STAT3 Involved in Cellular Vulnerability to Isoflurane

doi:10.21203/rs.3.rs-461611/v1

Download PDF

Research Article

STAT3 Involved in Cellular Vulnerability to Isoflurane

https://doi.org/10.21203/rs.3.rs-461611/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

STAT3 signaling is crucial during neural spontaneous death period, a restricted developmental time window in which the neonatal brain is vulnerable to isoflurane. Here, we designed experiments to assess whether isoflurane target STAT3 to deliver its cytotoxicity. Mice at postnatal day 7 or 21, primary cortical neurons cultured for 5 or 14 days and human neuroglioma U251 cells were treated with isoflurane. A plasmid containing human wild-type STAT3, STAT3 anti-sense oligonucleotide, STAT3 specific inhibitor STA21, proteasome inhibitor MG-132 and calcineurin inhibitor FK506 were utilized to evaluate the influence of STAT3 levels on isoflurane-induced cytotoxicity. In the present study, an upregulation of STAT3 parallel with a decline in calcineurin activity as well as a decrease in the ability of isoflurane to trigger calcineurin activity and neuroapoptosis were observed in more mature neuron or brain. STAT3 survival pathway was impaired after isoflurane exposure in U251 cells and exerted a prominent effect. STAT3 disruption exaggerated isoflurane-induced oxidative injury and apoptosis, whereas, STAT3 overexpression exhibited notable cellular protection. The blockage of calcineurin activity ameliorated neural apoptosis, dendritic spine impairment and cognitive dysfunction induced by isoflurane. Overall, these results indicated that specific regulation of STAT3 was closely related with the cellular vulnerability to isoflurane.

Cellular & Molecular Neuroscience

Isoflurane

Neurotoxicity

STAT3

Calcineurin

Ubiquitination

Accumulating evidence suggests that most commonly used clinical anesthetics causes widespread neuronal apoptosis in newborn animals [1,2], whereas the mature brain appears resistance [3,4]. Animal research further showed that anesthetics may trigger cell death in immature neurons of a specifically vulnerable age, when they require synaptic connections and neurotrophin such as NGF and BDNF for survival [5]. However, the underlying mechanism of this inherent vulnerability is largely unknown.

As a transcriptional factor and an intracellular signal transducer, signal transducer and activator of transcription-3 (STAT3) is increasingly recognized for its neuroprotective effect in various brain injuries [6]. During postnatal brain development, STAT3 is identified as a key mediator of the neurotrophin-induced survival pathway, and its protein level gradually increases as neurons pass through the developmental death period [7]. STAT3 knockdown in mature neurons induces neurotrophin dependency, whereas overexpression of STAT3 enables immature neurons to achieve resistance against neurotrophin deprivation.

Our previous study found that isoflurane impaired the STAT3-mediated survival pathway in the brain of neonatal mice. In the current study, we hypothesized that the impaired STAT3 pathway further sensitize cells to the toxicity of isoflurane. We designed gain and loss experiments to determine whether STAT3 signaling was involved in the cellular vulnerability to isoflurane.

Mice anesthesia and treatment

Animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and were approved by the Ethics Review Board for Animal Studies of Nanjing Drum Tower Hospital. The animal treatments were similar to that in our previous study [8]. C57/BL6 mice on postnatal day 7 or 21 (from Model Animal Research Center of Nanjing University, China) were anesthetized with 1.5% isoflurane for 6 hr in 100% oxygen at 37°C. Mice in the interaction group were injected intraperitoneally with a calcineurin inhibitor FK506 (5 mg/kg, InvivoGen, USA) 30 min before isoflurane exposure.

Primary neuron culture and treatment

The primary cortical neurons were cultured in vitro for 5 days (C5) or 14 days (C14) and treated with 1.5% isoflurane in a gas mixture of 21% O₂, 5% CO₂, and balanced nitrogen for 6 hr as we described previously [9]. In the interaction experiments, neurons were pretreated with FK506 (1mM) 30 min before isoflurane exposure.

U251 cell culture and treatment

We used U251 human neuroglioma cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) in the succeeding experiments. Cells incubated in serum-free media were exposed to 2% isoflurane for 6 hr, performed similarly as our previous study [8]. The wild-type pcDNA3-6×Myc-mSTAT3 vector [10] or an antisense oligonucleotide (ASO) of STAT3 (HSS186131, Invitrogen, USA) were transfected into cells using lipofectamine^TM 2000 (Invitrogen, USA). An AxyPrep Endofree Plasmid Miniprep Kit (Axygen Biosciences, USA) was used to extract the plasmids from bacterial culture. After the transfection, cells were incubated at 37°C in a CO₂ incubator for 48 hr prior to isoflurane exposure. The untransfected cells without isoflurane exposure and the empty vector-transfected cells were set as the controls. In the interaction studies, cells were pretreated with a proteasome inhibitor MG-132 (30mM, Calbiochem, Germany) [11], FK506 (1mM) or a STAT3 inhibitor STA21 (30mM, Enzo Life Sciences, Switzerland) [12] 30 min before isoflurane exposure.

