HindawiBioMed Research InternationalVolume 2020, Article ID 8868839, 8 pages ArticleKetamine and Propofol Protect Neuron Cells from OxygenGlucose Deprivation-Induced Injury through SAPK/JNKSignalling PathwayAihua Qi, Yiyun Cao, and Aizhong WangShanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, ChinaCorrespondence should be addressed to Aizhong Wang; [email protected] 24 September 2020; Revised 5 November 2020; Accepted 18 November 2020; Published 16 December 2020Academic Editor: Junyan LiuCopyright 2020 Aihua Qi et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Ketamine and propofol are commonly used anaesthetic reagents. Recent research revealed that ketamine and propofol have animportant role in cell survival. However, it remains unknown whether they affect the outcome of hypoxic-ischemic brain injury.To address this issue, we in this study investigated the effects of ketamine and propofol on the survival and proliferation ofneuronal PC12 cells after exposure to oxygen-glucose deprivation- (OGD-) induced injury. PC12 cells were maintained under a3-dimensional (3D) culture system to mimic a real physiological microenvironment. The cell injury was induced by 5% CO2and 95% N2 for a different time point. MTT assay was used for the cell proliferation assay. The cell apoptosis was evaluated byannexin V and propidium iodide (PI) labeling, immunofluorescence staining, transmission electron microscopy (TEM), flowcytometry, and Western blot, respectively. Our results showed that PC12 cell apoptosis was significantly increased for up to 70%after the cells were treated with OGD for 24 hours and reduced to baseline at the 72-hour time point. However, pretreatmentwith ketamine and propofol significantly protected the cells from OGD-induced cell apoptosis, as evidenced by more expressionof antiapoptotic Bcl-2 and lower expression of proapoptotic cleaved caspase-3, phosphor-SAPK/JNK, and phosphor-c-Jun thanthose of untreated control cells. Thus, we conclude that ketamine and propofol protected PC12 cells from OGD-induced cellapoptosis, at least partially through the SAPK/JNK signalling pathway.1. IntroductionIschemia and hypoxia often occur during surgical operationand can induce severe damage to multiple organs in patientsunder general anaesthesia. The brain is the mostly affectedorgan. Severe damage to neuron cells is observed during perioperative hypoxia-ischemia [1, 2]. Thus, it is essential to seekan effective approach in the prevention and treatment ofperioperative hypoxic-ischemic brain injury. Ketamine(KTM) and propofol (PPF) are commonly used anaesthesiareagents during surgery [3]. However, recent studies haveshown that ketamine and propofol have a protective functionin oxygen-glucose deprivation- (OGD-) induced neuroninjury [2, 4]. Recent reports suggested that ketamine and propofol may protect microglia against OGD/reoxygenationinduced cell death by binding to Toll-like receptor 4(TLR4) [5]. It was also reported that propofol can protectendothelial cells during intermittent hypoxia/reoxygenationthrough suppressing p38MAPK/NF-κB signalling [6].It is documented that SAPK/JNK signalling is involved inthe initiation of neuron cell activation and apoptosis [7, 8].SAPK/JNK inhibitor SP6000125 successfully reduced cytokine production in low-intensity microwave-exposed RAW264.7 macrophages [9]. However, little is known about therole and underlying molecular mechanisms of SAPK/JNKsignalling in ketamine and propofol-mediated neuron protection. It was documented that the 3-dimensional culture(3D) system mimics the physiological environment muchbetter than the 2-dimensional (2D) culture system. However,it is not well defined whether the 3D system improves neuroncell survival than the 2D system under oxidative stress condition. To address this issue, we in this study maintained neuron cells in both 3D and 2D culture systems. Then, the cellswere treated with OGD alone or in conjunction with

