Environmental Toxicology and Pharmacology 42 (2016) 85–91
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Lipid emulsion reverses bupivacaine-induced apoptosis of h9c2 cardiomyocytes: PI3K/Akt/GSK-3 signaling pathway Danni Lv a , Zhixia Bai b , Libin Yang c , Xiaohui Li a , Xuexin Chen b,∗ a b c
Ning Xia Medical University, Yin Chuan, China Department of Anesthesiology, Tumor Hospital, General Hospital of Ning Xia Medical University, Yin Chuan, China Department of Anesthesiology, First People’s Hospital, Shi Zui Shan, China
a r t i c l e
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Article history: Received 2 July 2015 Received in revised form 2 January 2016 Accepted 5 January 2016 Available online 9 January 2016 Keywords: Lipid emulsion Bupivacaine Cardiac cytotoxicity mPTP PI3K/Akt/GSK-3
a b s t r a c t Some findings have suggested that the rescue of bupivacaine (BPV)-induced cardiotoxicity by lipid emulsion (LE) is associated with inhibition of mitochondrial permeability transition pore (mPTP). However, the mechanism of this rescue action is not clearly known. In this study, the roles of phosphoinositide 3kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) in the molecular mechanism of LE-induced protection and its relationship with mPTP were explored. h9c2 cardiomyocytes were randomly divided into several groups: control, BPV, LE, BPV + LE. To study the effect of LE on mPTP, atractyloside (Atr, 20 M, mPTP opener) and cyclosporine A (CsA, 10 M, mPTP blocker) were used. To unravel whether LE protects heart through the PI3K/Akt/GSK-3 signaling pathway, cells were treated with LY294002 (LY, 30 M, PI3K blocker) or TWS119 (TWS 10 M, GSK-3 blocker). Later mitochondrial respiratory chain complexes, apoptosis, opening of mPTP and phosphorylation levels of Akt/GSK-3 were measured. LE significantly improved the mitochondrial functions in h9c2 cardiomyocytes. LE reversed the BPV-induced apoptosis and the opening of mPTP. The effect of LE was not only enhanced by CsA and TWS, but also abolished by Atr and LY. LE also increased the phosphorylation levels of Akt and GSK-3. These results suggested that LE can reverse the apoptosis in cardiomyocytes by BPV and a mechanism of its action is inhibition of mPTP opening through the PI3K/Akt/GSK-3 signaling pathway. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, researchers have fully demonstrated that lipid emulsion (LE) plays an important role in the treatment of bupivacaine (BPV)-induced cardiac toxicity (Weinberg et al., 2006; Litz et al., 2008; Lin and Aronson, 2010), but the exact mechanism is still not clear. Fatty acid oxidation is necessary for successful rescue of BPV-induced cardiotoxicity by LE (Partownavid et al., 2012; Weinberg, 2012a,b). This rescue action is associated with inhibition of mitochondrial permeability transition pore opening (mPTP) (Partownavid et al., 2012), which is located in the inner mitochondrial membrane, but its exact mechanism of action is unknown.
∗ Corresponding author at: Department of Anesthesiology, Tumor Hospital, General Hospital of Ning Xia Medical University, No. 804, Shengli Street (South), Xingqing District, Yin Chuan, China. E-mail addresses: [email protected] (D. Lv), [email protected] (Z. Bai), [email protected] (L. Yang), [email protected] (X. Li), [email protected] (X. Chen). http://dx.doi.org/10.1016/j.etap.2016.01.004 1382-6689/© 2016 Elsevier B.V. All rights reserved.
Akt is closely related to the survival of cells in many systems including the heart (Matsui et al., 2001; Miyamoto et al., 2005). Glycogen synthase kinase-3 (GSK-3) has been shown can promote apoptosis and activate apoptosis-related protein kinase, caspase family, and Bax gene, which eventually cause apoptosis (Jope and Johnson, 2004). GSK-3 is regulated by phosphoinositide 3-kinase (PI3K)/Akt signal transduction pathway in vivo. GSK-3 serves as an important role in the downstream event of Akt by mediating convergence of protection signaling to inhibit mPTP (Juhaszova et al., 2004) through interaction with component of mPTP and contributing to a cardioprotective effect (Nishihara et al., 2007). Rahman et al. have confirmed that postischemic administration of intralipid inhibits the opening of mPTP and the underlying mechanism of cardioprotection by LE is phosphorylation of GSK-3 mediates the PI3K/Akt/extracellular signal-regulated kinase (ERK) pathway (Rahman et al., 2011). Therefore, it can be hypothesized that LE reverses BPV-induced cardiotoxicity through the PI3K/Akt/GSK-3 signaling pathway to suppress the mPTP opening, thus regulating mitochondrial functions. In this study, a model of cardiotoxicity by BPV in h9c2
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cardiomyocytes was established to prove the relationship between PI3K/Akt/GSK-3 signaling pathway and mPTP. 2. Materials and methods 2.1. Materials The h9c2 cell lines were from Shanghai Institutes for Biological Sciences, China. BPV hydrochloride injection was purchased from Kangqi Pharmaceutical Co., Ltd. (Wuhu, China). LE injection (20%, MCT/LCT) was obtained from Libang Pharmaceutical Co., Ltd. (Xi’an, China). Cyclosporine A (CsA), an mPTP blocker, was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Atractyloside (Atr), which opens mPTP, was obtained from Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, China). Both TWS119 (TWS), a GSK-3 inhibitor, and LY294002 (LY), a PI3K inhibitor, were from Selleck Company. Other reagents used in this study were as follows: Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and penicillin-streptomycin solution (100×) (Gibco, Grand Island, NY, USA), fetal bovine serum (Hyclone), mitochondrial complex detection kits (Beijing Solarbio Technology Co., Ltd.), Calcein-AM (Sigma), TUNEL staining kit (BD, Shanghai, China), Akt, phosphoAkt (Ser473) antibody, and GSK-3␣/, phospho-GSK-3 (Ser9) (Cell Signaling Technology, MA, USA).