Mitochondria isolation

A mitochondria isolation kit for cultured cells (Thermo Scientific, USA) was used to evaluate the levels of mitochondria-located STAT3 and cytochrome C oxidase released from mitochondria into cytosol in U251 cells. After exposed to isoflurane, cells grown on 10-cm culture dishes (appropriately 1 ´ 10⁷ cells incubated in 9 ml of culture media per dish) were collected, centrifuged at 850×g for 2 min, and supernatant was then discarded. Subsequently, 800 μL of mitochondria isolation reagent A (with 100:1 protease inhibitors), provided in the kit, was added to each sample. The resulting mixtures were incubated on ice for exactly 2 min. Option A, which used the reagent-based method, was utilized in accordance with the manufacturer’s protocol.

Real-time PCR (RT-PCR)

U251 cells (n = 4 wells) grown in six-well plates were treated with lysis buffer provided by an Rneasy Mini Kit (QIAGEN, Hilden, Germany) immediately after isoflurane treatment. The frontal cortex was harvested, frozen in liquid nitrogen, and stored at −80°C before use. The tissues (n = 4) were ground in liquid nitrogen using a small mortar and then treated with lysis buffer. Retrotranscription and PCR analysis were carried out as we described previously [9]. STAT3, cyclin D1, Mcl-1, survivin, and Bcl-xl genes (3 repetitions per sample) were amplified using specific oligonucleotide primers (GenScript, USA; see table, Supplemental Digital Content , which is a table listing sequences of all primers used in this study). Housekeeping gene, b-actin, was used as internal standard. The no template sample was used as negative control.

Western blot analysis

The brain tissues were immediately removed and stored in -80℃ after anaesthesia . The frontal cortex were homogenized with lysis buffer containing 1% protease and phosphatase inhibitor cocktail (Thermo scientific, USA) on ice for 30 min. For the in vitro experiments, lysis buffer was added to six-well plates seeded with U251 cells or primary neurons immediately after 6-h isoflurane exposure. Protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA) and then each sample (25 μg) was separated by SDS-PAGE gel. Rabbit primary antibodies against STAT3 (1:1000; Epitomics, USA), Tyr705-phosphorylated STAT3 (pY705-STAT3, 1:1000; Epitomics), Bcl-xl (1:1000; Epitomics), survivin (1:1000; Epitomics), Mcl-1 (1:1000; Epitomics), cytochrome C (1:1000; Abcam, USA), MnSOD (1:1000; Abcam), activated caspase-3 (1:500; Bioworld), caspase-3 (1:500; Bioworld), Bim (1:1000; Epitomics), ubiquitin (1:1000; Thermo scientific, USA) and loading control GAPDH (1:2500; Bioworld) was used. GeneTools image analysis software was used for densitometric analysis of immunoblots.

Detection of intracellular ROS production

A fluorescent probe carboxy-H2DCFDA (Invitrogen, Eugene, USA) was used to assess the levels of intracellular reactive oxygen species (ROS). U251 cells grown in six-well plates were incubated with ROS detection solution for 30 min at 37 °C in the dark (n = 6 wells). Each sample was then washed thrice with Hank’s buffer and immediately observed under a Fluoview Fv10i confocal microscope (Olympus, Japan). A fluorescence spectrophotometer (F-7000, Hitachi, Japan) was also used to measure the cellular fluorescence of cells grown on 96-well plates (n = 6 wells).

Flow cytometric detection of apoptosis

A FITC Annexin V apoptosis detection kit (BD Biosciences, USA) was used to quantify the apoptotic rate in U251 cells (n = 6 wells). Cells grown on six-well plates were collected, washed twice with cold PBS, resuspended in 1 ´ binding buffer at a concentration of 1 ´ 10⁶ cells/ml, and then incubated with 5 μL FITC Annexin V as well as 5 μL PI for 15 min in the dark. The apoptotic rate was analyzed by a flow cytometer (BD Biosciences, USA) and expressed as the percentage of FITC Annexin V positive cells out of the total number of cells counted.

Immunofluorescence analysis

U251 cells or primary neurons grown on six-well plates were fixed in 4% PFA for 30 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% BSA for 1 hr (n = 3). Rabbit primary antibodies against STAT3 (1:100), Drebrin (1:100; Abcam) and Alexa Fluor 488 donkey anti-rabbit secondary antibodies (1:200; Molecular Probes, USA) were used for immunofluorescence analysis. Images (n = 3) were captured using a Fluoview Fv10i confocal microscope (Olympus, Japan) and analyzed using Image-Pro Plus analysis software (Media Cybernetics, Inc., Rockville, MD).

TUNEL staining

U251 cells or primary neurons grown on six-well plates were assayed through TUNEL staining by using the in situ cell death detection kit (Roche Applied Science, Germany) per the manufacturer’s protocol. After fixed in 4% PFA for 1 hr, cells were permeabilized with 0.1% Triton X-100 for 2 min on ice and stained with TUNEL for 1 hr at 37 °C in the dark. Representative images were obtained at 200´ magnification by using a TCS SP2 multiphoton confocal microscope (Leica, Germany).

Calcineurin activity assays

The activity of calcineurin was determined in the frontal cortex obtained from mice that were killed immediately after anesthesia or age-matched controls (n = 6). Fresh tissues were homogenized with lysis buffer containing 1% protease inhibitor (Thermo scientific, USA). A colorimetric calcineurin cellular activity assay kit (Calbiochem, Germany) was used to perform the test as we previously described [9].

