Vitexin is a bioactive compound extracted from hawthorn leaves, which reduces blood pressure and has anti-inflammatory and potential anticancer effects. However, the mechanisms underlying the protective effects of vitexin against isoflurane-induced neurotoxicity remain elusive. Therefore, the aim of the present study was to investigate these mechanisms further. Sprague Dawley rats received 1.4% isoflurane in a 100% oxygen environment for 2 h. Human PC12 pheochromocytoma neurosecretory cells were exposed to 2% isoflurane for 12 h before they were treated with 1, 10 or 100 µM vitexin for a further 24 h. Vitexin inhibited the isoflurane-induced cell cytotoxicity and weakened isoflurane-induced neuroinflammation and oxidative stress pathways in PC12 cells. In addition, treatment with vitexin suppressed isoflurane-induced caspase-3 activation and increased β-secretase 1 levels in PC12 cells. Furthermore, vitexin treatment decreased the levels of isoflurane-induced cytosolic calcium and reactive oxygen species, and downregulated the expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) and glutamate ionotropic receptor NMDA type subunit 2B (NR2B) protein expression in isoflurane-treated PC12 cells. These results suggest that vitexin mediates its protective effects against isoflurane-induced neurotoxicity by targeting the TRPV1 and NR2B signaling pathways.
Since the discovery of inhalation anesthetics and their clinical application, researchers have gained an improved understanding of ‘inhalation anesthetics and inhalation anesthesia’ (
Recent studies investigating the mechanisms of neuron damage and protection in the central nervous system have made significant progress (
The state of consciousness is dependent on the electrophysiological characteristics of the central nervous system (
The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is universally expressed across the central nervous system, including the hippocampus, cerebral cortex and thalamus (
Glutamate excitotoxity is considered to be the primary mechanism underlying neuron injury induced by cerebral ischemia (
The rosaceous hawthorn plant is used as a digestion aid in traditional Chinese medicine (
A total of 30 male Sprague Dawley rats (250–300 g) were obtained from the Experimental Animal Center of the Central Hospital of Cangzhou (Hebei, China). The rats were maintained in 12 h dark/light cycles at 23±2°C with 55±5% humidity, and provided with food and water
A total of 30 Sprague Dawley rats were separated at random into the following five equally-sized treatment groups: i) Control; ii) isoflurane-treated; and iii) 1 mg/kg; iv) 3 mg/kg; and v) 10 mg/kg vitexin-treated groups, respectively. The isoflurane and vitexin-treated groups were exposed to 1.4% isoflurane (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in a 100% oxygen environment for 2 h. Following isoflurane treatment, the vitexin-treated group additionally received 1, 3 and 10 mg/kg vitexin (Sigma-Aldrich; Merck Millipore) for 30 min. The rats were euthanized using decollation under anesthesia. Samples of rat brain tissue slices were fixed in 10% formalin buffer overnight and then dehydrated using 90% ethanol for 1 h and 100% ethanol for 2 h. They were subsequently cleared with xylene for 2 h and then embedded in paraffin at 60°C.
Human PC12 pheochromocytoma neurosecretory cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 9% heat-inactivated fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Thermo Fisher Scientific, Inc.), and were maintained in an incubator at 37°C in 5% CO2 and with 95% humidity.
PC12 cells were seeded at a density of 1×104 cells/well in 96-well plates before they were exposed to 2% isoflurane for 12 h and then cultured with 1, 10 and 100 µM vitexin for 24 h. MTT solution (Beyotime Institute of Biotechnology, Haimen, China) was added into each well at a final concentration of 0.5 mg/ml and cells were subsequently incubated at 37°C for 4 h. Dimethyl sulfoxide solution (98%; 150 µl; Sangon Biotech Co., Ltd., Shanghai, China) was then added to each well. The optical density (OD) was read at 570 nm using the Universal Microplate Reader (Elx800; BioTek instruments, Inc., Winooki, VT, USA).