2ketamine and propofol for the indicated time points. Ourresults revealed a better cell survival and proliferation of neuron cells cultured in the 3D system. In addition, we observedthat ketamine and propofol pretreatment significantlyimproved neuron survival and induced more extendedmonopolar neurite processes after OGD exposure. Theresults indicated that the 3D cell culture system is beneficialin promoting neuron cell proliferation, survival, and differentiation. It provided significant molecular bases for the roleof ketamine and propofol in preventing OGD-mediated neuron injury and has important implication in the treatment ofischemia and hypoxia during surgical operation.2. Materials and Methods2.1. 3D Cell Culture and Treatment. The PC12 cell line(ATCC, Manassas, VA) was seeded in a 3D system and maintained in RPMI1640 medium supplied with 10% fetal bovineserum (FBS) and nerve growth factor (NGF). Before experimental intervention, the cells were starved in RPMI1640medium without FBS. The cell injury was induced by maintaining cells in culture medium deficient glucose and undergas containing 0.5% O2, 5% CO2, and 95% N2. The cells werepretreated with 10 mM ketamine and 10 μM propofol 1minute prior to hypoxic exposure.2.2. Cell Viability Assay. Cell viability was measured by theMTT assay according to the manufacturer instruction.Briefly, the cell supernatants were collected after PC12 cellswere exposed to OGD for 0, 6, 12, 24, 48, or 72 hours. Products in cell supernatants were measured by an enzyme-linkedimmunosorbent assay, and absorbance density (OD) wasdetected at 570 nm.2.3. Electron Microscopy Analysis. 1 107 cells were plated onculture dishes. After 10 days of cell culture, the medium wasremoved, and cells were fixed with 2.5% glutaraldehyde for 2hours. Then, the cells were washed twice with PBS, dehydrated using ethanol gradient, embedded, solidified, and cutinto ultrathin sections. The sections were finally viewedunder electron microscopy.2.4. Apoptosis Assay. The cells were digested with 0.2% trypsin to generate a single cell suspension, washed with PBS, andtreated with annexin V and PI. The cells undergoing necrosisand apoptosis were visualized under a fluorescence microscope in a blinded manner (5 regions/group) and quantifiedby flow cytometry.2.5. Western Blot Analysis. Cell lysates were obtained fromthe treated cells and dissolved in 10% acrylamide/bis gelcontaining 0.1% SDS. After gel electrophoresis, protein ingels was transferred into nitrocellulose membranes. Theblots were incubated with primary antibody and followedby incubation with HRP-conjugated second antibody.After washing with TBST buffer, the blots were developedwith ECL substrate solution. Protein bands were quantifiedusing densitometric analysis software (Bio-Rad).BioMed Research International2.6. Statistical Analysis. All data was presented as means sem, and statistical analysis was performed using GraphPadPrism 5 software (GraphPad, USA). A value of p 0:05 wasconsidered statistically significantly different.3. Results3.1. 3D-Matrigel Culture Promoted Neurite Outgrowth ofPC12 Cells. Because the current 2D culture system is limitedfor low efficiency in cell proliferation and differentiation, wein this study cultured neuron cells, PC12, in the 3D system tomimic the cell growth environment in vivo. In parallelculture to the 2D system, we observed more cell growth inthe 3D system than in the 2D system, as evidenced by theincreased cell number and clusters 48 hours after the cellswere cultured in the 3D system, compared to the cells inthe 2D system (Figure 1(a)). H&E staining showed theincreased monopolar neurites of cells in the 3D system(Figure 1(b)). Therefore, we conclude that the 3D culturesystem significantly improved neuron cell growth anddifferentiation.3.2. OGD Induced PC12 Cell Apoptosis That Was Reversed byPretreatment with Ketamine and Propofol. To investigate theeffects of ketamine and propofol on the survival of PC12 cells,we at first established a neuron cell injury model, respectively, in 2D and 3D systems by exposing to OGD. Ourresults showed that OGD effectively reduced cell viability ina time-dependent manner. However, there was more cellviability cultured in the 3D system than in the 2D system,indicating the beneficial role of the 3D system in neuron cellculture (Figure 2(a)). Further electron microscopy showedthat OGD treatment resulted in lower growth of cell neurites(Figure 2(b)) and induced more cell shrinkage, indicative ofcell apoptosis (Figure 2(c)).To further define whether ketamine (KTM) and propofol(PPF) can reverse OGD-induced cell apoptosis, we pretreatedthe neuron cells with ketamine or propofol prior to OGDexposure. The results showed that ketamine or propofolpretreatment effectively suppressed the OGD-inducedannexin V PI apoptotic cells (Figure 3(a)). There wasaround 