Intellicage test

Mice assigned for behavior assessment were allowed to recover in 100% oxygen for 20 min after isoflurane exposure and reunited with their mothers. Mice were housed in a temperature-controlled (21± 1 °C) room with a 12-h light/dark cycle and free access to food and water. Four weeks after isoflurane exposure, place learning was performed in the IntelliCage system (NewBehavior Inc., Zurich, Switzerland) as we described before [8]. Briefly, the test started from an adaptation period of 6 days with ad libitum access to obtain water in all the corners. The least preferred corner for each mouse was chosen, and for the following 4 days, mice could obtain access to water only in their assigned correct corner. A brief air puff (1 bar/2 s) was triggered as a punishment if the mice visit an incorrect corner. When the test turned to the reversal learning phase, the opposite corner to the initial correct corner was reassigned as correct corner to each mouse for the next 4 days. Place learning was assessed as a ratio of number of visits in correct corner to all corners.

Statistics

Data are expressed as mean ± SD. The Western blot results, levels of mRNA and intracellular ROS, apoptotic rate, and activity of calcineurin were analyzed using one-way ANOVA for overall differences among groups followed by Bonferroni post hoc test for multiple comparisons. Unpaired Student’s t-test was used for comparisons between 2 groups. Repeated measures ANOVA followed by Bonferroni post hoc test was used to examine statistical comparisons among the four groups on correct visit ratios at different time point in Intellicage test. The sample sizes used were based on previous experience. All analyses were performed using SPSS 13.0 software (IBM Corporation, Armonk, USA). P < 0.05 (2-tailed) was considered as significant difference.

STAT3 levels were closely related with neural vulnerability to isoflurane

In previous study we have found that isoflurane exposure increased the activity of calcineurin, which specifically promotes STAT3 degradation. To determine whether calcineurin-mediated degradation of STAT3 is also time-restricted as the neurotoxicity of isoflurane to the brain [13], mice on postnatal day 7 (P7) and day 21 (P21) were anesthetized with 1.5% isoflurane for 6 hr. Our study showed that isoflurane markedly increased the level of activated caspase-3 in the frontal cortex of P7 mice (Figure 1A; n = 4, 1.50 ± 0.16 vs 0.91 ± 0.09, P < 0.001), but not in P21 mice (n = 4, 1.13 ± 0.19 vs 0.99 ± 0.08, P = 0.96). In line with this observation, we found that the protein level of STAT3 in the frontal cortex of P21 mice were obviously elevated (n = 4, 1.87 ± 0.15 vs 1.05 ± 0.04, P < 0.001) when compared with that in P7 mice, whereas, the calcineurin activity was significantly lower (Figure 1B; n = 6, 0.87 ± 0.16 vs 2.10 ± 0.34, P < 0.001). Moreover, there was a 2-fold increase in calcineurin activity in P7 mice after isoflurane exposure (n = 6, 4.40 ± 0.70 vs 2.10 ± 0.34, P < 0.001), but the increase of calcineurin activity in P21 mice was minimal (n = 6, P = 0.21).

The in vitro system exhibited a similar developmental regulation. Compared with the primary cortical neurons cultured for 5 days (C5), a significant upregulation in STAT3 protein level (Figure 1C; n = 4, 1.70 ± 0.13 vs 0.99 ± 0.08, P < 0.001) was observed in C14 neurons, at the instance when isoflurane did not induce apoptosis (P = 1.00). Moreover, a notable decline of STAT3 protein levels (n = 4, 0.62 ± 0.08 vs 0.99 ± 0.08, P = 0.002) that coincident with an increase in calcineurin activity (Figure 1D; n = 6, 3.85 ± 0.63 vs 1.87 ± 0.34, P < 0.001) after isoflurane exposure was observed in C5 but not in more mature C14 neurons (n = 4, 1.47 ± 0.13 vs 1.70 ± 0.13, P = 0.06 for STAT3 protein levels; n = 6, 0.89 ± 0.25 vs 0.54 ± 0.16, P = 0.76 for calcineurin activity). Collectively, these findings suggested that calcineurin-mediated STAT3 degradation may involved in neural vulnerability to isoflurane.

Isoflurane exposure impaired STAT3 survival pathway in U251 cells

To determine whether STAT3 is crucial in cellular vulnerability to isoflurane, we tested the role of ectopic STAT3 in isoflurane-induced apoptosis. We employed U251 human neuroglioma cells, which showed caspase-3 activation and ROS accumulation after a 6-h isoflurane exposure in our previous study. The present study showed that decreased protein levels of STAT3, pY705-STAT3 and its downstream survival targets were observed in U251 cells after isoflurane exposure (Figure 2A; n = 4, P < 0.001 for STAT3 and pY705-STAT3, P = 0.001 for Bcl-xl and survivin). Immunofluorescence showed that isoflurane exposure induced a 30% reduction of STAT3 staining (Figure 2B; n = 6, P < 0.001) in U251 cells, indicating that this cell model may be used for the study of STAT3 function in isoflurane-induced cytotoxicity in vitro.