When the degree of cell fusion was 80%, the cells were treated with drugs as planned except control group. After treatment with drugs for 24 h, the activity of mitochondrial respiratory complexes was evaluated. First, cells were digested with 0.25% trypsin. The cells were collected after centrifugation at 1000 rpm for 5 min and then homogenized with an ice bath after adding reagents. The supernatant was transferred to another centrifuge tube after centrifugation (600 g, 5 min, 4 ◦ C) to further centrifuge at 4 ◦ C (11,000 g, 10 min). After reagents were added, the sediment was broken by sonication (20% power, 3 s, interval of 10 s, and repeated 30 times), and then detected its activity. Machine was zeroed and calibrated with distilled water at appropriate wavelengths (corresponding wavelengths of mitochondrial respiratory complexes I, II, III, IV respectively were 340 nm, 605 nm, 550 nm, 550 nm). Furthermore, cells were incubated at 37 ◦ C for 5 min with both enzyme solution and work solution based on corresponding instructions, and then mixed after adding corresponding reagents. The initial absorbance (A1) was immediately recorded at the corresponding wavelength. The second absorbance (A2) was recorded after reaction for 2 min at 37 ◦ C. The activity of complexes was measured. 2.4. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay
h9c2 cells were recoveried and cultured in high glucose DMEM containing 10% fetal bovine serum in a humidified 5% CO2 incubator at 37 ◦ C. The medium was changed every 2–3 days. Cells were transferred into 96-well cell plates with a density of 105 mL–1 and were used for experiments once the degree of cell growth reached 80–90%. BPV (0.75%) and Intralipid (20%) were diluted in a serum medium, the final concentration respectively were 1 mM and 1%. The experimental classification consisted of groups control, 1 mM BPV, 1% LE, 1 mM BPV + 1% LE, 1 mM BPV + 1% LE + 20 M Atr or 10 M CsA, 1 mM BPV + 1% LE + 30 M LY or 10 M TWS.
The cells were rinsed three times after drugs treatment, fixed with paraformaldehyde for 1 h, and 0.1% sodium citrate solution was added. The cells were rinsed three times with phosphate-buffered saline (PBS) after incubation for 2 min. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction solution was added to the cells. The h9c2 cardiomyocytes were incubated at 37 ◦ C moist and dark environment for 60 min and were rinsed three times. Subsequently, the cells were examined and photographed under a fluorescence microscope at excitation and emission wavelengths of 450–500 nm and 515–565 nm, respectively. Bright red fluorescence was positive cells. TUNEL-positive nuclei and the total cell nuclei by Hoechst 33,342 staining were counte manually by researchers in four nonoverlapping fields per cover slip. The apoptotic rate was expressed as a percentage of apoptotic cells to the total cells.
2.3. Measurement of mitochondrial respiratory chain complexes
2.5. Western blot assay
The activity of mitochondrial respiratory chain complex I, also known as nicotinamide adenine dinucleotide (NADH)-coenzyme Q (CoQ) reductase or NADH dehydrogenase, can reflect respiratory electron-transport chain. Complex I is capable of catalyzing NADH, then it converts to NAD+ by dehydrogenation. The activity of enzyme can be calculated by measuring the NADH oxidation rate at 340 nm. Mitochondrial respiratory chain complex II, also known as succinate-CoQ reductase. CoQ, complex II catalytic product, can be further reduced to 2,6-dichloro-indoxyl, which has a characteristic absorption peak at 605 nm. Hence the activity of enzyme is calculated by detecting the rate of reduction of 2,6-dichloroindoxyl. Mitochondrial respiratory chain complex III, also called CoQ-cytochrome C reductase, is responsible for transferring the hydrogen of reduced CoQ to cytochrome C to further generate reduced cytochrome C. Reduced cytochrome C has a characteristic light absorption at 550 nm; therefore, the increase of light absorption rate can reflect the activity of mitochondrial complex III. Mitochondrial respiratory chain complex IV, also known as cytochrome C oxidase, is responsible for the oxidation of reduced cytochrome C and finally transferring the electron to oxygen, resulting in water. Mitochondrial complex IV can catalyze reductive cytochrome C to generate oxidizable cytochrome C. Hence the decrease of light absorption rate at 550 nm can reflect the activity of mitochondrial complex IV.
As described earlier, total proteins were extracted after treatment. h9c2 cells in six-well plates were added to 500 L lysates (1000 LRIPA Lysis Buffer + 10 L phenylmethylsulfonyl fluoride) on ice for 30 min. Later, lysates were transferred to 1.5 mL centrifuge tubes (performed on ice) for centrifugation at 12,000 rpm for 5 min at 4 ◦ C. Concentration of protein was detected based on instructions of Biyuntian BCA assay kit. Protein samples (30 g) were separated by gel electrophoresis, electrotransferred to PVDF membranes, blocked with Tris-buffered saline and Tween-20 (TBST) solution containing 5% bovine serum albumin (BSA) for 1 h, immunoblotted with anti-Akt, anti-pAkt, anti-GSK3 or anti-pGSK3 antibody diluted at 1:1000 with TBST containing 3% BSA for overnight at 4 ◦ C. Primary antibody was washed three times (10 min each time), with TBST on a shaker. The membranes were incubated with anti-rabbit immunoglobulin at a dilution of 1:2000 for 1 h at room temperature. After rinsing, the membranes were exposed to X-ray film. Quantification of bands was performed by scanning the films.
2.2. Cell and experimental classification
2.6. Opening of mPTP After treatment, cells were cultured for 24 h and then rinsed three times with PBS. Calcein-AM was dissolved in dimethylsulfoxide and then diluted with DMEM, a final concentration was
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1 mol/L. The cells were incubated for 10 min at 37 ◦ C in the dark after addition of 1 mL calcein-AM. Cobalt chloride (4 mmol/L) was added to the well plates to quench calcein-AM in the cytoplasm for 60 min. When mPTP opened, a fluorescent substance calcein entered from the mitochondria to the cytosol. The cells were dropped on a slide glass after loaded with calcein. The fluorescence intensity in the mitochondria was assessed using confocal microscopy with excitation and emission wavelengths at 488 and 515 nm. The more green fluorescent light, the higher the density. 2.7. Statistical analysis SPSS, version 17.0, was used to analyze the data. For continuous data, results were summarized using mean ± standard error. For normally distributed and homogeneous variance data, single-factor analysis of variance was used; otherwise nonparametric rank sum test was used. Multiple comparison tests were performed by LSD. P < 0.05 was considered to be statistically significant. 3. Results 3.1. LE reversed the effect of BPV on mitochondrial respiratory chain complexes As shown in Fig. 1, after treatment with BPV, the activity of mitochondrial respiratory chain complexes (I, II, III, IV) decreased compared with control group (P < 0.001, P < 0.05, P < 0.001, P < 0.01). In LE group, the activity of mitochondrial respiratory chain complexes did not show significant change compared with control group. The activity of complex III was increased (P < 0.05). The activity of mitochondrial respiratory chain complexes of group BPV + LE was higher at different degrees than that of group BPV (P < 0.01,
Fig. 2. Picture represented the effect on the activity of mitochondrial respiratory chain complexes by drugs. The function of mitochondria respiratory chain complexes was measured with CsA or Atr after administration of BPV and LE. The data was shown as Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 compared with BPV + LE group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV + LE + CsA group.
