About BES
Aims & Scope
SCI and IF
Editor in Chief
Editorial Board
Editorial Office
Articles
Advance Publication
Current Issues
Archive
Authors and Reviewer
Notice of Charge
Instruction for authors
Online Submission
Submission:FAQ
Revise:FAQ
Author Login
Reviewer Login
Editor-in-chief Login
Office Login
Subscribe
How to Subscribe
Links
NHFPC
China CDC
China doi
doi
PubMed
Elsevier
WANFANG data
CNKI
VIP data
Full-Text PDF: 116-126.pdf
Ginsenoside Rg1 Attenuates Isoflurane-induced Caspase-3 Activation via Inhibiting Mitochondrial Dysfunction
 

Original Article

Ginsenoside Rg1 Attenuates Isoflurane-induced Caspase-3 Activation via Inhibiting Mitochondrial Dysfunction*

MIAO Hui Hui1, ZHEN Yu1, DING Guan Nan1, HONG Fang Xiao1, XIE Zhong Cong2, and TIAN Ming1,#

1. Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; 2. Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard MedicalSchool, Charlestown, MA 02129-2060, USA

*This work was supported by 2013 Beijing NOVA Program (Z131107000413044).

#Correspondence should be addressed to Professor TIAN Ming, Tel: 86-10-63138362, E-mail: tian_mz@139.com

Biographical note of the first author: MIAO Hui Hui, female, born in 1982, Master’s degree candidate, attending physician, majoring in postoperative cognitive dysfunction.

 

Abstract

Objective  The inhalation anesthetic isoflurane has been shown to induce mitochondrial dysfunction and caspase activation, which may lead to learning and memory impairment. Ginsenoside Rg1 is reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rg1 can attenuate isoflurane-induced caspase activation via inhibiting mitochondrial dysfunction.

Methods  We investigated the effects of ginsenoside Rg1 at concentrations of 12.5, 25, and 50 μmol/L and pretreatment times of 12 h and 24 h on isoflurane-induced caspase-3 activation in H4 na?ve and stably transfected H4 human neuroglioma cells that express full-length human amyloid precursor protein (APP) (H4-APP cells). For mitochondrial dysfunction, we assessed mitochondrial permeability transition pore (mPTP) and adenosine-5’-triphosphate (ATP) levels. We employed Western blot analysis, chemiluminescence, and flowcytometry.

Results  Here we show that pretreatment with 50 μmol/L ginsenoside Rg1 for 12 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in H4-APP cells, while pretreatment with 25 and 50 μmol/L ginsenoside Rg1 for 24 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in both H4 na?ve and H4-APP cells.

Conclusion  These data suggest that ginsenoside Rg1 may ameliorate isoflurane-induced caspase-3 activation by inhibiting mitochondrial dysfunction. Pending further studies, these findings might recommend the use of ginsenoside Rg1 in preventing and treating isoflurane-induced neurotoxicity.

Key words: Ginsenoside Rg1; Isoflurane; Neurotoxicity; Mitochondrial dysfunction

Biomed Environ Sci, 2015; 28(2): 116-126        doi: 10.3967/bes2015.014         ISSN: 0895-3988

www.besjournal.com (full text)                CN: 11-2816/Q      Copyright ?2015 by China CDC

 

 

INTRODUCTION

A

fter surgery and anesthesia, some patients show a decline in cognitive function. This condition is called postoperative cognitive dysfunction (POCD), and is an impairment of memory, concentration, language comprehension, and social integration[1]. A distressing complication after surgery, POCD is independently associated with poor short-term and long-term outcomes. Anesthesia and surgery have been reported to induce cognitive dysfunction, to which patients with Alzheimer’s disease (AD) or older patients are susceptible[2], but it can develop in all age groups. However, there is still a lack of effective treatments for POCD, and many studies aim to find new and novel drugs to treat or prevent POCD[3].

       The exact pathogenesis of POCD is complex and multifactorial; however, it appears to share certain pathological markers with AD including amyloid β deposition, caspase activation, and cell apoptosis[4-5]. Animal models have shown that anesthetics, particularly inhalational anesthetics, can increase the development of these pathological markers in the brain[4]. Isoflurane, a common inhalational anesthetic, could induce caspase activation and apoptosis through the mitochondrial dependent apoptosis pathway[6], and current studies suggest that caspase activation (without apoptosis) can induce microglia activation, contributing to neuropathogenesis of AD[7]. Mitochondrial dysfunction can lead to caspase activation and apoptosis, potentially through the opening of mPTP, reductions in mitochondrial membrane potential, and decreases in ATP concentration, leading to cytotoxicity and impairment of learning and memory[8-10].