In contrast with the reduction of STAT3 protein, the mRNA level of STAT3 in U251 cells was increased remarkably after 6-h isoflurane exposure (Figure 2C; n = 4, 2.34 ± 0.14 vs 1.04 ± 0.08, P < 0.001), indicating that isoflurane-induced STAT3 reduction may be caused by a post-transcriptional mechanism. Protein degradation is known to be achieved mostly through the ubiquitin (Ub)–proteasome pathway (UPP) [14]. To determine the role of STAT3 degradation in isoflurane, a proteasome inhibitor MG132 was introduced (Figure 2D). Interestingly, our data showed that isoflurane significantly decreased the levels of total ubiquitinated proteins in U251 cells (n = 4, P < 0.001). MG132 prevented this downregulation of ubiquitinated proteins (P < 0.001), so as STAT3 (P = 0.012). However, MG-132 coincubation did not lead to any further enhancement of protein ubiquitination (P = 0.14), indicating that isoflurane accelerated the degradation of ubiquitinated proteins, but not protein ubiquitination. Moreover, this MG132 effect enhanced the levels of Bim (P < 0.001), which is an essential initiator of apoptosis, with an increased caspase-3 activation (P < 0.001) after isoflurane treatment. In contrast, pretreatment with FK506, a calcineurin-specific inhibitor, prevented the degradation of STAT3 (Figure 2E, n = 4; P < 0.001) and cleavage in caspase-3 (P < 0.001), without significant influence to the decreased levels of total ubiquitinated proteins (P = 1.0) after isoflurane exposure. These findings highlighted the prominent importance of STAT3 in isoflurane-induced cytotoxicity.

Ectopic STAT3 protected cells from isoflurane-induced cytotoxicity in vitro

U251 cells were then transiently transfected with a wild-type STAT3 gene-containing vector STAT3-pcDNA3. Western blot analysis confirmed that the cells transfected with STAT3-pcDNA3 expressed high levels of STAT3 protein (Figure 3A; n = 4, P < 0.001) and its downstream anti-apoptotic factors, e.g. Mcl-1(P = 0.006) and survivin (P = 0.008), at 48 hr post-transfection. Real-time PCR showed that the transcript of STAT3 target genes e.g. cyclin D1 (Figure 3B; n = 4, P < 0.001), Mcl-1 (P < 0.001), survivin (P = 0.014) and Bcl-xl (P = 0.003) were remarkably upregulated, when compared to those of controls.

Next, we found that STAT3 overexpression was able to restore isoflurane-induced decline of STAT3 and its downstream anti-apoptotic proteins (Figure 3C; n = 4, P = 0.011 for Mcl-1, P = 0.006 for survivin) as well as mitochondria-located STAT3 (Figure 3D; n = 4, P < 0.001), which has been previously confirmed to play a major role in modulating mitochondrial respiration and antioxidative stress [15]. Simultaneously, cytochrome c that released from mitochondria into cytoplasm after isoflurane exposure was also prevented (Figure 3D; n = 4, P < 0.001).

The protective effects of STAT3 involved an antioxidative stress mechanism

ROS accumulation was reported to be a critical event in triggering the cytotoxicity of isoflurane [16]. To determine whether the cellular protective function of STAT3 involved an antioxidative stress mechanism, we used an antisense oligonucleotide (ASO) to knockdown STAT3 and a specific inhibitor STA21 to hinder STAT3 dimerization, together with the STAT3 overexpression assay. Western blot and immunofluorescence analyses showed that STAT3-ASO efficiently knocked down STAT3 expression in U251 cells (Figure 3A; n = 3, P < 0.001), meanwhile 6-h STA-21 treatment markedly inhibited the nuclear translocation of STAT3, without significant influence on its total protein level. STAT3 knockdown or its nuclear-translocation disruption resulted in a more than 10-fold increase of ROS in U251 cells after isoflurane exposure, when compared with that of controls (Figure 3B; n = 6, P < 0.001). The apoptotic rates after isoflurane exposure (Figure 3C & D; n = 6, 12.42 ± 2.23%) were obviously augmented in cells with STAT3 knockdown (24.78 ± 4.65%, P < 0.001) or STA21 treatment (19.10 ± 3.31%, P < 0.001). By contrast, STAT3 overexpression mitigated isoflurane-induced ROS accumulation (Figure 3B; n = 6, P < 0.001) and apoptosis (Figure 3C & D; n = 6, 7.51 ± 1.33%, P = 0.002). These protective effects of STAT3 were also confirmed by TUNEL staining (Figure 3E; 3 wells per group, 6 images per well) and Western blot analysis of cleaved caspase-3 (Figure 3F & G; n = 4). Notably, STAT3 disruption or overexpression did not affect the levels of ROS and apoptosis in isoflurane-untreated cells.

Mechanistically, the antioxidative effect of STAT3 was linked to its canonical activity as a nuclear transcription factor. We found the protein level of manganese-containing superoxide dismutase (MnSOD or SOD2), a critical cellular antioxidant enzyme and a direct target of STAT3 [17,18], was decreased (Figure 3F & G; n = 4, P < 0.001) after isoflurane exposure. The decline was further aggravated in cells with STAT3 disruption, but was restored by STAT3 overexpression (P < 0.001), suggesting that isoflurane-induced ROS accumulation is at least partially ascribed to impaired ROS scavenging.