P < 0.05, P < 0.001, P < 0.01), but did not show significant difference than that of group control. 3.2. LE adjusted the activity of mitochondrial respiratory chain complexes I and IV by mPTP The activity of mitochondrial respiratory complexes I and IV was slightly lower in the BPV + LE group than Atr group (P < 0.001, P < 0.01), but noticeably higher than CsA group (P < 0.05, P < 0.05) (Fig. 2). These results strongly demonstrated that LE had an effect on the activity of partial mitochondrial respiratory complexes by adjusting mPTP. 3.3. Reversal of BPV-induced apoptosis by LE via mPTP, its effect increased by TWS, but attenuated by LY After treatment with BPV alone, apoptotic rate was significantly increased compared with control group. Apoptotic rate in the BPV + LE group fell approximately half compared with BPV group, but was still higher compared with control group, indicating that LE can partially reverse the apoptosis induced by BPV. Apoptosis was significantly reduced after addition of BPV + LE + CsA, but increased together with Atr compared with BPV + LE group, suggesting that LE likely reduced the apoptosis by regulating the opening of mPTP. Apoptosis of that group in the presence of TWS was significantly lower than that of group BPV + LE (P < 0.001), however, it was higher when LY was used (P < 0.001). (Fig. 3A and B). 3.4. Expression of pAkt and pGSK-3ˇ
Fig. 1. Lipid emulsion reversed the effect of bupivacaine on mitochondrial respiratory chain complexes. In all groups (n = 3), the results are presented as Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. LE group.
As shown in Fig. 4A, phosphorylation of Akt after treatment with LE alone increased about threefold compared with control group (P < 0.001). The level of pAkt was markedly decreased in BPV group (P < 0.001). The Akt phosphorylation was higher in BPV + LE group than in BPV group (P < 0.001). LE-induced cardioprotection was associated with increase of Akt phosphorylation,
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Fig. 3. The apoptosis was measured by TUNEL staining. Fig. (A) Comparison of the effect of drugs. (B) Immunofluorescent detection of DNA damage (TUNEL) in H9c2 cells. After the drug treatment, cells were fixed and processed for TUNEL (red) assay. The nuclei were stained with DAPI (blue). All data were shown as Mean ± S.E in all groups. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. BPV + LE group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The proteins expression of pAkt/Akt and pGSK-3/GSK-3 in each group. The concentrations of the bands were measured with a densitometer. A and B. Western Blot proteins quantification of pAkt to total Akt (A) and pGSK-3 to total GSK-3 (B) of control ratios in all groups ((Mean ± S.E), n = 3). C and D representative immunoblots of pAkt and total Akt (C), pGSK-3 and total GSK-3(D). *P < 0.05, **P < 0.01, ***P < 0.001 compared with Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 compared with BPV + LE group.
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Fig. 5. Depicts the effect of drugs on the opening of mPTP. (A) Representative Calcein fluorescence intensity after treament with excepted drugs. All data were shown as Mean ± S.E (n = 5). (B) Representative fluorescence images by confocal microscopy. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 compared with BPV + LE group.
which was reversed when LE was used together with BPV and Atr or LY (1.572 ± 0.077 in BPV + LE vs. 0.914 ± 0.032 in BPV + LE + Atr vs. 1.268 ± 0.032 in BPV + LE + LY), but was enhanced when added together with TWS or CsA (1.572 ± 0.077 in BPV + LE vs. 1.808 ± 0.054 in BPV + LE + TWS and 2.266 ± 0.062 in BPV + LE + CsA). The phosphorylation level of GSK-3 was lower in BPV group compared with control group,but in BPV + LE group as high as control group (P > 0.05), suggesting that LE has an effect on pGSK-3. The GSK-3 phosphorylation was also significantly increased after addition of BPV with both LE and CsA or TWS compared with BPV + LE (1.548 ± 0.049, P < 0.001 and 1.348 ± 0.031, P < 0.001 vs. 1.086 ± 0.069); in contrast, it was lower in groups BPV + LE and Atr or LY (0.775 ± 0.027, P < 0.05 and 0.599 ± 0.018, P < 0.001 vs. 1.086 ± 0.070). These data strongly support the role of PI3K/Akt/GSK-3 in LE-induced cardioprotection against cardiotoxicity by BPV in h9c2 cardiomyocytes. 3.5. Opening of mPTP As shown Fig. 5A and B, in BPV group the calcein intensity had a dramatic decrease (fivefold) compared with control group. The calcein intensity did not show significant difference between BPV + LE group and control group. After addition of LE, the intensity of
calcein markedly increased, indicating that LE partially reversed the reduction of calcein intensity by BPV. It was shown that addition of CsA to BPV + LE group resulted in significant improvement in calcein intensity, while it was greatly reduced in the presence of Atr. It can be observed that in group BPV + LE + TWS the intensity was higher than that of group control, but in group BPV + LE + LY LY was lower. 4. Discussion Szstard et al. have shown that BPV inhibited the activity of mitochondrial complexes I and II causing disorder of mitochondrial energy metabolism of myocardial cells (Szstard et al., 1998). Sztark et al. found that BPV suppressed mitochondrial respiratory chain enzyme activity, especially the NADH synthase (Sztark et al., 2000). A study has shown that the production of reactive oxygen species (ROS) by L-BPV reduced the activity of complexes I and III in murine cardiomyoblasts (h9c2) and inhibition of function of mitochondrial respiratory chain complexes enhanced the production of ROS, which leading to a vicious cycle (Cela et al., 2010). Others have found that BPV-induced toxicity of muscles was directly related to the opening of mPTP (Irwin et al., 2002). These results suggested that BPV induced mitochondrial dysfunction, resulting
