous infusion at 10 mg/kg/h), and Lipid group (10% Lipofundin® intravenous infusion at 100 mg/kg/h) (n = 10, each group). 1% Propofol (Maruishi) or Lipofundin was infused at 1 mL/kg/h for 72 h. The solvent of 1% propofol (Maruishi) is a 10% lipid emulsion. Lipofundin consists of 50% long-chain triacylglycerols and 50% medium-chain triacylglycerols, similar to the propofol solvent. Plasma concentrations of creatine kinase and myoglobin, superoxide production level, and 4-hydroxynonenal and malondialdehyde expression in the gastrocnemius muscle were evaluated 72 h after the interventions. RESULTS: Plasma concentrations of creatine kinase and myoglobin in the Lipid group were significantly higher than those in the other three groups. The superoxide production level, and 4-hydroxynonenal and malondialdehyde expression in the Lipid group were also significantly higher than in the other three groups. CONCLUSION: Lipofundin induces skeletal muscle damage via lipid peroxidation, and 1% propofol (Maruishi) conversely suppresses the muscle damage via antioxidant effects.

Potential role of the cAMP/PKA/CREB signalling pathway in hypoxic preconditioning and effect on propofol­induced neurotoxicity in the hippocampus of neonatal rats.

Hypoxic preconditioning (HPC) is neuroprotective against ischaemic brain injury; however, the roles of potential anti­apoptotic signals in this process have not been assessed. To elucidate the molecular mechanisms involved in HPC­induced neuroprotection, the effects of HPC on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element­binding protein (CREB) signalling pathway and apoptosis in Sprague­Dawley pups (postnatal day 7) treated with propofol were investigated. Western blot and histological analyses demonstrated that HPC exerts multiple effects on the hippocampus, including the upregulation of cAMP and phosphorylation of CREB. These effects were partially blocked by intracerebroventricular injection of the protein kinase antagonist H89 (5 µmol/5 µl). Notably, the level of cleaved caspase­3 was significantly downregulated by treatment with the cAMP agonist Sp­cAMP (20 nmol/5 µl). The results indicate that propofol increased the level of cleaved caspase­3 and Bax by suppressing the activity of cAMP­dependent proteins and Bcl­2; thus, HPC prevents propofol from triggering apoptosis via the cAMP/PKA/CREB signalling pathway.

Overexpression of phosphoprotein enriched in astrocytes 15 reverses the damage induced by propofol in hippocampal neurons.

Propofol is a general anesthetic used in surgical operations. Phosphoprotein enriched in astrocytes 15(PEA15) was initially identified in astrocytes. The present study examined the role of PEA15 in the damage induced by propofol in hippocampal neurons. A model of hippocampal neuron damage was established using 50 µmol/l propofol. Cell viability, proliferation and apoptosis of hippocampal neurons were tested by Cell Counting Kit­8 and flow cytometry. Western blotting and reverse transcription­quantitative polymerase chain reaction analysis were performed to measure the expression levels of PEA15, and additional factors involved in apoptosis or in the signaling pathway downstream of PEA15. The present results suggested that propofol significantly decreased PEA15 expression levels in hippocampal neurons. Furthermore, overexpression of PEA15 significantly increased the cell viability and cell proliferation of cells treated with propofol. Additionally, PEA15 overexpression decreased apoptosis, which was promoted by propofol. Treatment with propofol significantly decreased the protein expression levels of pro­caspase­3, B­cell lymphoma-2, phosphorylated extracellular signal­regulated kinases (ERK)1/2, ribosomal S6 kinase 2 (RSK2) and phosphorylated cAMP responsive element binding protein 1 (CREB1). However, propofol upregulated active caspase­3 and Bax expression levels. Notably, PEA15 overexpression was able to reverse the effects of propofol. Collectively, overexpression of PEA15 was able to attenuate the neurotoxicity of propofol in rat hippocampal neurons by increasing proliferation and repressing apoptosis via upregulation of the ERK­CREB­RSK2 signaling pathway.