       Ginseng root has been used for several thousand years as a highly valued herb to treat weakness and fatigue, especially in China. The major active components of ginseng are ginsenosides, a diverse group of steroidal saponins, which target myriad tissues, producing an array of pharmacological responses[11]. Ginsenosides include Rb1, Rb2, Rc, Rd, Re, Rg1, and Rg2, with Rg1 being one of the most studied components. Ginsenoside Rg1 exerts a neuroprotective effect and is beneficial in AD models in vivo and in vitro[12-13]. Ginsenoside Rg1, used as a small-molecule drug, can improve learning and memory in animals[14-15], inhibit apoptosis induced by amyloid β[16], alleviate oxidative stress[17], inhibit β-secretase activity[13], maintain neuron activity at a normal level in the hippocampus of amouse model of amyloid-β-induced dementia[18], and improve neuralplasticity[19]. Moreover, ginsenoside Rg1 has recently been used to attenuate oligomeric amyloid β1-42-induced mitochondrial dysfunction[20]. Ginsenoside Rg1 may be a candidate neuroprotective agent for treating AD or aging and memory deterioration[19]. Having few side effects, it can also be used for prevention or treatment for all age groups. However, whether ginsenoside Rg1 has any protective effect on isoflurane-induced neurotoxicity and mitochondrial dysfunction has not yet been reported. In this study, we assessed the effects of different doses and pretreatment times of ginsenoside Rg1 on isoflurane-induced caspase-3 activation in H4 na?ve and H4-APP cells. In addition, we performed mechanistic studies to determine whether ginsenoside Rg1 can attenuate isoflurane-induced caspase-3 activation via inhibiting mPTP opening and increasing ATP concentration.

MATERIALS AND METHODS

Cells

We employed H4 na?ve and H4-APP cells. The cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) containing 9% heat-inactivated fetal calfserum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L-glutamine; H4-APP cells were supplemented with 220 μg/mL G418.

Treatments for H4 Na?ve and H4-APP Cells

Isoflurane was delivered from an anesthesia machine to a sealed plastic box in a 37 °C incubator containing six-well plates seeded with one million cells in 1.5 mL cell culture media. A compact anesthesia monitor (Datex-Ohmeda, GE Healthcare, Finland) was used to continuously monitor the delivered concentrations of CO2, O2, and isoflurane. Cells were treated with 2% isoflurane plus 21% O2 and 5% CO2 for 6 h, as described by Zhang et al.[21], for the purpose of measuring caspase-3 activation. The cultured cells were treated for 3 h in the studies, to measure mPTP opening and ATP levels without cell death, as described by Zhang et al.[22]. Ginsenoside Rg1 was obtained from the National Institutes for Food and Drug Control (Beijing, China) in the form of white powder-like crystals, and has a molecular weight of 800 and general formula C42H72O14. In the interaction experiments, ginsenoside Rg1 was dissolved in DMEM without serum at 4 °C. Cells were pretreated with 12.5, 25, or 50 μmol/L ginsenoside Rg1 for 12 h or 24 h or DMEM as control before isoflurane treatment, as well as during isoflurane treatment.

Cell Lysis and Protein Amount Quantification

Cell pellets were detergent-extracted on ice using an immunoprecipitation buffer (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A). The lysates were collected, and centrifuged at 13,000 rpm for 15 min. Total protein content was determined using a bicinchoninic acid protein assay kit (Thermo scientific, Rockford, USA).

Western Blot Analysis

The harvested H4 na?ve and H4-APP cells were subjected to Western blot analyses as described by Zhang et al.[22]. A caspase-3 antibody (1:1000 dilution; Cell Signaling Technology, Inc., Danvers, MA) was used to recognize full-length (FL) caspase-3 (35-40 kDa) and caspase-3 fragment (17-20 kDa) resulting from cleavage at aspartate position 175. Antibody anti-β-actin(1:10,000, Sigma, St. Louis, MO) was used to detect β-actin (42 kDa). Results were obtained from three independent experiments. The intensity of the signals was analyzed using the Quantity One program (version 4.62). Caspase-3 normalization was performed by determining the ratio of caspase-3 fragment to FL caspase-3, and changes in levels of caspase-3 in treated cells were presented as percentages of the corresponding levels in control cells.

ATP Measurement

We employed an ATP determination kit (Invitrogen, Carlsbad, CA) in experiments to detect ATP levels according to a protocol provided by the company. Briefly, cells were incubated in six-well plates for the desired pretreated times with ginsenoside Rg1. The cells were then exposed to isoflurane treatment. At the end of the treatment, the amount of fluorescence was measured and the levels of ATP in the experimental samples were calculated by comparison with a standard curve, made from samples containing known concentrations of ATP.

Flowcytometric Analysis of mPTP Opening

Cells were treated with 2% isoflurane for 3 h. The opening of mPTP was determined by flowcytometry, using a MitoProbe? transition pore assay kit (Invitrogen, Carlsbad, CA). In normal conditions, non-fluorescent acetoxymethyl ester (AM) of calcein dye (calcein AM) and cobalt can enter the cell. The AM groups are cleaved from calcein via non-specific esterase, and calcein then fluoresces in both the cytosol and mitochondria. Cobalt can quench the cytosolic calcein fluorescence. However, cobalt cannot enter healthy mitochondria freely, and therefore cannot quench the mitochondrial calcein signal. When the mPTP open, cobalt enters through it and subsequently quenches the mitochondrial calcein signal. Flowcytometry was used to detect the amount of cells that exhibit quenched calcein signals inside the mitochondria. The position of the curves indicates the amount of such cells, and thus indicates the opening of mPTP. Ionomycin was used as a positive control for the opening of mPTP. Dead cells and debris were excluded from analysis by setting gates on forward and side angle light scatter.