2-fold reduction of apoptotic cells pretreated withketamine or propofol, compared to the OGD treatment alone(Figure 3(b), p 0:05 and p 0:01, n 3). Accordingly, thecell viability was significantly increased in the ketamine(Figure 3(c))- and propofol (Figure 3(d))- pretreated cellsunder a concentration-dependent manner, compared to theuntreated cells. Consistent with the finding in Figure 2(a),we also observed better cell viability in the 3D system thanin the 2D system when the cells were pretreated withketamine and propofol. Thus, ketamine and propofol werebeneficial in preventing OGD-induced neuron cell apoptosisin vitro.3.3. Pretreatment with Ketamine and Propofol Reduced theExpression of p-SAPK/JNK and Cleaved Caspase-3 in PC12Cells. Whether the reduced cell apoptosis by ketamine or propofol pretreatment is associated with the altered expressionof apoptosis relevant molecules was further investigated.

BioMed Research International324 hrs48 hrs2D culture3D culture(a)H&E staining(b)Figure 1: 3D-matrigel culture promoted neurite outgrowth of PC12 cells. (a) PC12 neuron cells were cultured in 2D and 3D culture systems,respectively, for 24 hrs and 48 hrs. The pictures were taken under a contrast microscope at a magnification of 200x. (b) Representative H&Estaining of PC12 cells cultured in the 3D system. Representative photograph was shown.Our further analysis by Western blot showed that OGDsignificantly increased the expression of p-SAPK/JNK andp-c-Jun. However, pretreatment with ketamine and propofol effectively reversed the OGD-induced upregulation ofp-SAPK/JNK and p-c-Jun (Figures 4(a) and 4(b)). In addition, we observed the increased expression of apoptoticcleaved caspase-3 but reduced expression of antiapoptoticBcl-2 in the OGD-treated cells. However, ketamine andpropofol pretreatment effectively reversed the altered expression of cleaved caspase-3 and Bcl-2 (Figures 4(c) and 4(d)).The results were also further confirmed by immunostaininganalysis, in which ketamine and propofol pretreatment significantly reversed the OGD-induced upregulation of cleavedcaspase-3 in the neuron cells (Figure 4(e)). Therefore,ketamine and propofol both exerted antiapoptotic functionthrough the SAPK/JNK signalling pathway in the OGDtreated neuron cells.4. DiscussionHypoxemia is a common cause of acute neuron injury. In theinjured neuron area, massive inflammation and a largeamount of proinflammatory cytokines are expressed, due tohypoxemia-induced oxidative stress and cell damage [10,11]. Treatment with anti-inflammatory reagents and neurotrophic factors becomes essential in the prevention and treatment of acute neuron injury in vivo [10, 12]. Because it waspreviously reported that 3D in vitro culture promoted moreefficient neuronal differentiation of stem cells, inducing moreperipheral nerve regeneration than 2D in vitro culture [13],we in this study compared neuron cell growth in the 3Dsystem and 2D system. After 3 days in culture, we observedhigher cell growth and viability in the 3D cell culture systemthan the 2D cell culture system. In addition, more monopolarneurites were observed in the 3D system, indicating thebeneficial role of the 3D culture model in improving neuronproliferation and differentiation.By using this system, we further investigated the effects ofketamine and propofol in OGD-induced neuron injury. Ourresults showed that OGD can effectively induce acute neuroncell injury, as indicated by the higher number of cell apoptosis and lower cell survival starting 6 hours after OGD exposure. However, we observed a significantly reduced neuroncell apoptosis and death after neuron cells were cotreated

4BioMed Research International150125Viability (%)100⁎⁎75⁎⁎502506122448Time (hrs)722D3D(a)Electron microscope1500 5000 OGDControl200 (b)0OGD(c)Figure 2: OGD induced PC12 cell apoptosis. (a) PC12 cells cultured in 2D and 3D systems were exposed to OGD for a different time point.The cell viability was measured by MTT assay. Data was presented as the mean standard error, n 3. p 0:05 vs. the cells at 0 time point.(b) PC12 cells cultured in the 3D system were treated with OGD or untreated for 48 hrs, and the cell morphology was identified by an electronmicroscope at magnification of 200x, 1500x, and 5000x. The OGD-treated cells had low neurite growth. (c) The apoptotic PC12 cells under anelectron microscope. Representative photograph was shown.