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in disruption of energy metabolism. As mentioned earlier, the data from the present study showed that BPV reduced the activity of mitochondrial respiratory chain complexes (I, II, III, IV); however, the results were different from previous studies, perhaps as a consequence of different methods of measurement. Different complex enzyme kits were used to detect the activity of complex enzymes. Our previous results showed that LE reduced the generation of ROS (Yang et al., 2015), thus weakening the effect on mitochondrial respiratory chain complexes. Akt, an intersection of a number of mitochondria-mediated apoptotic pathways, directly or indirectly regulates apoptosis through phosphorylation or interaction with cell death-promoting factors. The activity of GSK-3 is inversely related to the phosphorylation status of serine-9 (Dajani et al., 2001). Postischemic treatment with intralipid increased the phosphorylation levels of GSK-3 and Akt through the PI3K/Akt/ERK pathways to inhibit the opening of mPTP and then reduced the myocardial infarct size in rat model (Rahman et al., 2011). BPV suppressed the activation of Akt, which promoted apoptosis in cultured mouse C2C12 myoblasts (Maurice et al., 2010). Serine 9 phosphorylation of GSK-3 plays an important role in cardioprotection by postconditioning and likely acts by inhibiting the opening of mPTP at the time of reperfusion in a CypD-independent manner (Gomez et al., 2008). In the present study, fatty acid oxidation was necessary for the successful rescue of BPV-induced cardiotoxicity by LE (Partownavid et al., 2012; Weinberg, 2012a,b) and this rescue action was associated with the inhibition of mPTP opening (Partownavid et al., 2012). Our previous evidence has shown that LE reversed the apoptosis induced by BPV in h9c2 cardiomyocytes, increased the production of Bcl-2 (an antiapoptotic factor), and decreased the production of Bax (a proapoptotic factor), caspase-3, and caspase-9 (Yang et al., 2015). LE regulates mPTP by reducing the production of ROS to protect the heart (Partownavid et al., 2012). Therefore, it was assumed that LE rescues the BPV-induced cardiotoxicity through the PI3K/Akt/GSK3 signaling pathway in mPTP. To examine whether it works through this pathway, LY and TWS were used. LY weakened the reversal of LE, but TWS strengthened. Hence, the hypothesis is correct. A few studies have suggested that dexamethasone and lithium could increase the activation of Akt and decreased BPV-induced neuronal cell injury (Ma et al., 2010; Wang et al., 2012). Recent studies have proposed several other possible mechanisms for the reversal of BPV-induced cardiotoxicity by LE. A previous study has shown that LE accelerated the clearance of BPV from the cardiac tissue, while recovery speedly from BPV-induced asystole (Weinberg et al., 2006; Chen et al., 2010), which supports the concept of “lipid sink” (Weinberg et al., 2006). Another study showed the effect of lipid rescue on the single-cell level toxicity by BPV and provided evidence for lipid-sink mechanism (Wanger et al., 2014). Mottram et al. came up with a possible mechanism of action by LE that was the salutary effects of it may be, in part, as a result of direct adjustive effect of fatty acids on cardiac sodium channels (Mottram et al., 2011). Weinberg proposed the activation of voltage-dependent calcium channel as another possible mechanism of rescue cardiotoxicity (Weinberg, 2012a,b) and calcium homeostasis was involved in the rescue of BPV-mediated cardiotoxicity by LE (Weinberg, 2012a,b). A recently published study demonstrated that use of LE accelerated the elimination of BPV and promoted its metabolism on pharmacokinetics in rats (Shi et al., 2013) to protect the heart. According to the results of previous studies,we found that concentrations of BPV were 0.77–1 mM could generate significant pro-apoptotic effects, so 1 mM BPV was chosen to observe the occurrence of apoptosis (Maurice et al., 2010; Perez-Castro et al., 2009; Yang et al., 2015.). Different concentrations of lipid emulsion had no effect on the growth of h9c2 cardiomyocytes, and even a slight pro-cell proliferation,1% LE more better (Yang et al., 2015;
Bai, 2013). After treated with drugs cells were cultivated for 24 h, because each drugs achieved the best effects at this time (Yang et al., 2015; Bai, 2013). Some limitations of this study should be noted. First, antiapoptotic effect of PI3K-Akt signaling pathway is also influenced by a number of other cell-specific enzymes or proteins and Akt regulates the cell activity not only by adjusting GSK-3, but also by acting on Forkhead, BAD, or other proteins, which are downstream signaling targets of Akt (Carnero and Paramio, 2014). Furthermore, although TWS is a specific blocker of GSK-3, it also has a slight effect on GSK-3␣. Third, the conclusions drawn from this in vitro experiment cannot be used to predict in vivo experiment, which needs to be further studied. Last, according to instructions of CsA, Atr, TWS and LY, its own had no effects on the cells, so we did not design related groups to exclude the impact. These need to be proved in the future. The impact of these factors on the experimental results cannot be ruled out. In summary, the present study suggests that LE mainly reduces the opening of mPTP by regulating the activity of the mitochondrial ion channel or signaling pathway, reducing the apoptosis factors or the generation of ROS, and increasing the expression of antiapoptotic factors to protect the heart. These results provide a novel insight into the molecular mechanisms underlying cardiac cytotoxicity by BPV and several possible new targets for the development of drugs. 5. Conclusions The data indicated that LE can reverse the apoptosis in cardiomyocytes by BPV and a mechanism of its action is by inhibition of mPTP opening through the PI3K/Akt/GSK-3 signaling pathway. Authors contributions Danni Lv helped conduct the study, analyze the data and prepare the manuscript and also attests to the integrity of the data and approves the final manuscript. She is the archival author. Zhixia Bai helped design the study and modified the manuscript Attestation and also attests to the integrity of the data and approves the final manuscript. Libin Yang helped collect the data and analyze the data and also attests to the integrity of the data and approves the final manuscript. Xiaohui Li helped collect the data and analyze the data and also attests to the integrity of the data and approves the final manuscript. Chen helped design the study and also attests to the integrity of the data and approves the final manuscript. Conflict of interest The authors declare no conflicts of interest. Funding This work was funded by the National Natural Science Foundation of China (No. 81241140). Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81241140).