Statistical Analysis

All numerical results were expressed as mean±standard deviation. Data were analyzed by analysis of variance (ANOVA) followed by analysis of significance (least significant difference test) for multiple time points groups. Probability values <0.05 were considered statistically significant. Statistical analysis was carried out using SPSS (version 17.0 for Windows).

RESULTS

Effect of Ginsenoside Rg1 with 12 h Pretreatment on Isoflurane-induced Caspase-3 Activation in H4-APP and H4 Na?ve Cells

The H4-APP cells were treated with 12.5, 25, 50 μmol/L ginsenoside Rg1 or DMEM for 12 h followed by 2% isoflurane or control condition for 6 h. The cells were harvested at the end of the experiment and were subjected to Western blot analysis. Quantification of the Western blot (Figure 1B) revealed that isoflurane (Bar 5) led to caspase-3 activation, as compared with the control condition (Bar 1) in H4-APP cells: 7.23 vs. 1.00 fold (*P=0.000). Treatment with 50 μmol/L Rg1 (Bar 8) attenuated isoflurane-induced caspase-3 activation (Bar 5): 4.02 vs. 7.23 fold (#P=0.029). These findings suggest that pretreatment with 50 μmol/L ginsenoside Rg1 for  12 h might mitigate isoflurane-induced caspase-3 activation in H4-APP cells.

Next, we assessed whether ginsenoside Rg1 pretreated for 12 h could also attenuate isoflurane-induced caspase-3 activation in H4 na?ve cells, which have lower amyloid β levels than H4-APP cells. Quantification of the Western blot (Figure 1D) showed that isoflurane (Bar 5) led to caspase-3 activation, as compared with the control condition (Bar 1) in H4 na?ve cells: 4.75 vs. 1.00 fold (*P=0.000). Treatment with 12.5, 25, and 50 μmol/L Rg1 (Bars 6, 7, and 8) did not attenuate isoflurane-induced caspase-3 activation (Bar 5) in H4 na?ve cells: 4.47, 4.29, and 4.21, respectively, vs. 4.75 fold (P=0.60, 0.45, and 0.31, respectively). These results showed that ginsenoside Rg1 did not mitigate isoflurane-induced caspase-3 activation in H4 na?ve cells. Taken together, these findings suggest that pretreatment with 50 μmol/L ginsenoside Rg1 for  12 h might have different effects on isoflurane-induced caspase-3 activation in H4-APP and H4 na?ve cells.

Effect of Ginsenoside Rg1 with 24 h Pretreatment on Isoflurane-induced Caspase-3 Activation in H4-APP and H4 Na?ve Cells We performed relevance studies by pretreatment with 12.5, 25, and 50 μmol/L ginsenoside Rg1 for 24 h in H4-APP cells. Quantification of the Western blot (Figure 2B) showed that isoflurane (Bar 5) led to caspase-3 activation, as compared with the control condition (Bar 1) in H4-APP cells: 8.16 vs. 1.00 fold (*P=0.001). Treatment with 12.5 μmol/L Rg1 (Bar 6) did not attenuate isoflurane-induced caspase-3 activation (Bar 5): 5.21 vs. 8.16 fold (P=0.10), but treatment with 25, and 50 μmol/L Rg1 (Bars 7 and 8) attenuated isoflurane-induced caspase-3 activation: 4.30 and 2.99, respectively, vs. 8.16 fold (#P=   0.036, 0.007, respectively). These findings suggest that pretreatment with 25 and 50 μmol/L ginsenoside Rg1 for 24 h might mitigate isoflurane-induced caspase-3 activation in H4-APP cells.

 

Figure 1. Effect of ginsenoside Rg1 with 12 h pretreatment on isoflurane-induced caspase-3 activation in H4-APP and H4 na?ve cells. (A) Western blotting shows that treatment with 2% isoflurane for 6 h (Lane 5) induces caspase-3 activation as compared with the control condition (Lane 1) in H4-APP cells. 50 μmol/L Rg1 treatment attenuates isoflurane-induced caspase-3 activation (Lane 8) as compared with isoflurane treatment (Lane 5) in H4-APP cells. (B) Quantification of the Western blot shows that isoflurane (Bar 5) induces caspase-3 activation, as compared with the control condition (Bar 1) in H4-APP cells. Treatment with 50 μmol/L Rg1 (Bar 8) attenuates isoflurane-induced caspase-3 activation, as compared with isoflurane treatment (Bar 5) in H4-APP cells. (C) Western blotting shows that treatment with 2% isoflurane for 6 h (Lane 5) induces caspase-3 activation, as compared with the control condition (Lane 1) in H4 na?ve cells, but that treatment with 12.5, 25, or 50 μmol/L Rg1 does not attenuate isoflurane-induced caspase-3 activation (Lanes 6, 7, and 8), as compared with isoflurane treatment (Lane 5) in H4 na?ve cells. (D) Quantification of the Western blot shows that isoflurane (Bar 5) induces caspase-3 activation, as compared with the control condition (Bar 1) in H4 na?ve cells, but that treatment with 12.5, 25, and 50 μmol/L Rg1 (Bars 6, 7, and 8) does not attenuate isoflurane-induced caspase-3 activation, as compared with isoflurane treatment (Bar 5) in H4 na?ve cells. *vs. control group, P<0.05, #vs. isoflurane treatment group, P<0.05.