BioMed Research International5150⁎#100100##⁎500KTM (mM):Viability (%)Viability 1%PPF/OGD103B21.4%102102102PI GD103##0PPF 10.2%B22.1%B395.5%B41.9%100101102103Annexin V(c)Annexin V cells (%)40⁎30⁎⁎201000KTMPPFOGD(d)Figure 3: Ketamine and propofol pretreatment improved the PC12 cell survival and suppressed cell apoptosis. PC12 cells cultured in 2D and3D systems were pretreated with different concentrations of ketamine (KTM) (a) and propofol (PPF) (b) for 1 minute before OGD exposure.The treated cells were analyzed for cell viability by MTT at 48 hours after OGD exposure. Data was presented as the mean standard error,n 3. #p 0:05 vs. the cells treated with 0/OGD, p 0:05 vs. the cells cultured in the 2D system. (c) The cells were pretreated with 10 mMketamine and 10 μM propofol 1 minute before RGD treatment for 48 hours. Flow cytometry analysis for apoptotic cells. The annexin V cellswere identified as apoptotic cells. One representative dot plot of three independent experiments was shown. (d) Quantitative analysis ofapoptotic cells in the treated cells. p 0:05, p 0:01 vs. the cells treated with 0/OGD, n 3.with both OGD and ketamine or propofol, compared to thecells treated with OGD alone. The results indicated the therapeutic role of ketamine and propofol in suppressinghypoxemia-induced neuron cell injury. Ketamine and propofol are commonly used anaesthesia reagents in clinical practice. Recent study revealed that both ketamine and propofol

6BioMed Research InternationalOGD00KTM2.0PPF54kDap-SAPK/JNKRelative lative mber of cleaved caspase-3 cells/field(c)80604020⁎⁎KTMPPF000OGDOGDCleaved caspase-3Bcl-2(d)(e)Figure 4: Pretreatment with ketamine and propofol reduced the expression of p-SAPK/JNK and cleaved caspase-3 in PC12 cells. (a) Theexpression of p-SAPK/JNK and p-Jun in the treated cells was analyzed by Western blot analysis. One representative blot was shown. (b)Quantitative analysis of p-SAPK/JNK and p-Jun expression in the treated cells by ImageJ software; data was presented as the ratio ofphosphorylated protein to total protein in each lane. (c) Representative blot for the expression of caspase-3 and Bcl-2 in the treated cells; (d)quantitative analysis of the cleaved caspase-3 and Bcl-2 expression by ImageJ software. Data was presented as the ratio of cleaved caspase-3to procaspase-3, or the ratio of Bcl-2 to internal control GAPDH in each lane. n 3. p 0:05, p 0:01 vs. the cells treated with 0/OGDcontrol. (e) The expression of cleaved caspase-3 in the treated cells was detected by immunostaining. The positive cells were quantitativelyanalyzed, and data was presented as the mean number of cleaved caspase-3 cells per field standard error. n 6 field/treatment.