Calcineurin inhibition alleviated the neurotoxicity of isoflurane

Since STAT3 levels were closely related with cellular vulnerability to isoflurane, we next examined whether calcineurin-specific inhibitor FK506 have a long-term protective effect against the neurotoxicity of isoflurane. STAT3 degradation (Figure 4A; n = 4, P < 0.001) and apoptosis (Figure 4B; 4 wells per group, 6 images per well) induced by isoflurane were prevented by FK506 pretreatment in C5 neurons. After exposed to isoflurane, C5 neurons were maintained in culture for 5 days and then dendritic spines were stained using a neuronal F-actin marker, drebrin. Some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction [19]. In the present study, we observed that neurons in the control group exhibited extensive and overlapping neurites. A significant reduction in the number of dendritic spines was detected in neurons exposed to isoflurane. Pretreatment with FK506 attenuated the isoflurane-induced loss of dendritic spines (Figure 4C; 4 wells per group, 6 images per well).

The post-transcriptional mechanism was also involved in isoflurane-induced STAT3 downregulation in the brain of P7 mice. The protein levels of STAT3 were not significantly decreased until isoflurane exposure for 4 hr (Figure 4D; n = 4, P < 0.001). In contrast, the mRNA level of STAT3 was increased remarkably after 1-h isoflurane exposure (Figure 4E; n = 4, P < 0.001). Intriguingly, the protein level of STAT3 was elevated initially, and the underlying mechanism remained to be determined.

Finally, we used the Intellicage system to assess the long-term effect of FK506 on isoflurane-induced cognitive dysfunction. As we described before, the cognitive impaired induced by isoflurane exposure was displayed in a more demanding task. During the first three days of learning phase, the isoflurane-treated mice did not show a significant disability in recognizing the correct corner. When test turned to the reversal learning phase, as compared to mice in the control group, the ratio for making a correct visit was approximately 20% lower in the isoflurane-treated mice (Figure 4F; n = 8, P < 0.01 at day 4-8), reflecting that they had not learned to drink successfully in their new assigned corner. Mice pretreated with FK506 showed significant improvement in spatial memory as compared to that in the isoflurane group (Figure 4F; n = 8, P < 0.01 at day 4-7, P < 0.05 at day 8).

In the present study, we propose the hypothesis that STAT3 signaling is involved in the cellular vulnerability to isoflurane. Our results showed that calcineurin-mediated degradation of STAT3 is closely related with the cytotoxicity of isoflurane in the brain of mice and cultured primary cortical neurons. Subsequently, we used the U251 cell line to determine the underlying mechanism and found that STAT3 is of particular importance in isoflurane-induced neurotoxicity. STAT3 disruption exaggerated, while STAT3 overexpression mitigated isoflurane-induced oxidative injury and apoptosis.

Calcineurin is a calcium and calmodulin dependent serine/threonine protein phosphatase that is highly expressed in brain [20]. Recently, it has also been shown that calcineurin promotes the degradation of STAT3 and controls the critical developmental death of neurons in newborn brain [21]. The present study found that the isoflurane exposure resulted in a 2-fold increase of calcineurin activity in the frontal cortex of P7 mice, but not in P21 mice. Moreover, the calcineurin activity of P21 mice was significantly lower than that of P7 mice, accompanied by a marked elevation in STAT3 protein levels in the frontal cortex. A similar developmental regulation of STAT3 was exhibited in primary cortical neurons cultured in vitro. Compared with C14 neurons, an upregulation of STAT3 parallel with a decline in calcineurin activity as well as with a decrease in the ability of isoflurane to trigger calcineurin activity were observed in more mature C14 neurons. This finding revealed that calcineurin-mediated STAT3 degradation may be closely related with developmental stage-related vulnerability of the brain to isoflurane stress. Given previous research showing that isoflurane induce neurodegeneration by activating inositol 1,4,5-trisphosphate (IP3) receptors [22,23], it is possible that the upstream trigger of calcineurin activity could be the excessive release of calcium from the endoplasmic reticulum.

The ubiquitin-proteasome pathway is the major intracellular non-lysozymal mechanism for degrading proteins and is involved in the regulation of a number of key signaling pathways. Proteins destined for degradation are conjugated by an ubiquitin chain and the ubiquitinated proteins are subsequently targeted for degradation by proteasome [24]. Previous studies have shown that ubiquitin metabolism affects growth inhibition of isoflurane in yeast [25,26]. A recent research revealed that isoflurane preconditioning decreased ubiquitin-conjugated protein aggregation and prevented free ubiquitin depletion in the CA1 region of hippocampal after global cerebral ischemia in mice [27]. It is noteworthy that we found isoflurane exposure triggered a 40% decrease in total ubiquitin-conjugated proteins levels in U251 cells, while when co-incubation with a proteasome inhibitor MG132, the ubiquitin-conjugated proteins levels were not increased, indicating that isoflurane accelerated the degradation of ubiquitin-conjugated proteins, but not promote ubiquitin conjugate to protein. In addition, our study found that MG132 result in aggravated apoptosis after isoflurane exposure, hence it is essential to alternative techniques to specifically regulate ubiquitination and proteasomal degradation of the key protein.