Alzheimer's Disease Presenilin-1 Mutation Sensitizes Neurons to Impaired Autophagy Flux and Propofol Neurotoxicity: Role of Calcium Dysregulation.

BACKGROUND: Disruption of intracellular Ca2+ homeostasis and associated autophagy dysfunction contribute to neuropathology in Alzheimer's disease (AD). OBJECTIVE: To study the effects of propofol on cell viability via its effects on intracellular Ca2+ homeostasis, and the impact of autophagy, in a neuronal model of presenilin-mutated familial AD (FAD). METHODS: We treated PC12 cells, stably transfected with either mutated presenilin-1 (L286V) or wild type (WT) controls, with propofol at different doses and durations, in the presence or absence of extracellular Ca2+, antagonists of inositol trisphosphate receptors (InsP3R, xestospongin C) and/or ryanodine receptors (RYR, dantrolene), or an inhibitor of autophagy flux (Bafilomycin). We determined cell viability, cytosolic Ca2+ concentrations ([Ca2+]c), vATPase protein expression, and lysosomal acidification. RESULTS: The propofol dose- and time-dependently decreased cell viability significantly more in L286V than WT cells, especially at the pharmacological dose (>50µM), and together with bafilomycin (40 nM). Clinically used concentrations of propofol (<20µM) tended to increase cell viability. Propofol significantly increased [Ca2+]c more in L286V than in WT cells, which was associated with decrease of vATPase expression and localization to the lysosome. Both toxicity and increased Ca2+ levels were ameliorated by inhibiting InsP3R/RYR. However, the combined inhibition of both receptors paradoxically increased [Ca2+]c, by inducing Ca2+ influx from the extracellular space, causing greater cytotoxicity. CONCLUSION: Impairment in autophagy function acts to deteriorate cell death induced by propofol in FAD neuronal cells. Cell death is ameliorated by either RYR or InsP3R antagonists on their own, but not when both are co-administered.

Propofol Regulates Neural Stem Cell Proliferation and Differentiation via Calmodulin-Dependent Protein Kinase II/AMPK/ATF5 Signaling Axis.

BACKGROUND: Propofol can cause degeneration of developing brain cells and subsequent long-term learning or memory impairment. However, at the early stage of embryonic development, the molecular mechanism of propofol-induced inhibition in neural stem cells (NSCs) neurogenesis is still unclear. The aim of this study was to determine the role of propofol in NSCs neurogenesis and, more importantly, to explore the underlying mechanism. METHODS: First, a single intraperitoneal injection of propofol was performed in pregnant mice, and 6 hours after administration of propofol, the hippocampus RNA and the protein of the embryos' brains was extracted to analyze the expression of neuron-specific markers. Second, the primary NSCs were isolated from the hippocampus of mouse embryonic brain and then treated with propofol for cell viability, immunostaining, and transwell assays; more importantly, we performed RNA sequencing (RNA-seq) and q-reverse transcription polymerase chain reaction assays to identify genes regulated by propofol; the Western blot, small interfering RNA (SiRNA), and luciferase reporter assays were used to study the effects of propofol on calmodulin-dependent protein kinase (CaMk) II/5' adenosine monophosphate-activated protein kinase (AMPK)/activating transcription factor 5 (ATF5) signaling pathway. RESULTS: Our results indicated that propofol treatment could inhibit the proliferation, migration, and differentiation of NSCs. The results of RNA-seq assays showed that propofol treatment resulted in downregulation of a group of Ca-dependent genes. The following mechanism studies showed that propofol regulates the proliferation, differentiation, and migration of NSCs through the CaMkII/phosphorylation of serine at amino acid position 485 (pS485)/AMPK/ATF5 signaling pathway. CONCLUSIONS: The results from study demonstrated that propofol inhibits the proliferation, differentiation, and migration of NSCs, and these effects are partially mediated by CaMkII/pS485/AMPK/ATF5 signaling pathway.

Histamine H3 receptor antagonist Clobenpropit protects propofol-induced apoptosis of hippocampal neurons through PI3K/AKT pathway.