BioMed Research Internationalhave anti-inflammatory function in glial cells [14, 15]. Basedon our results, ketamine and propofol should be consideredpromising adjuvants in the treatment of hypoxemiainduced acute neuron injury and suppressing local inflammation. More studies in the animal model will be performedto define their therapeutic benefits in the future.However, it is still not clear how ketamine and propofolexert anti-inflammatory function and promote neuronsurvival in this study. Previously reported results showed thatketamine and propofol can suppress LPS-induced upregulation of IL-1beta and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation in glia cells and brains [15,16]. IL-1beta is a proinflammatory cytokine and criticallyparticipates in cell activation and death of macrophage pyroptosis [17, 18]. TNF-alpha, tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), and IFN-gamma are apotent inducer of neuron, microglia, and astroglia apoptosisafter hypoxia-ischemia and inflammation [1, 19]. However,inhibition of the JAK/STAT1 pathway in IFN-gammainjected animals effectively reduced microglial activationand microglia-mediated neuroinflammation and neurogenicinjury [20]. The results displayed the critical role of thesecytokines in neuronal inflammation and injury. Ketamineand propofol may protect neuron cells from oxidativestress-induced injury through suppressing these signallingpathways and production of proinflammatory cytokines.Our further study in vitro also revealed the increasedactivation of SAPK/JNK signalling in the OGD-treatedneuron cells, but the activated SAPK/JNK signalling wassignificantly suppressed in the OGD/ketamine- or OGD/propofol-cotreated cells, indicating the involvement ofSAPK/JNK signalling in ketamine and propofol-mediatedneuron survival. It was previously reported that SAPK/JNKsignalling was critically involved in cell activation and apoptosis under oxidative stress, because SAPK/JNK inhibitorSP600125 can reverse porcine epidemic diarrhea virusinduced Vero cell apoptosis [8]. In addition, SP6000125treatment can effectively suppress cytokine production inlow-intensity microwave-exposed macrophages. Theseresults indicated the important role of SAPK/JNK signallingin cell activation, apoptosis, and inflammation [9]. Therefore,SAPK/JNK signalling was associated with neuron activationand apoptosis. Ketamine and propofol may prevent neuroncells from OGD-induced acute injury and neuron cellapoptosis through suppressing the SAPK/JNK signallingpathway.In summary, the results indicated that 3D culture significantly improved neuron cell growth and survival. Ketamineand propofol exerted significant activities against OGDinduced neuron cell injury. The protective effects are at leastpartially associated with suppressing the SAPK/JNK signalling pathway. Therefore, ketamine and propofol not onlyinduced anaesthesia and sedation but also had neuroprotective effects.Data AvailabilityAll data, models, and code generated or used during thestudy appear in the submitted article.7Conflicts of InterestThe authors declare that they have no conflicts of interest.References[1] A. Kichev, C. I. Rousset, A. A. Baburamani et al., “Tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) signaling and cell death in the immature central nervous systemafter hypoxia-ischemia and inflammation,” Journal of Biological Chemistry, vol. 289, no. 13, pp. 9430–9439, 2014.[2] B. Sun, H. Ou, F. Ren et al., “Propofol inhibited autophagythrough Ca2 /CaMKKβ/AMPK/mTOR pathway in OGD/Rinduced neuron injury,” Molecular Medicine, vol. 24, no. 1,p. 58, 2018.[3] H. Gulec, S. Sahin, E. Ozayar, S. Degerli, F. Bercin, andO. 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SAPK/JNK inhibitor SP6000125 successfully reduced cyto-kine production in low-intensity microwave-exposed RAW 264.7 macrophages [9]. However, little is known about the role and underlying molecular mechanisms of SAPK/JNK signalling in ketamine and propofol-mediated neuron pro-tection. It was documented that the 3-dimensional culture