 

 

Next, we performed the same treatment in H4 na?ve cells. Quantification of the Western blot (Figure 2D) showed that isoflurane (Bar 5) led to caspase-3 activation, as compared with the   control condition (Bar 1) in H4 na?ve cells: 4.57 vs. 1.00 fold (*P=0.000). Treatment with 12.5 μmol/L Rg1 (Bar 6) did not attenuate isoflurane-induced caspase-3 activation (Bar 5): 4.09 vs. 4.57 fold (P=0.53), but treatment with 25 and 50 μmol/L Rg1 (Bars 7 and 8) attenuated isoflurane-induced caspase-3 activation (Bar 5): 2.95 and 2.70, respectively, versus 4.57 fold (#P=0.044 and 0.024, respectively). Taken together, these findings suggest that pretreatment with 25 and 50 μmol/L ginsenoside Rg1 for 24 h might have similar effects in affecting isoflurane-induced caspase-3 activation between the H4-APP and H4 na?ve cells.

Effect of Ginsenoside Rg1 with 12 h Pretreatment on Isoflurane-induced Mitochondrial Dysfunction in H4-APP and H4 Na?ve Cells Ginsenoside Rg1 can attenuate isoflurane- induced caspase-3 activation, and isoflurane-induced caspase-3 activation may result from isoflurane-induced mitochondrial dysfunction. Next, we asked whether ginsenoside Rg1 can attenuate isoflurane-induced caspase-3 activation through the mitochondrial pathway. To determine the effect of Rg1 on mitochondrial function and properties, we measured the ATP level and mPTP opening.

 

Figure 2. Effect of ginsenoside Rg1 with 24 h pretreatment on isoflurane-induced caspase-3 activation in H4-APP and H4 na?ve cells. (A) Western blotting shows that treatment with 2% isoflurane for 6 h (Lane 5) induces caspase-3 activation, as compared with the control condition (Lane 1) in H4-APP cells. Treatment with 25 and 50 μmol/L Rg1 attenuates isoflurane-induced caspase-3 activation (Lanes 7 and 8), as compared with isoflurane treatment (Lane 5) in H4-APP cells. (B) Quantification of the Western blot shows that treatment with 25 and 50 μmol/L Rg1 (Bars 7 and 8) attenuates the isoflurane-induced caspase-3 activation (Bar 5). (C) Western blotting shows that treatment with 2% isoflurane for 6 h (Lane 5) induces caspase-3 activation, as compared with the control condition (Lane 1) in H4 na?ve cells. Treatment with 25 and 50 μmol/L Rg1 attenuates isoflurane-induced caspase-3 activation (Lanes 7 and 8), as compared with isoflurane treatment (Lane 5) in H4 na?ve cells. (D) Quantification of the Western blot shows that treatment with 25 and 50 μmol/L Rg1 (Bars 7 and 8) attenuates isoflurane-induced caspase-3 activation, as compared with isoflurane treatment. *vs. control group, P<0.05, #vs. isoflurane treatment group, P<0.05.

 

The H4-APP cells were treated with 12.5, 25, and 50 μmol/L ginsenoside Rg1 or DMEM for 12 h followed by 2% isoflurane or control conditions for 3 h, which would decrease ATP levels without cell death[22]. As shown in Figure 3A, ATP generation was decreased for cells treated with isoflurane (Bar 5), as compared with the control group (Bar 1): 0.41 vs. 1.00 fold (*P=0.000). However, after pretreatment with Rg1 for 12 h, the ATP levels were increased compared with the isoflurane-treated group, especially for cells pretreated with 50 μmol/L Rg1 (Bar 8): 0.68 vs. 0.41 fold (#P=0.019). Second, we measured mPTP opening. Flow cytometry of calcein AM and cobalt showed that treatment with 50 μmol/L Rg1 (Figure 4A, Peak 5) led to reductions in isoflurane-induced mPTP opening (Figure 4A, Peak 2), as evidenced by the right shift of the curve, whereas treatment with 12.5 and       25 μmol/L Rg1 (Figure 4A, Peaks 3 and 4) did not affect the opening of mPTP. Quantification of the fluorescence intensity (Figure 4B) showed that     50 μmol/L Rg1 (Bar 5) led to reductions in isoflurane-induced mPTP opening: 3.25 vs. 1.39 (#P=0.000). The change of mPTP opening correlated with the change of ATP level in H4-APP cells. Overall, the change of mitochondrial function correlated with the change of caspase-3 activation for 50 μmol/L ginsenoside Rg1 pretreatment in H4-APP cells.