OBJECTIVE: The aim of the study was to explore whether histamine H3 receptor antagonist Clobenpropit could protect propofol-induced neurotoxicity in hippocampal neurons. MATERIALS AND METHODS: Hippocampal neurons were extracted from neonatal rats and induced with propofol. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect apoptotic rate of neurons. Western blot was conducted to detect protein levels of cleaved-caspase-3 and Bax/Bcl2. After LY294002 treatment, the PI3K pathway antagonist was applied in neurons, protein levels of cleaved-caspase-3 and Bax/Bcl2 were detected by Western blot as well. RESULTS: Propofol treatment remarkably induced neuronal apoptosis. Clobenpropit alleviated cell apoptosis induced by propofol. Protein expressions of cleaved-caspase-3 and Bax/Bcl2 were remarkably downregulated in neurons treated with Clobenpropit. LY294002 induction remarkably reverses the protective role of Clobenpropit in neuronal apoptosis, manifesting as downregulated PI3K and p-AKT after LY294002 treatment. CONCLUSIONS: Clobenpropit protects propofol-induced neuronal apoptosis through activating PI3K/AKT pathway.

Hypoxic preconditioning reduces propofol-induced neuroapoptosis via regulation of Bcl-2 and Bax and downregulation of activated caspase-3 in the hippocampus of neonatal rats.

OBJECTIVE:  Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning (HPC) was previously demonstrated to provide neuroprotection and brain recovery from either acute or chronic neurodegeneration in several cellular and animal models. Therefore, the present study was designed to investigate the protective effects of hypoxic preconditioning on apoptosis caused by propofol in neonatal rats. METHODS: Propofol (100 mg/kg) was given to 7-day-old (P7) Sprague Dawley pups. Before the propofol injection, hypoxic preconditioning was administered by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2), then 2 h of room air. We detected neuronal structure changes and apoptosis by hematoxylin and eosin (HE) staining and TUNEL assay, respectively. Bcl-2, Bax and cleaved-caspase-3 levels were quantified using Western blotting and immunohistochemistry. RESULT:  After treatment with propofol, Bcl-2 levels decreased and Bax and cleaved-caspase-3 levels increased. However, our results suggest that hypoxic preconditioning could reverse this change. Conclusion: Our results indicate that pretreatment with hypoxic preconditioning prevents propofol-induced neuroapoptosis by increasing the levels of Bcl-2 and decreasing the levels of Bax and cleaved-caspase-3.

PKA-CREB-BDNF signaling pathway mediates propofol-induced long-term learning and memory impairment in hippocampus of rats.

Studies have found that propofol can induce widespread neuroapoptosis in developing brains, which leads to cause long-term learning and memory abnormalities. However, the specific cellular and molecular mechanisms underlying propofol-induced neuroapoptosis remain elusive. The aim of the present study was to explore the role of PKA-CREB-BDNF signaling pathway in propofol-induced long-term learning and memory impairment during brain development. Seven-day-old rats were randomly assigned to control, intralipid and three treatment groups (n = 5). Rats in control group received no treatment. Intralipid (10%, 10 mL/kg) for vehicle control and different dosage of propofol for three treatment groups (50, 100 and 200 mg/kg) were administered intraperitoneally. FJB staining, immunohistochemistry analysis for neuronal nuclei antigen and transmission electron microscopy were used to detect neuronal apoptosis and structure changes. MWM test examines the long-term spatial learning and memory impairment. The expression of PKA, pCREB and BDNF was quantified using western blots. Propofol induced significant increase of FJB-positive cells and decrease of PKA, pCREB and BDNF protein levels in the immature brain of P7 rats. Using the MWM test, propofol-treated rats demonstrated long-term spatial learning and memory impairment. Moreover, hippocampal NeuN-positive cell loss, long-lasting ultrastructural abnormalities of the neurons and synapses, and long-term down-regulation of PKA, pCREB and BDNF protein expression in adult hippocampus were also found. Our results indicated that neonatal propofol exposure can significantly result in long-term learning and memory impairment in adulthood. The possible mechanism involved in the propofol-induced neuroapoptosis was related to down-regulation of PKA-CREB-BDNF signaling pathway.