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Miyamoto, S., Howes, A.L., Adams, J.W., Dorn, G.W., Brown, J.H., 2005. Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT. J. Biol. Chem. 280, 38505–38512. Mottram, A.R., Valdivia, C.R., Makielski, J.C., 2011. Fatty acids antagonize bupivacaine -induced I (Na) blockade. Clin. Toxicol. (Phila.) 49, 729–733. Nishihara, M., Miura, T., Miki, T., Miki, T., Tanno, M., Yano, T., Naitoh, K., Ohori, K., Hotta, H., Terashima, Y., Shimamoto, K., 2007. Modulation of the mitochondrial permeability transition pore complex in GSK-3-mediated myocardial protection. J. Mol. Cell Cardiol. 43, 564–570. Partownavid, P., Umar, S., Li, J., Rahman, S., Eghbali, M., 2012. Fatty-acid oxidation and calcium homeostasis are involved in the rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. Crit. Care Med. 40, 2431–2437. Perez-Castro, R., Patel, S., Garavito-Aguilar, Z.V., Rosenberg, A., Recio-Pinto, E., Zhang, J., Blanck, T.J., Xu, F., 2009. Cytotoxicity of local anesthetics in human neuronal cells. Anesth. Analg. 108, 997–1007. Rahman, S., Li, J., Bopassa, J.C., Umar, S., Iorga, A., Partownavid, P., Eghbali, M., 2011. Phosphorylation of GSK-3 mediates intralipid-induced cardioprotection against ischemia/reperfusion injury. Anesthesiology 115, 242–253. Shi, K., Xia, Y., Wang, Q., Wu, Y., Dong, X., Chen, C., Tang, W., Zhang, W., Luo, M., Wang, X., Papadimos, T.J., Xu, X., 2013. The effect of lipid emulsion on pharmacokinetics and tissue distribution of bupivacaine in rats. Anesth. Analg. 117, 536. Szstard, F., Malgat, M., Dabadie, P., Mazat, J.P., 1998. Comparison of the effect of bupivacaine and ropivacaine on heart cell mitochondrial bioenergetics. Anesthesiology 88, 1340. Sztark, F., Nouette-Gaulain, K., Malgat, M., Dabadie, P., Mazat, J.P., 2000. Absence of stereospecific effects of bupivacaine isomerson heart mitochondrial bioenergetics. Anesthesiology 93, 456–462. Wang, Z., Shen, J., Wang, J., Lu, T., Li, C., Zhang, X., Liu, L., Ding, Z., 2012. Lithium attenuates bupivacaine-induced neurotoxicity in vitro through phosphatidylinositol-3-kinase/threonine-serine protein kinase B- and extracellular signal-regulated kinase-dependent mechanisms. Neuroscience 206, 190–200. Wanger, M., Zausig, Y.A., Ruf, S., Rudakova, E., Gruber, M., Graf, B.M., Volk, T., 2014. Lipid rescue reverses the bupivacaine-induced block of the fast Na current (INa) in cardiomyocytes of the rat left ventricle. Anesthesiology 120, 724–736. Weinberg, G.L., Ripper, R., Murphy, P., Edelman, L.B., Hoffman, W., Strichartz, G., Feinstein, D.L., 2006. Lipid infusion accelerates removal of bupivacaine and recovery from bupivacaine toxicity in the isolated rat heart. Reg. Anesth. Pain Med. 31, 296–303. Weinberg, G.L., 2012a. Lipid emulsion infusion: resuscitation for local anesthetic and other drug overdose. Anesthesiology 117, 180–187. Weinberg, G.L., 2012b. Lipid resuscitation: more than a sink. Crit. Care Med. 40, 2521–2523. Yang, L., Bai, Z., Lv, D., Liu, H., Li, X., Chen, X., 2015. Rescue effect of lipid emulsion on bupivacaine-induced cardiac toxicity in cardiomyocytes. Mol. Med. Rep. 12, 3739–3747.
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Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Lipid emulsion reverses bupivacaine-induced apoptosis of h9c2 cardiomyocytes: PI3K/Akt/GSK-3 signaling pathway Danni Lv a , Zhixia Bai b , Libin Yang c , Xiaohui Li a , Xuexin Chen b,∗ a b c
Ning Xia Medical University, Yin Chuan, China Department of Anesthesiology, Tumor Hospital, General Hospital of Ning Xia Medical University, Yin Chuan, China Department of Anesthesiology, First People’s Hospital, Shi Zui Shan, China
a r t i c l e
i n f o
Article history: Received 2 July 2015 Received in revised form 2 January 2016 Accepted 5 January 2016 Available online 9 January 2016 Keywords: Lipid emulsion Bupivacaine Cardiac cytotoxicity mPTP PI3K/Akt/GSK-3
a b s t r a c t Some findings have suggested that the rescue of bupivacaine (BPV)-induced cardiotoxicity by lipid emulsion (LE) is associated with inhibition of mitochondrial permeability transition pore (mPTP). However, the mechanism of this rescue action is not clearly known. In this study, the roles of phosphoinositide 3kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) in the molecular mechanism of LE-induced protection and its relationship with mPTP were explored. h9c2 cardiomyocytes were randomly divided into several groups: control, BPV, LE, BPV + LE. To study the effect of LE on mPTP, atractyloside (Atr, 20 M, mPTP opener) and cyclosporine A (CsA, 10 M, mPTP blocker) were used. To unravel whether LE protects heart through the PI3K/Akt/GSK-3 signaling pathway, cells were treated with LY294002 (LY, 30 M, PI3K blocker) or TWS119 (TWS 10 M, GSK-3 blocker). Later mitochondrial respiratory chain complexes, apoptosis, opening of mPTP and phosphorylation levels of Akt/GSK-3 were measured. LE significantly improved the mitochondrial functions in h9c2 cardiomyocytes. LE reversed the BPV-induced apoptosis and the opening of mPTP. The effect of LE was not only enhanced by CsA and TWS, but also abolished by Atr and LY. LE also increased the phosphorylation levels of Akt and GSK-3. These results suggested that LE can reverse the apoptosis in cardiomyocytes by BPV and a mechanism of its action is inhibition of mPTP opening through the PI3K/Akt/GSK-3 signaling pathway. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, researchers have fully demonstrated that lipid emulsion (LE) plays an important role in the treatment of bupivacaine (BPV)-induced cardiac toxicity (Weinberg et al., 2006; Litz et al., 2008; Lin and Aronson, 2010), but the exact mechanism is still not clear. Fatty acid oxidation is necessary for successful rescue of BPV-induced cardiotoxicity by LE (Partownavid et al., 2012; Weinberg, 2012a,b). This rescue action is associated with inhibition of mitochondrial permeability transition pore opening (mPTP) (Partownavid et al., 2012), which is located in the inner mitochondrial membrane, but its exact mechanism of action is unknown.
∗ Corresponding author at: Department of Anesthesiology, Tumor Hospital, General Hospital of Ning Xia Medical University, No. 804, Shengli Street (South), Xingqing District, Yin Chuan, China. E-mail addresses: [email protected] (D. Lv), [email protected] (Z. Bai), [email protected] (L. Yang), [email protected] (X. Li), [email protected] (X. Chen). http://dx.doi.org/10.1016/j.etap.2016.01.004 1382-6689/© 2016 Elsevier B.V. All rights reserved.
Akt is closely related to the survival of cells in many systems including the heart (Matsui et al., 2001; Miyamoto et al., 2005). Glycogen synthase kinase-3 (GSK-3) has been shown can promote apoptosis and activate apoptosis-related protein kinase, caspase family, and Bax gene, which eventually cause apoptosis (Jope and Johnson, 2004). GSK-3 is regulated by phosphoinositide 3-kinase (PI3K)/Akt signal transduction pathway in vivo. GSK-3 serves as an important role in the downstream event of Akt by mediating convergence of protection signaling to inhibit mPTP (Juhaszova et al., 2004) through interaction with component of mPTP and contributing to a cardioprotective effect (Nishihara et al., 2007). Rahman et al. have confirmed that postischemic administration of intralipid inhibits the opening of mPTP and the underlying mechanism of cardioprotection by LE is phosphorylation of GSK-3 mediates the PI3K/Akt/extracellular signal-regulated kinase (ERK) pathway (Rahman et al., 2011). Therefore, it can be hypothesized that LE reverses BPV-induced cardiotoxicity through the PI3K/Akt/GSK-3 signaling pathway to suppress the mPTP opening, thus regulating mitochondrial functions. In this study, a model of cardiotoxicity by BPV in h9c2
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cardiomyocytes was established to prove the relationship between PI3K/Akt/GSK-3 signaling pathway and mPTP. 2. Materials and methods 2.1. Materials The h9c2 cell lines were from Shanghai Institutes for Biological Sciences, China. BPV hydrochloride injection was purchased from Kangqi Pharmaceutical Co., Ltd. (Wuhu, China). LE injection (20%, MCT/LCT) was obtained from Libang Pharmaceutical Co., Ltd. (Xi’an, China). Cyclosporine A (CsA), an mPTP blocker, was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Atractyloside (Atr), which opens mPTP, was obtained from Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, China). Both TWS119 (TWS), a GSK-3 inhibitor, and LY294002 (LY), a PI3K inhibitor, were from Selleck Company. Other reagents used in this study were as follows: Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose and penicillin-streptomycin solution (100×) (Gibco, Grand Island, NY, USA), fetal bovine serum (Hyclone), mitochondrial complex detection kits (Beijing Solarbio Technology Co., Ltd.), Calcein-AM (Sigma), TUNEL staining kit (BD, Shanghai, China), Akt, phosphoAkt (Ser473) antibody, and GSK-3␣/, phospho-GSK-3 (Ser9) (Cell Signaling Technology, MA, USA).