       The H4 na?ve cells were treated in the same way. As shown in Figure 3B, ATP generation treated with isoflurane (Bar 5) decreased, as compared with the control group (Bar 1): 0.43 vs. 1.00 fold (*P=0.002) in H4 na?ve cells. However, pretreatment with 12.5, 25, and 50 μmol/L Rg1 (Bars 6, 7, and 8) for 12 h did not affect the ATP levels, as compared with the isoflurane-treated group (Bar 5): 0.52, 0.58, and 0.65, respectively, versus 0.43 fold (P=0.56, 0.35, and 0.17, respectively). Flow cytometry of calcein AM and cobalt showed that the treatment with 12.5 and   25 μmol/L Rg1 (Figure 4C, Peaks 3 and 4) did not lead to reductions in the isoflurane-induced mPTP opening (Figure 4C, Peak 2), whereas pretreatment with 50 μmol/L Rg1 (Figure 4C, Peak 5) led to a right shift of the curve. Quantification of the fluorescence intensity (Figure 4D) showed that 50 μmol/L Rg1 (Bar 5) led to reductions in isoflurane-induced mPTP opening (Bar 2): 3.13 vs. 1.23 (#P=0.000) in H4 na?ve cells. The change of ATP level correlated with the change of caspase-3 activation, but the change of mPTP opening was different from the changes of ATP level and caspase-3 activation for 50 μmol/L ginsenoside Rg1 pretreatment in H4 naive cells.

Effect of Ginsenoside Rg1 with 24 h Pretreatment on Isoflurane-induced Mitochondrial Dysfunction in H4-APP and H4 Na?ve Cells We performed relevance studies by pretreatment with 12.5, 25, and 50 μmol/L ginsenoside Rg1 for 24 h in H4-APP cells. As can   be seen in Figure 5A, ATP generation treated    with isoflurane (Bar 5) decreased, as compared with the control group (Bar 1): 0.13 vs. 1.00 fold (*P=0.001). When cells were pretreated with 25 and 50 μmol/L Rg1 for 24 h, ATP levels (Bars 7 and 8) increased, as compared with the isoflurane-treated group (Bar 5): 0.72 and 0.89, respectively, versus 0.13 fold (#P=0.01 and 0.002, respectively). Flow cytometry of calcein AM and cobalt (Figure 6A) showed that treatment with 25 and 50 μmol/L Rg1 (Figure 6A, Peaks 4 and 5) led to a reduction in isoflurane-induced mPTP opening (Figure 6A, Peak 2). Quantification of fluorescence intensity (Figure 6B) showed that 25 and 50 μmol/L Rg1 (Bars 4 and 5) led to reductions in isoflurane-induced mPTP opening (Bar 2): 4.88 and 6.49 versus 1.37 (#P=0.002 and 0.000, respectively).

 

 

 

Figure 3. Effect of 12 h pretreatment with ginsenoside Rg1 on ATP levels in H4-APP and H4 na?ve cells. (A) Isoflurane (Bar 5) reduces ATP level, as compared with the control condition (Bar 1) in H4-APP cells. Pretreatment with 50 μmol/L Rg1 for 12h (Bar 8) increases the ATP level, as compared with the isoflurane-treated group (Bar 5) in H4-APP cells. (B) Isoflurane (Bar 5) reduces ATP level as compared with the control condition (Bar 1) in H4 na?ve cells. Pretreatment with 12.5, 25, and 50 μmol/L Rg1 for 12 h (Bars 6, 7, and 8) does not increase ATP levels, as compared with the isoflurane-treated group (Bar 5) in H4 na?ve cells. *vs. control group, P<0.05, #vs. isoflurane treatment group, P<0.05.

 

 

For H4 na?ve cells (Figure 5B), ATP generation decreased after treatment with isoflurane (Bar 5), as compared with the control group (Bar 1): 0.25 vs. 1.00 fold (*P=0.000). After pretreatment with 25 and 50 μmol/L Rg1 for 24 h, ATP levels (Bars 7 and 8) were increased, compared with that of the isoflurane-treated group (Bar 5) in H4 na?ve cells: 0.62 and 0.82, respectively, versus 0.25 fold (#P=0.021 and 0.001, respectively). Flow cytometry of calcein AM and cobalt (Figure 6C) showed that treatment with 25 and 50 μmol/L Rg1 (Figure 6C, Peaks 4 and 5) led to reductions in isoflurane- induced mPTP opening (Figure 6C, Peak 2) in H4 na?ve cells. Quantification of the fluorescence intensity (Figure 6D) showed that 25 and 50 μmol/L Rg1 (Bars 4 and 5) led to reductions in isoflurane- induced mPTP opening(Bar 2) in H4 na?ve cells: 4.51 and 5.98, respectively, versus 1.37 (#P=0.000 and 0.000, respectively). Taken together, these findings suggest that pretreatment with 25 and 50 μmol/L ginsenoside Rg1 for 24 h might have the same effects on ATP level and mPTP opening for H4-APP and H4 na?ve cells. The changes of mitochondrial function also correlated with the changes of caspase-3 activation in H4-APP and H4 na?ve cells.