As a transcription factor, STAT3 is suggested to play an instructive role in brain development. STAT3-mediated cytokine signaling regulates gliogenesis as well as neurogenesis during brain development [28]. Neurons in newborn brain are under selection through a process of developmental death, depend on neurotrophin for survival. Overexpression of STAT3 completely attenuated neurotrophin dependency in this state, suggesting STAT3 is a key mediator of survival pathway that neurons acquire in this vulnerable period [29]. Moreover, it has been reported that calcineurin mediate STAT3 degradation via directing STAT3 to the E3 ubiquitin ligase complex [21]. To identify whether STAT3 is a key mediator of the cellular vulnerability to isoflurane, we performed gain- and loss-of-function studies in U251 cells. We confirmed that the levels of STAT3, p-STAT3 and its downstream anti-apoptotic proteins were significantly reduced after isoflurane exposure in U251 cells. In comparison with global blockade the degradation of ubiquitinated proteins by MG132 aggravated apoptosis after isoflurane exposure, specifically blocking the degradation of STAT3 by calcineurin-inhibitor FK506 prevented caspase-3 activation after isoflurane exposure, revealing the particular importance of STAT3 in isoflurane-induced cytotoxicity.

This notion was further confirmed by the result that STAT3 overexpression mitigated isoflurane-induced ROS accumulation, caspase-3 activation and apoptosis in U251 cells, whereas cells with STAT3 knockdown exhibited more sensitive to the toxicity of isoflurane, mimicking the cellular phenomenon of immature neurons with relatively lower levels of STAT3. Moreover, STAT3 knockdown or overexpression did not show significant influence on the basal levels of ROS and apoptotic rate in isoflurane-untreated cells, suggesting that STAT3 was functioning in a stress-related protective mechanism.

Oxidative injury was regarded as a key event in isoflurane-induced cell apoptosis [30,31]. Previous studies have reported both enhanced ROS production [32] and impaired ROS scavenging were involved in the ROS accumulation after isoflurane exposure [33]. It has been demonstrated that isoflurane impaired SOD but not catalase activity, while it remains unclear whether the impaired ROS scavenging capability is an initial event or a result of excess ROS accumulation. ROS is generated when electrons released from the mitochondrial electron transport chain (ETC) incompletely reduce O2, mainly form complexes I and III. STAT3 has recently been identified to colocalize with complex I and shown essential for optimal function of the electron transfer chain [15,34,35]. Mitochondrial STAT3 appeared to exert protection against certain diseases [18,36,37] by inhibiting oxidative stress or mitochondrial permeability transition pore (mPTP) opening [38,39]. Notably, the present study showed that mitochondria-located STAT3 was also decreased after isoflurane exposure and was able to be restored by STAT3 overexpression. Meanwhile, we found that inhibiting the nuclear translocation of STAT3 by STA21 greatly aggravated isoflurane-induced ROS accumulation and apoptosis as well as STAT3 knockdown. Thus, STAT3 overexpression may contribute to cytoprotection not only through its transcriptional activity such as upregulating anti-oxidative and anti-apoptotic proteins, but also through its mitochondrial function that reducing ROS generation.

Taken together, the present study demonstrated that STAT3-mediated survival pathway as well as its anti-oxidative effect were impaired after isoflurane exposure and contributed to the cellular vulnerability to isoflurane. The connections between calcineurin-mediated STAT3 degradation and neuronal vulnerability to isoflurane reported here point out novel therapeutic target for the prevention and treatment of isoflurane-induced neurotoxicity, not only to the immature brain, but also the aged brain, in which a marked decrease of STAT3 was also reported [40]. In addition to STAT3, a marked decrease in the amount of ubiquitin-conjugated proteins protein was found after isoflurane exposure. Continued analysis of additional proteins relevant to isoflurane-response degradation is imperative in future studies.

Funding

This study was supported by the National Natural Science Foundation of China (81600932, 81971044, 81730033, 81771142), China Postdoctoral Science Foundation（2017M621730）, Jiangsu Planned Projects for Postdoctoral Research Funds（1701006A）, and Nanjing Medical Science and technique Development Foundation（QRX17137）.

Conflict of Interest

The authors have no relevant financial or non-financial interests to disclose.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The software application and custom code are available from the corresponding author upon reasonable request.

Authors’ contributions

All authors contributed and critically reviewed and approved the manuscript. Q.G., Z.M., X.G., H.W. and Y.Y. conceived and designed the experiments. Y.Y., S.S., Y.L., W.Z., J.Z., Y.S., and J.H. performed the experiments. Y.Y., S.S., and X.Y. analysed the data. Z.M., Q.G., X.G., H.W. and Y.Y. prepared the manuscript.