When the degree of cell fusion was 80%, the cells were treated with drugs as planned except control group. After treatment with drugs for 24 h, the activity of mitochondrial respiratory complexes was evaluated. First, cells were digested with 0.25% trypsin. The cells were collected after centrifugation at 1000 rpm for 5 min and then homogenized with an ice bath after adding reagents. The supernatant was transferred to another centrifuge tube after centrifugation (600 g, 5 min, 4 ◦ C) to further centrifuge at 4 ◦ C (11,000 g, 10 min). After reagents were added, the sediment was broken by sonication (20% power, 3 s, interval of 10 s, and repeated 30 times), and then detected its activity. Machine was zeroed and calibrated with distilled water at appropriate wavelengths (corresponding wavelengths of mitochondrial respiratory complexes I, II, III, IV respectively were 340 nm, 605 nm, 550 nm, 550 nm). Furthermore, cells were incubated at 37 ◦ C for 5 min with both enzyme solution and work solution based on corresponding instructions, and then mixed after adding corresponding reagents. The initial absorbance (A1) was immediately recorded at the corresponding wavelength. The second absorbance (A2) was recorded after reaction for 2 min at 37 ◦ C. The activity of complexes was measured. 2.4. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay
h9c2 cells were recoveried and cultured in high glucose DMEM containing 10% fetal bovine serum in a humidified 5% CO2 incubator at 37 ◦ C. The medium was changed every 2–3 days. Cells were transferred into 96-well cell plates with a density of 105 mL–1 and were used for experiments once the degree of cell growth reached 80–90%. BPV (0.75%) and Intralipid (20%) were diluted in a serum medium, the final concentration respectively were 1 mM and 1%. The experimental classification consisted of groups control, 1 mM BPV, 1% LE, 1 mM BPV + 1% LE, 1 mM BPV + 1% LE + 20 M Atr or 10 M CsA, 1 mM BPV + 1% LE + 30 M LY or 10 M TWS.
The cells were rinsed three times after drugs treatment, fixed with paraformaldehyde for 1 h, and 0.1% sodium citrate solution was added. The cells were rinsed three times with phosphate-buffered saline (PBS) after incubation for 2 min. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction solution was added to the cells. The h9c2 cardiomyocytes were incubated at 37 ◦ C moist and dark environment for 60 min and were rinsed three times. Subsequently, the cells were examined and photographed under a fluorescence microscope at excitation and emission wavelengths of 450–500 nm and 515–565 nm, respectively. Bright red fluorescence was positive cells. TUNEL-positive nuclei and the total cell nuclei by Hoechst 33,342 staining were counte manually by researchers in four nonoverlapping fields per cover slip. The apoptotic rate was expressed as a percentage of apoptotic cells to the total cells.
2.3. Measurement of mitochondrial respiratory chain complexes
2.5. Western blot assay
The activity of mitochondrial respiratory chain complex I, also known as nicotinamide adenine dinucleotide (NADH)-coenzyme Q (CoQ) reductase or NADH dehydrogenase, can reflect respiratory electron-transport chain. Complex I is capable of catalyzing NADH, then it converts to NAD+ by dehydrogenation. The activity of enzyme can be calculated by measuring the NADH oxidation rate at 340 nm. Mitochondrial respiratory chain complex II, also known as succinate-CoQ reductase. CoQ, complex II catalytic product, can be further reduced to 2,6-dichloro-indoxyl, which has a characteristic absorption peak at 605 nm. Hence the activity of enzyme is calculated by detecting the rate of reduction of 2,6-dichloroindoxyl. Mitochondrial respiratory chain complex III, also called CoQ-cytochrome C reductase, is responsible for transferring the hydrogen of reduced CoQ to cytochrome C to further generate reduced cytochrome C. Reduced cytochrome C has a characteristic light absorption at 550 nm; therefore, the increase of light absorption rate can reflect the activity of mitochondrial complex III. Mitochondrial respiratory chain complex IV, also known as cytochrome C oxidase, is responsible for the oxidation of reduced cytochrome C and finally transferring the electron to oxygen, resulting in water. Mitochondrial complex IV can catalyze reductive cytochrome C to generate oxidizable cytochrome C. Hence the decrease of light absorption rate at 550 nm can reflect the activity of mitochondrial complex IV.
As described earlier, total proteins were extracted after treatment. h9c2 cells in six-well plates were added to 500 L lysates (1000 LRIPA Lysis Buffer + 10 L phenylmethylsulfonyl fluoride) on ice for 30 min. Later, lysates were transferred to 1.5 mL centrifuge tubes (performed on ice) for centrifugation at 12,000 rpm for 5 min at 4 ◦ C. Concentration of protein was detected based on instructions of Biyuntian BCA assay kit. Protein samples (30 g) were separated by gel electrophoresis, electrotransferred to PVDF membranes, blocked with Tris-buffered saline and Tween-20 (TBST) solution containing 5% bovine serum albumin (BSA) for 1 h, immunoblotted with anti-Akt, anti-pAkt, anti-GSK3 or anti-pGSK3 antibody diluted at 1:1000 with TBST containing 3% BSA for overnight at 4 ◦ C. Primary antibody was washed three times (10 min each time), with TBST on a shaker. The membranes were incubated with anti-rabbit immunoglobulin at a dilution of 1:2000 for 1 h at room temperature. After rinsing, the membranes were exposed to X-ray film. Quantification of bands was performed by scanning the films.