 

 

 

Figure 4. Effect of ginsenoside Rg1 with 12 h pretreatment on isoflurane-induced mPTP opening in H4-APP and H4 na?ve cells. (A) Flow cytometry shows changes in calcein levels in mitochondria of H4-APP cells stained with calcein AM plus cobalt, indicating the opening of mPTP. Peak 1: treatment with ionomycin (positive control of mPTP opening); Peak 2: treatment with isoflurane; Peak 3: treatment with isoflurane plus 12.5 μmol/L Rg1; Peak 4: treatment with isoflurane plus 25 μmol/L Rg1; Peak 5: treatment with isoflurane plus 50 μmol/L Rg1. Treatment with 50 μmol/L Rg1 attenuates isoflurane-induced opening of mPTP in H4-APP cells, as demonstrated by the right shift of the curve. (B) Quantification of fluorescence intensity shows that 50 μmol/L Rg1 (Bar 5) leads to a reduction in isoflurane-induced mPTP opening (Bar 2) in H4-APP cells. (C) Flow cytometry shows changes in calcein levels in mitochondria of H4 na?ve cells. Peak 1: treatment with ionomycin; Peak 2: treatment with isoflurane; Peak 3: treatment with isoflurane plus 12.5 μmol/L Rg1; Peak 4: treatment with isoflurane plus 25 μmol/L Rg1; Peak 5: treatment with isoflurane plus 50 μmol/L Rg1. Treatment with 50 μmol/L Rg1 attenuates isoflurane-induced opening of mPTP in H4 na?ve cells, as demonstrated by the right shift of the curve. (D) Quantification of fluorescence intensity shows that 50 μmol/L Rg1 (Bar 5) leads to a reduction in isoflurane-induced mPTP opening (Bar 2) in H4 na?ve cells. #vs. isoflurane treatment group, P<0.05.

 

 

DISCUSSION

Current studies have shown that the common inhalation anesthetic isoflurane might induce caspase-3 activation and neurotoxicity in vitro[23] and in vivo[24], which may lead to learning and memory impairment in mice[25] and cognitive dysfunction in human beings[26]. Our results are consistent with the published observation that isoflurane results in caspase-3 activation. Mitochondria function as ‘cellular power plants’ because they generate most of the cellular ATP, which is used as a source of chemical energy. Specifically, isoflurane may induce caspase-3 activation through the mitochondrial pathway. Zhang et al.[22] suggest that isoflurane-induced opening of mPTP could be among the underlying mechanisms by which isoflurane induces caspase-3 activation and neurotoxicity. Cyclosporin A, a blocker of mPTP opening[27-29], can attenuate isoflurane-induced mitochondrial dysfunction and caspase-3 activation. However, in the search for a strategy to prevent and treat isoflurane neurotoxicity, cyclosporin A is not routinely used in patients, owing to its nephrotoxicity, hepatotoxicity, and cardiotoxicity side effects[30]. Therefore, it is important to assess whether other mPTP inhibitors can also attenuate isoflurane-induced neurotoxicity.

As one of the main active ingredients in ginseng, Rg1 is generally regarded to be beneficial for neurodegenerative diseases, with few side effects. Previous studies have demonstrated that ginsenoside Rg1 has effective anti-amnesic, anti-aging, and anti-oxidant effects[19,31], but it is unclear whether Rg1 has a protective effect on isoflurane-induced mitochondrial dysfunction. We have found that ginsenoside Rg1 can attenuate isoflurane-induced caspase-3 activation in vitro. These results suggest that ginsenoside Rg1 might attenuate isoflurane-induced neurotoxicity. In our mechanistic studies, we have shown that ginsenoside Rg1 can inhibit isoflurane-induced decreases in ATP concentration and mPTP opening. Zhang et al.[22] have revealed that isoflurane might induce caspase activation, apoptosis, and learning and memory impairment by inducing mitochondrial dysfunction (e.g., mPTP opening and decrease in ATP concentrations). Collectively, these findings suggest that ginsenoside Rg1 might mitigate isoflurane- induced caspase-3 activation by inhibiting mitochondrial dysfunction, although further studies are required. Note that treatment with 2% isoflurane for a short duration (e.g., 3 h) induces opening of mPTP and reduction of ATP without caspase-3 activation and cell death, whereas treatment with 2% isoflurane for a long duration (e.g., 6 h) induces caspase-3 activation and cell death[32]. These results suggest that isoflurane-induced mitochondrial dysfunction (especially mPTP opening) might precede isoflurane-induced cytotoxicity. For the outcomes of H4 na?ve cells, 50 μmol/L ginsenoside Rg1 treated for 12 h did not attenuate caspase-3 activation or ATP level, but the change in mPTP had already occurred, whereas extending the treatment to 24 h induced changes in caspase-3 activation and ATP levels. It is interesting that pretreatment with 50 μmol/L ginsenoside Rg1 for 12 h have different effects on isoflurane-induced caspase-3 activation in H4-APP and H4 na?ve cells, so 50 μmol/L ginsenoside of the curve. (D) Quantification of fluorescence intensity shows that 25 and 50 μmol/L Rg1 (Bars 4 and 5) lead to reductions in isoflurane-induced mPTP opening (Bar 2) in H4 na?ve cells. #versus isoflurane treatment group, P<0.05.