Ethics approval

Animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Review Board for Animal Studies of Nanjing Drum Tower Hospital.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW (2011) Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114 (3):578-587. doi:10.1097/ALN.0b013e3182084a70
Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H (2010) Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112 (6):1325-1334. doi:10.1097/ALN.0b013e3181d94da5
Stratmann G, Sall JW, May LD, Bell JS, Magnusson KR, Rau V, Visrodia KH, Alvi RS, Ku B, Lee MT, Dai R (2009) Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110 (4):834-848. doi:10.1097/ALN.0b013e31819c463d
Zhu C, Gao J, Karlsson N, Li Q, Zhang Y, Huang Z, Li H, Kuhn HG, Blomgren K (2010) Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents. J Cereb Blood Flow Metab 30 (5):1017-1030. doi:10.1038/jcbfm.2009.274 jcbfm2009274 [pii]
Hofacer RD, Deng M, Ward CG, Joseph B, Hughes EA, Jiang C, Danzer SC, Loepke AW (2013) Cell age-specific vulnerability of neurons to anesthetic toxicity. Ann Neurol 73 (6):695-704. doi:10.1002/ana.23892
Ng YP, Cheung ZH, Ip NY (2006) STAT3 as a downstream mediator of Trk signaling and functions. The Journal of biological chemistry 281 (23):15636-15644. doi:10.1074/jbc.M601863200
Dziennis S, Alkayed NJ (2008) Role of signal transducer and activator of transcription 3 in neuronal survival and regeneration. Rev Neurosci 19 (4-5):341-361
Yang Y, Chen X, Min H, Song S, Zhang J, Fan S, Yi L, Wang H, Gu X, Ma Z, Gao Q (2017) Persistent mitoKATP Activation Is Involved in the Isoflurane-induced Cytotoxicity. Molecular neurobiology 54 (2):1101-1110. doi:10.1007/s12035-016-9710-z
Yang Y, Song S, Min H, Chen X, Gao Q (2016) STAT3 degradation mediated by calcineurin involved in the neurotoxicity of isoflurane. Neuroreport 27 (2):124-130. doi:10.1097/WNR.0000000000000509
Nie Y, Erion DM, Yuan Z, Dietrich M, Shulman GI, Horvath TL, Gao Q (2009) STAT3 inhibition of gluconeogenesis is downregulated by SirT1. Nat Cell Biol 11 (4):492-500. doi:10.1038/ncb1857 ncb1857 [pii]
Selvendiran K, Koga H, Ueno T, Yoshida T, Maeyama M, Torimura T, Yano H, Kojiro M, Sata M (2006) Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids. Cancer research 66 (9):4826-4834. doi:10.1158/0008-5472.CAN-05-4062
Song H, Wang R, Wang S, Lin J (2005) A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl Acad Sci U S A 102 (13):4700-4705. doi:0409894102 [pii] 10.1073/pnas.0409894102
Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V (2005) Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135 (3):815-827. doi:S0306-4522(05)00308-8 [pii] 10.1016/j.neuroscience.2005.03.064
Lecker SH, Goldberg AL, Mitch WE (2006) Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J Am Soc Nephrol 17 (7):1807-1819. doi:ASN.2006010083 [pii] 10.1681/ASN.2006010083
Wegrzyn J, Potla R, Chwae YJ, Sepuri NB, Zhang Q, Koeck T, Derecka M, Szczepanek K, Szelag M, Gornicka A, Moh A, Moghaddas S, Chen Q, Bobbili S, Cichy J, Dulak J, Baker DP, Wolfman A, Stuehr D, Hassan MO, Fu XY, Avadhani N, Drake JI, Fawcett P, Lesnefsky EJ, Larner AC (2009) Function of mitochondrial Stat3 in cellular respiration. Science 323 (5915):793-797. doi:10.1126/science.1164551
Boscolo A, Starr JA, Sanchez V, Lunardi N, DiGruccio MR, Ori C, Erisir A, Trimmer P, Bennett J, Jevtovic-Todorovic V (2012) The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: the importance of free oxygen radicals and mitochondrial integrity. Neurobiol Dis 45 (3):1031-1041. doi:10.1016/j.nbd.2011.12.022 S0969-9961(11)00397-4 [pii]
Guo Z, Jiang H, Xu X, Duan W, Mattson MP (2008) Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization. J Biol Chem 283 (3):1754-1763. doi:M703753200 [pii] 10.1074/jbc.M703753200
Negoro S, Kunisada K, Fujio Y, Funamoto M, Darville MI, Eizirik DL, Osugi T, Izumi M, Oshima Y, Nakaoka Y, Hirota H, Kishimoto T, Yamauchi-Takihara K (2001) Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase. Circulation 104 (9):979-981
Sekino Y, Kojima N, Shirao T (2007) Role of actin cytoskeleton in dendritic spine morphogenesis. Neurochemistry international 51 (2-4):92-104. doi:10.1016/j.neuint.2007.04.029
Jiang H, Xiong F, Kong S, Ogawa T, Kobayashi M, Liu JO (1997) Distinct tissue and cellular distribution of two major isoforms of calcineurin. Molecular immunology 34 (8-9):663-669
Murase S (2013) Signal transducer and activator of transcription 3 (STAT3) degradation by proteasome controls a developmental switch in neurotrophin dependence. The Journal of biological chemistry 288 (28):20151-20161. doi:10.1074/jbc.M113.470583
Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, Wang S, Eckenhoff RG (2008) The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5-trisphosphate receptors. Anesthesiology 108 (2):251-260. doi:10.1097/01.anes.0000299435.59242.0e
Zhao Y, Liang G, Chen Q, Joseph DJ, Meng Q, Eckenhoff RG, Eckenhoff MF, Wei H (2010) Anesthetic-induced neurodegeneration mediated via inositol 1,4,5-trisphosphate receptors. The Journal of pharmacology and experimental therapeutics 333 (1):14-22. doi:10.1124/jpet.109.161562
Roos-Mattjus P, Sistonen L (2004) The ubiquitin-proteasome pathway. Annals of medicine 36 (4):285-295
Wolfe D, Reiner T, Keeley JL, Pizzini M, Keil RL (1999) Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast. Molecular and cellular biology 19 (12):8254-8262
Keil RL, Wolfe D, Reiner T, Peterson CJ, Riley JL (1996) Molecular genetic analysis of volatile-anesthetic action. Molecular and cellular biology 16 (7):3446-3453
Zhang HP, Yuan LB, Zhao RN, Tong L, Ma R, Dong HL, Xiong L (2010) Isoflurane preconditioning induces neuroprotection by attenuating ubiquitin-conjugated protein aggregation in a mouse model of transient global cerebral ischemia. Anesthesia and analgesia 111 (2):506-514. doi:10.1213/ANE.0b013e3181e45519
Deverman BE, Patterson PH (2009) Cytokines and CNS development. Neuron 64 (1):61-78. doi:10.1016/j.neuron.2009.09.002
Murase S, Kim E, Lin L, Hoffman DA, McKay RD (2012) Loss of signal transducer and activator of transcription 3 (STAT3) signaling during elevated activity causes vulnerability in hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 32 (44):15511-15520. doi:10.1523/JNEUROSCI.2940-12.2012
Zhang Y, Dong Y, Wu X, Lu Y, Xu Z, Knapp A, Yue Y, Xu T, Xie Z (2010) The mitochondrial pathway of anesthetic isoflurane-induced apoptosis. The Journal of biological chemistry 285 (6):4025-4037. doi:10.1074/jbc.M109.065664
Cheng B, Zhang Y, Wang A, Dong Y, Xie Z (2015) Vitamin C Attenuates Isoflurane-Induced Caspase-3 Activation and Cognitive Impairment. Molecular neurobiology 52 (3):1580-1589. doi:10.1007/s12035-014-8959-3
Hirata N, Shim YH, Pravdic D, Lohr NL, Pratt PF, Jr., Weihrauch D, Kersten JR, Warltier DC, Bosnjak ZJ, Bienengraeber M (2011) Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: implications for preconditioning. Anesthesiology 115 (3):531-540. doi:10.1097/ALN.0b013e31822a2316
Boscolo A, Milanovic D, Starr JA, Sanchez V, Oklopcic A, Moy L, Ori CC, Erisir A, Jevtovic-Todorovic V (2013) Early exposure to general anesthesia disturbs mitochondrial fission and fusion in the developing rat brain. Anesthesiology 118 (5):1086-1097. doi:10.1097/ALN.0b013e318289bc9b
Elschami M, Scherr M, Philippens B, Gerardy-Schahn R (2013) Reduction of STAT3 expression induces mitochondrial dysfunction and autophagy in cardiac HL-1 cells. Eur J Cell Biol 92 (1):21-29. doi:10.1016/j.ejcb.2012.09.002 S0171-9335(12)00158-6 [pii]
Bernier M, Paul RK, Martin-Montalvo A, Scheibye-Knudsen M, Song S, He HJ, Armour SM, Hubbard BP, Bohr VA, Wang L, Zong Y, Sinclair DA, de Cabo R (2011) Negative regulation of STAT3 protein-mediated cellular respiration by SIRT1 protein. J Biol Chem 286 (22):19270-19279. doi:10.1074/jbc.M110.200311 M110.200311 [pii]
Jung JE, Kim GS, Narasimhan P, Song YS, Chan PH (2009) Regulation of Mn-superoxide dismutase activity and neuroprotection by STAT3 in mice after cerebral ischemia. J Neurosci 29 (21):7003-7014. doi:10.1523/JNEUROSCI.1110-09.2009 29/21/7003 [pii]
Sarafian TA, Montes C, Imura T, Qi J, Coppola G, Geschwind DH, Sofroniew MV (2010) Disruption of astrocyte STAT3 signaling decreases mitochondrial function and increases oxidative stress in vitro. PLoS One 5 (3):e9532. doi:10.1371/journal.pone.0009532
Boengler K, Hilfiker-Kleiner D, Heusch G, Schulz R (2010) Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion. Basic Res Cardiol 105 (6):771-785. doi:10.1007/s00395-010-0124-1
Di Lisa F, Bernardi P (2009) A CaPful of mechanisms regulating the mitochondrial permeability transition. J Mol Cell Cardiol 46 (6):775-780. doi:10.1016/j.yjmcc.2009.03.006 S0022-2828(09)00112-6 [pii]
De-Fraja C, Conti L, Govoni S, Battaini F, Cattaneo E (2000) STAT signalling in the mature and aging brain. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 18 (4-5):439-446

SupplementaryMaterial.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

STAT3 Involved in Cellular Vulnerability to Isoflurane

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Supplementary Files

Status:

Version 1