2.2. Cell and experimental classification
2.6. Opening of mPTP After treatment, cells were cultured for 24 h and then rinsed three times with PBS. Calcein-AM was dissolved in dimethylsulfoxide and then diluted with DMEM, a final concentration was
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1 mol/L. The cells were incubated for 10 min at 37 ◦ C in the dark after addition of 1 mL calcein-AM. Cobalt chloride (4 mmol/L) was added to the well plates to quench calcein-AM in the cytoplasm for 60 min. When mPTP opened, a fluorescent substance calcein entered from the mitochondria to the cytosol. The cells were dropped on a slide glass after loaded with calcein. The fluorescence intensity in the mitochondria was assessed using confocal microscopy with excitation and emission wavelengths at 488 and 515 nm. The more green fluorescent light, the higher the density. 2.7. Statistical analysis SPSS, version 17.0, was used to analyze the data. For continuous data, results were summarized using mean ± standard error. For normally distributed and homogeneous variance data, single-factor analysis of variance was used; otherwise nonparametric rank sum test was used. Multiple comparison tests were performed by LSD. P < 0.05 was considered to be statistically significant. 3. Results 3.1. LE reversed the effect of BPV on mitochondrial respiratory chain complexes As shown in Fig. 1, after treatment with BPV, the activity of mitochondrial respiratory chain complexes (I, II, III, IV) decreased compared with control group (P < 0.001, P < 0.05, P < 0.001, P < 0.01). In LE group, the activity of mitochondrial respiratory chain complexes did not show significant change compared with control group. The activity of complex III was increased (P < 0.05). The activity of mitochondrial respiratory chain complexes of group BPV + LE was higher at different degrees than that of group BPV (P < 0.01,
Fig. 2. Picture represented the effect on the activity of mitochondrial respiratory chain complexes by drugs. The function of mitochondria respiratory chain complexes was measured with CsA or Atr after administration of BPV and LE. The data was shown as Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 compared with BPV + LE group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV + LE + CsA group.
P < 0.05, P < 0.001, P < 0.01), but did not show significant difference than that of group control. 3.2. LE adjusted the activity of mitochondrial respiratory chain complexes I and IV by mPTP The activity of mitochondrial respiratory complexes I and IV was slightly lower in the BPV + LE group than Atr group (P < 0.001, P < 0.01), but noticeably higher than CsA group (P < 0.05, P < 0.05) (Fig. 2). These results strongly demonstrated that LE had an effect on the activity of partial mitochondrial respiratory complexes by adjusting mPTP. 3.3. Reversal of BPV-induced apoptosis by LE via mPTP, its effect increased by TWS, but attenuated by LY After treatment with BPV alone, apoptotic rate was significantly increased compared with control group. Apoptotic rate in the BPV + LE group fell approximately half compared with BPV group, but was still higher compared with control group, indicating that LE can partially reverse the apoptosis induced by BPV. Apoptosis was significantly reduced after addition of BPV + LE + CsA, but increased together with Atr compared with BPV + LE group, suggesting that LE likely reduced the apoptosis by regulating the opening of mPTP. Apoptosis of that group in the presence of TWS was significantly lower than that of group BPV + LE (P < 0.001), however, it was higher when LY was used (P < 0.001). (Fig. 3A and B). 3.4. Expression of pAkt and pGSK-3ˇ
Fig. 1. Lipid emulsion reversed the effect of bupivacaine on mitochondrial respiratory chain complexes. In all groups (n = 3), the results are presented as Mean ± S.E. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. LE group.
As shown in Fig. 4A, phosphorylation of Akt after treatment with LE alone increased about threefold compared with control group (P < 0.001). The level of pAkt was markedly decreased in BPV group (P < 0.001). The Akt phosphorylation was higher in BPV + LE group than in BPV group (P < 0.001). LE-induced cardioprotection was associated with increase of Akt phosphorylation,
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Fig. 3. The apoptosis was measured by TUNEL staining. Fig. (A) Comparison of the effect of drugs. (B) Immunofluorescent detection of DNA damage (TUNEL) in H9c2 cells. After the drug treatment, cells were fixed and processed for TUNEL (red) assay. The nuclei were stained with DAPI (blue). All data were shown as Mean ± S.E in all groups. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. BPV + LE group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The proteins expression of pAkt/Akt and pGSK-3/GSK-3 in each group. The concentrations of the bands were measured with a densitometer. A and B. Western Blot proteins quantification of pAkt to total Akt (A) and pGSK-3 to total GSK-3 (B) of control ratios in all groups ((Mean ± S.E), n = 3). C and D representative immunoblots of pAkt and total Akt (C), pGSK-3 and total GSK-3(D). *P < 0.05, **P < 0.01, ***P < 0.001 compared with Control group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 compared with BPV + LE group.
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Fig. 5. Depicts the effect of drugs on the opening of mPTP. (A) Representative Calcein fluorescence intensity after treament with excepted drugs. All data were shown as Mean ± S.E (n = 5). (B) Representative fluorescence images by confocal microscopy. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with BPV group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 compared with BPV + LE group.