 

 

 

Figure 5. Effect of pretreatment with ginsenoside Rg1 for 24 h on ATP levels in H4-APP and H4 na?ve cells. (A) Isoflurane (Bar 5) reduces ATP level, as compared with the control condition (Bar 1) in H4-APP cells. Pretreatment with 25 and 50 μmol/L Rg1 for 24 h (Bars 7 and 8) increases ATP levels, as compared with the isoflurane-treated group (Bar 5) in H4-APP cells. (B) Isoflurane (Bar 5) reduces ATP level, as compared with the control condition (Bar 1) in H4 na?ve cells. Pretreatment with 25 and 50 μmol/L Rg1 for 24 h (Bars 7 and 8) increases ATP levels, as compared with the isoflurane-treated group (Bar 5) in H4 na?ve cells. *vs. control group, P<0.05, #vs. isoflurane treatment group, P<0.05.

 

 

Figure 6. Effect of pretreatment with ginsenoside Rg1 for 24h on isoflurane-induced mPTP opening in H4-APP and H4 na?ve cells. (A) Flow cytometry shows changes in H4-APP cells. Peak 1: treatment with ionomycin; Peak 2: treatment with isoflurane; Peak 3: treatment with isoflurane plus 12.5 μmol/L Rg1; Peak 4: treatment with isoflurane plus 25 μmol/L Rg1; Peak 5: treatment with isoflurane plus 50 μmol/L Rg1. Treatment with 25 and 50 μmol/L Rg1 attenuates isoflurane-induced opening of mPTP in H4-APP cells, as demonstrated by the right shift of the curve. (B) Quantification of fluorescence intensity shows that 25 and 50 μmol/L ginsenoside Rg1 (Bars 4 and 5) lead to reductions in isoflurane-induced mPTP opening (Bar 2) in H4-APP cells. (C) Flow cytometry shows changes in H4 na?ve cells. Peak 1: treatment with ionomycin; Peak 2: treatment with isoflurane; Peak 3: treatment with isoflurane plus 12.5 μmol/L Rg1; Peak 4: treatment with isoflurane plus 25 μmol/L Rg1; Peak 5: treatment with isoflurane plus 50μmol/L Rg1. Treatment with 25 and 50 μmol/L Rg1 attenuates isoflurane-induced opening of mPTP in H4 na?ve cells, as demonstrated by the right shift

 

Rg1 for 12 h may only attenuate the isoflurane induced caspase-3 activation when levels of Aβ are already elevated (H4-APP cells). In our study, ginsenoside Rg1 treatment resulted in dose- and time-dependent changes in caspase-3 activation and mitochondrial function. Ginsenoside Rg1 treatment affects both H4 na?ve and H4-APP cells, so might affect not only patients with AD or older patients but also all age groups.

The studies have a few limitations. First, we only performed the research in vitro without using animal models or clinical trials, and we did not assess whether ginsenoside Rg1 can ameliorate isoflurane- induced learning and memory impairment. However, findings from current studies show that ginsenoside Rg1 inhibited isoflurane-induced mitochondrial dysfunction and neurotoxicity would establish a system for future studies in animals and human beings. Second, in this study, we only measured caspase-3 activation. This is because studies have already shown that isoflurane can induce caspase-3 activation, apoptosis, amyloid β accumulation, and neuroinflammation[23-24,33-34]. In addition, a recent study by Burguillos et al.[7] has shown that caspase activation alone without apoptosis might still contribute to AD neuropathogenesis.

In conclusion, we have found that ginsenoside Rg1 can attenuate the common inhalation anesthetic isoflurane-induce caspase-3 activation in vitro. Furthermore, we have found that ginsenoside Rg1 can attenuate isoflurane-induced opening of mPTP and decreases in ATP concentration, and is both dose- and time-dependent.

These findings should lead to additional studies to determine the potential effects of anesthetics on POCD and AD neuropathogenesis, the underlying mechanisms, and strategies for prevention and treatment. This study demonstrates that ginsenoside Rg1, one of the main components of ginseng, has a protective effect in the maintenance of mitochondrial function, in addition to neuronal protection. The detailed effects of Rg1 on isoflurane-induced mitochondrial and neuronal dysfunction require further investigation. Our results provide significant evidence that this ancient herb may indeed hold potential benefit in preventing and treating POCD.

Received: September 27, 2014;

Accepted: December 19, 2014

References

1.  Xie G, Zhang W, Chang Y, et al. Relationship between perioperative inflammatory response and postoperative cognitive dysfunction in the elderly. Med Hypotheses, 2009; 73, 402-3.

2.  Bittner EA, Yue Y, Xie Z. Brief review: anesthetic neurotoxicity in the elderly, cognitive dysfunction and Alzheimer’s disease. Can J Anaesth, 2011; 58, 216-23.

3.  Ologunde R, Ma D. Do inhalational anesthetics cause cognitive dysfunction? Acta Anaesthesiol Taiwan, 2011; 49, 149-53.

4.  Wan Y, Xu J, Meng F, et al. Cognitive decline following major surgery is associated with gliosis, β-amyloid accumulation, and τ phosphorylation in old mice. Crit Care Med, 2010; 38, 2190-8.

5.  Dong Y, Zhang G, Zhang B, et al. The common inhalational anesthetic sevoflurane induces apoptosis and increases β-amyloid protein levels. Arch Neurol, 2009; 66, 620-31.

6.  Zhang Y, Dong Y, Wu X, et al. The mitochondrial pathway of anesthetic isoflurane-induced apoptosis. J Biol Chem, 2010; 285, 4025-37.

7.  Burguillos MA, Deierborg T, Kavanagh E, et al. Caspase signalling controls microglia activation and neurotoxicity. Nature, 2011; 472, 319-24.

8.  Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev, 2007; 87, 99-163.

9.  Sullivan PG, Rabchevsky AG, Waldmeier PC, et al. Mitochondrial permeability transition in CNS trauma: cause or effect of neuronal cell death? J Neurosci Res, 2005; 79, 231-9.

10.Mitchell P, Moyle J. Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphatase systems of rat liver mitochondria. Nature, 1965; 208, 147-51.

11.Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol, 1999; 58, 1685-93.

12.Fang F, Chen X, Huang T, et al. Multi-faced neuroprotective effects of Ginsenoside Rg1 in an Alzheimer mouse model. BiochimBiophys Acta, 2012; 1822, 286-92.

13.Wang YH, Du GH. Ginsenoside Rg1 inhibits β-secretase activity in vitro and protects against Aβ-induced cytotoxicity in PC12 cells. J Asian Nat Prod Res, 2009; 11, 604-12.

14.Mook-Jung I, Hong HS, Boo JH, et al. Ginsenoside Rb1 and Rg1 improve spatial learning and increase hippocampal synaptophysin level in mice. J Neurosci Res, 2001; 63, 509-15.

15.Shi YQ, Huang TW, Chen LM, et al. Ginsenoside Rg1 attenuates amyloid-β content, regulates PKA/CREB activity, and improves cognitive performance in SAMP8 mice. J Alzheimers Dis, 2010; 19, 977-89.

16.Gong L, Li SL, Li H, et al. Ginsenoside Rg1 protects primary cultured rat hippocampal neurons from cell apoptosis induced by β-amyloid protein. Pharm Biol, 2011; 49, 501-7.

17.Liu Q, Kou JP, Yu BY. Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-kB activation. Neurochem Int, 2011; 58, 119-25.

18.Chen YB, Zhang DP, Feng M, et al. Effects of ginsenoside Rg1 on nuclear factor-κ B activity in β amyloid protein-treated neural cells. Neural Regen Res, 2009; 4, 590-6.

19.Cheng Y, Shen LH, Zhang JT. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action. Acta Pharmacol Sin, 2005; 26, 143-9.

20.Huang T, Fang F, Chen L, et al. Ginsenoside Rg1 attenuates oligomeric Aβ(1-42)-induced mitochondrial dysfunction. Curr Alzheimer Res, 2012; 9, 388-95.

21.Zhang G, Dong Y, Zhang B, et al. Isoflurane-induced caspase-3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci, 2008; 28, 4551-60.

22.Zhang Y, Xu Z, Wang H, et al. Anesthetics isoflurane and desflurane differently affect mitochondrial function, learning, and memory. Ann Neurol, 2012; 71, 687-98.

23.Xie Z, Dong Y, Maeda U, et al. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid β protein levels. Anesthesiology, 2006; 104, 988-94.

24.Xie Z, Culley DJ, Dong Y, et al. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid β-protein level in vivo. Ann Neurol, 2008; 64, 618-27.

25.Bianchi SL, Tran T, Liu C, et al. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiol Aging, 2008; 29, 1002-10.

26.Zhang B, Tian M, Zhen Y, et al. The effects of isoflurane and desflurane on cognitive function in humans. Anesth Analg, 2012; 114, 410-15.

27.Nicolli A, Basso E, Petronilli V, et al. Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel. J Biol Chem, 1996; 271, 2185-92.

28.Norman KG, Canter JA, Shi M, et al. Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients. Mitochondrion, 2010; 10, 94-101.

29.Alessandri B, Rice AC, Levasseur J, et al. Cyclosporin A improves brain tissue oxygen consumption and learning/memory performance after lateral fluid percussion injury in rats. J Neurotrauma, 2002; 19, 829-41.

30.Rezzani R. Exploring cyclosporine A-side effects and the protective role-played by antioxidants: the morphological and immunohistochemical studies. Histol Histopathol, 2006; 21, 301-16.

31.Chen XC, Zhou YC, Chen Y, et al. Ginsenoside Rg1 reduces MPTP-induced substantia nigra neuron loss by suppressing oxidative stress. Acta Pharmacol Sin, 2005; 26, 56-62.

32.Wei H, Kang B, Wei W, et al. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res, 2005; 1037, 139-47.

33.Xie Z, Dong Y, Maeda U, et al. The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid β-protein accumulation. J Neurosci, 2007; 27, 1247-54.

34.Wu X, Lu Y, Dong Y, et al. The inhalation anesthetic isoflurane increases levels of proinflammatory TNF-α, IL-6, and IL-1β. Neurobiol Aging, 2012; 33, 1364-78.

 

 

 
Full-Text PDF: 116-126.pdf
Biomedical and Environmental Sciences ISSN 0895-3988 CN 11-2816/Q
Publish Under the Auspices of Chinese Center for Disease Control and Prevention
Distributed by Chinese Center for Disease Control and Prevention
All Rights Reserved by Chinese Center for Disease Control and Prevention
京ICP备11024750