which was reversed when LE was used together with BPV and Atr or LY (1.572 ± 0.077 in BPV + LE vs. 0.914 ± 0.032 in BPV + LE + Atr vs. 1.268 ± 0.032 in BPV + LE + LY), but was enhanced when added together with TWS or CsA (1.572 ± 0.077 in BPV + LE vs. 1.808 ± 0.054 in BPV + LE + TWS and 2.266 ± 0.062 in BPV + LE + CsA). The phosphorylation level of GSK-3 was lower in BPV group compared with control group,but in BPV + LE group as high as control group (P > 0.05), suggesting that LE has an effect on pGSK-3. The GSK-3 phosphorylation was also significantly increased after addition of BPV with both LE and CsA or TWS compared with BPV + LE (1.548 ± 0.049, P < 0.001 and 1.348 ± 0.031, P < 0.001 vs. 1.086 ± 0.069); in contrast, it was lower in groups BPV + LE and Atr or LY (0.775 ± 0.027, P < 0.05 and 0.599 ± 0.018, P < 0.001 vs. 1.086 ± 0.070). These data strongly support the role of PI3K/Akt/GSK-3 in LE-induced cardioprotection against cardiotoxicity by BPV in h9c2 cardiomyocytes. 3.5. Opening of mPTP As shown Fig. 5A and B, in BPV group the calcein intensity had a dramatic decrease (fivefold) compared with control group. The calcein intensity did not show significant difference between BPV + LE group and control group. After addition of LE, the intensity of
calcein markedly increased, indicating that LE partially reversed the reduction of calcein intensity by BPV. It was shown that addition of CsA to BPV + LE group resulted in significant improvement in calcein intensity, while it was greatly reduced in the presence of Atr. It can be observed that in group BPV + LE + TWS the intensity was higher than that of group control, but in group BPV + LE + LY LY was lower. 4. Discussion Szstard et al. have shown that BPV inhibited the activity of mitochondrial complexes I and II causing disorder of mitochondrial energy metabolism of myocardial cells (Szstard et al., 1998). Sztark et al. found that BPV suppressed mitochondrial respiratory chain enzyme activity, especially the NADH synthase (Sztark et al., 2000). A study has shown that the production of reactive oxygen species (ROS) by L-BPV reduced the activity of complexes I and III in murine cardiomyoblasts (h9c2) and inhibition of function of mitochondrial respiratory chain complexes enhanced the production of ROS, which leading to a vicious cycle (Cela et al., 2010). Others have found that BPV-induced toxicity of muscles was directly related to the opening of mPTP (Irwin et al., 2002). These results suggested that BPV induced mitochondrial dysfunction, resulting
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in disruption of energy metabolism. As mentioned earlier, the data from the present study showed that BPV reduced the activity of mitochondrial respiratory chain complexes (I, II, III, IV); however, the results were different from previous studies, perhaps as a consequence of different methods of measurement. Different complex enzyme kits were used to detect the activity of complex enzymes. Our previous results showed that LE reduced the generation of ROS (Yang et al., 2015), thus weakening the effect on mitochondrial respiratory chain complexes. Akt, an intersection of a number of mitochondria-mediated apoptotic pathways, directly or indirectly regulates apoptosis through phosphorylation or interaction with cell death-promoting factors. The activity of GSK-3 is inversely related to the phosphorylation status of serine-9 (Dajani et al., 2001). Postischemic treatment with intralipid increased the phosphorylation levels of GSK-3 and Akt through the PI3K/Akt/ERK pathways to inhibit the opening of mPTP and then reduced the myocardial infarct size in rat model (Rahman et al., 2011). BPV suppressed the activation of Akt, which promoted apoptosis in cultured mouse C2C12 myoblasts (Maurice et al., 2010). Serine 9 phosphorylation of GSK-3 plays an important role in cardioprotection by postconditioning and likely acts by inhibiting the opening of mPTP at the time of reperfusion in a CypD-independent manner (Gomez et al., 2008). In the present study, fatty acid oxidation was necessary for the successful rescue of BPV-induced cardiotoxicity by LE (Partownavid et al., 2012; Weinberg, 2012a,b) and this rescue action was associated with the inhibition of mPTP opening (Partownavid et al., 2012). Our previous evidence has shown that LE reversed the apoptosis induced by BPV in h9c2 cardiomyocytes, increased the production of Bcl-2 (an antiapoptotic factor), and decreased the production of Bax (a proapoptotic factor), caspase-3, and caspase-9 (Yang et al., 2015). LE regulates mPTP by reducing the production of ROS to protect the heart (Partownavid et al., 2012). Therefore, it was assumed that LE rescues the BPV-induced cardiotoxicity through the PI3K/Akt/GSK3 signaling pathway in mPTP. To examine whether it works through this pathway, LY and TWS were used. LY weakened the reversal of LE, but TWS strengthened. Hence, the hypothesis is correct. A few studies have suggested that dexamethasone and lithium could increase the activation of Akt and decreased BPV-induced neuronal cell injury (Ma et al., 2010; Wang et al., 2012). Recent studies have proposed several other possible mechanisms for the reversal of BPV-induced cardiotoxicity by LE. A previous study has shown that LE accelerated the clearance of BPV from the cardiac tissue, while recovery speedly from BPV-induced asystole (Weinberg et al., 2006; Chen et al., 2010), which supports the concept of “lipid sink” (Weinberg et al., 2006). Another study showed the effect of lipid rescue on the single-cell level toxicity by BPV and provided evidence for lipid-sink mechanism (Wanger et al., 2014). Mottram et al. came up with a possible mechanism of action by LE that was the salutary effects of it may be, in part, as a result of direct adjustive effect of fatty acids on cardiac sodium channels (Mottram et al., 2011). Weinberg proposed the activation of voltage-dependent calcium channel as another possible mechanism of rescue cardiotoxicity (Weinberg, 2012a,b) and calcium homeostasis was involved in the rescue of BPV-mediated cardiotoxicity by LE (Weinberg, 2012a,b). A recently published study demonstrated that use of LE accelerated the elimination of BPV and promoted its metabolism on pharmacokinetics in rats (Shi et al., 2013) to protect the heart. According to the results of previous studies,we found that concentrations of BPV were 0.77–1 mM could generate significant pro-apoptotic effects, so 1 mM BPV was chosen to observe the occurrence of apoptosis (Maurice et al., 2010; Perez-Castro et al., 2009; Yang et al., 2015.). Different concentrations of lipid emulsion had no effect on the growth of h9c2 cardiomyocytes, and even a slight pro-cell proliferation,1% LE more better (Yang et al., 2015;
Bai, 2013). After treated with drugs cells were cultivated for 24 h, because each drugs achieved the best effects at this time (Yang et al., 2015; Bai, 2013). Some limitations of this study should be noted. First, antiapoptotic effect of PI3K-Akt signaling pathway is also influenced by a number of other cell-specific enzymes or proteins and Akt regulates the cell activity not only by adjusting GSK-3, but also by acting on Forkhead, BAD, or other proteins, which are downstream signaling targets of Akt (Carnero and Paramio, 2014). Furthermore, although TWS is a specific blocker of GSK-3, it also has a slight effect on GSK-3␣. Third, the conclusions drawn from this in vitro experiment cannot be used to predict in vivo experiment, which needs to be further studied. Last, according to instructions of CsA, Atr, TWS and LY, its own had no effects on the cells, so we did not design related groups to exclude the impact. These need to be proved in the future. The impact of these factors on the experimental results cannot be ruled out. In summary, the present study suggests that LE mainly reduces the opening of mPTP by regulating the activity of the mitochondrial ion channel or signaling pathway, reducing the apoptosis factors or the generation of ROS, and increasing the expression of antiapoptotic factors to protect the heart. These results provide a novel insight into the molecular mechanisms underlying cardiac cytotoxicity by BPV and several possible new targets for the development of drugs. 5. Conclusions The data indicated that LE can reverse the apoptosis in cardiomyocytes by BPV and a mechanism of its action is by inhibition of mPTP opening through the PI3K/Akt/GSK-3 signaling pathway. Authors contributions Danni Lv helped conduct the study, analyze the data and prepare the manuscript and also attests to the integrity of the data and approves the final manuscript. She is the archival author. Zhixia Bai helped design the study and modified the manuscript Attestation and also attests to the integrity of the data and approves the final manuscript. Libin Yang helped collect the data and analyze the data and also attests to the integrity of the data and approves the final manuscript. Xiaohui Li helped collect the data and analyze the data and also attests to the integrity of the data and approves the final manuscript. Chen helped design the study and also attests to the integrity of the data and approves the final manuscript. Conflict of interest The authors declare no conflicts of interest. Funding This work was funded by the National Natural Science Foundation of China (No. 81241140). Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81241140).
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