PC12 cells were seeded at a density of 1×104 cells/well in 96-well plates before they were exposed to 2% isoflurane for 12 h and then treated with 1, 10 and 100 µM vitexin for 24 h. PC12 cells were immediately collected and centrifuged at 4,000 ×
PC12 cells were seeded at a density of 2×106 cells/well in 6-well plates before they were exposed to 2% isoflurane for 12 h and then treated with 1, 10 and 100 µM vitexin for 24 h. PC12 cells were subsequently harvested in RIPA Lysis Buffer with protease inhibitors (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and total protein was extracted by centrifuging at 12,000 ×
PC12 cells were seeded at a density of 1×104 cells/well in 96-well plates before they were exposed to 2% isoflurane for 12 h and treated with 1, 10 and 100 µM vitexin for 24 h. PC12 cells were cultured with 2′,7′-dichlorofluorescein diacetate for 6 h, then incubated with cell lysis buffer (OxiSelect ROS assay kit; Cell Biolabs, Inc., San Diego, CA, USA) for 5 min at room temperature. The OD was read at 480/530 nm using the aforementioned microplate reader (Bio-Tek instruments, Inc.).
The levels of cytosolic calcium were determined as described previously (
Data are expressed as the mean ± standard deviation. Experiments for each treatment group was conducted in triplicate. Statistical analyses were performed using the Student's t-test and P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of vitexin is shown in
To determine whether the neuroprotective effects of vitexin following isoflurane treatment involves cell growth, the growth of human PC12 pheochromocytoma neurosecretory cells following treatment with isoflurane and vitexin was investigated. As shown in
Considering the key role of induced neuroinflammation in aged rodents (
Considering the key role of oxidative stress in isoflurane-induced PC12 cells, the neuroprotective effects of vitexin against isoflurane-induced GSH and SOD protein expression levels were investigated. As shown in
In order to investigate the neuroprotective effects of vitexin against isoflurane-induced caspase-3 activation, the protein expression levels of caspase-3 in isoflurane and vitexin-treated PC12 cells were determined using western blot analysis. Caspase-3 protein expression levels in isoflurane-treated PC12 cells were significantly reduced following treatment with 10 and 100 µM vitexin (P=0.0069 and 0.0033, respectively;
A previous study demonstrated that BACE promotes amyloid beta peptide production and affects the inhibition of hypomnesis (
To investigate the role of vitexin in preventing the isoflurane-induced increase in cytosolic calcium levels, the levels of cytosolic calcium in isoflurane-induced PC12 cells following vitexin treatment were investigated. As demonstrated in
In order to further investigate the neuroprotective role of vitexin in isoflurane-treated PC12 cells, TRPV1 protein expression levels were examined in isoflurane-treated PC12 cells following treatment with vitexin using western blot analysis. As shown in
The NR2B subunit is a fundamental regulatory subunit of the NMDA receptor and serves an important role in its structure and function (
Consistent with other inhaled anesthetics of the halogen family, isoflurane-mediated hyperpolarization of neurocytes decreases the excitability of the neural network (
Inhalation anesthetics used at concentrations higher than the clinical range may induce the following effects: Inhibition of the voltage susceptibility of Na+, K+ and Ca2+, thereby reducing the transmission of harmful ostensive stimuli; promote the hyperpolarization of the cell membranes; relieve the overload of Ca2+; analgesia; anesthesia; and protective cerebral functions (
Ca2+ influx, mediated by the voltage-gated calcium channel, is an important mechanism for activating the presynaptic membrane (
In the nervous tissues of mammals, the functional N-methyl-D-aspartate (NMDA) receptor consists of NR1 and NR2 subunits (
In conclusion, the present study demonstrated that vitexin mediates neuroprotective effects against isoflurane-induced neurotoxicity by targeting the TRPV1 and NR2B signaling pathways. This suggests that vitexin may be a strong candidate as a neurotoxicity drug. However, these results require validation in further studies.
The chemical structure of vitexin.
Vitexin protects against isoflurane-induced neurotoxicity in rat brain tissue slices. Tissue slices of (A) Control (B) isoflurane-treated, and isoflurane plus (C) 1 mg/kg (D) 3 mg/kg and (E) 10 mg/kg vitexin-treated groups (magnification, ×200).
Vitexin increases the growth of isoflurane-treated PC12 cells. MTT assay of PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group.
Vitexin protects against isoflurane-induced activation of pro-inflammatory cytokines. The protein concentrations of (A) TNF-α and (B) IL-6 in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin as determined by ELISA. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group. TNF-α, tumor necrosis factor-α; IL-6, interleukin-6.
Vitexin protects against isoflurane-induced oxidative stress. The protein concentrations of (A) GSH and (B) SOD as determined by ELISA, and the (C) cellular ROS levels in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin-treated PC12 cells. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group. GSH, glutathione synthetase; SOD, superoxide dismutase; ROS, reactive oxygen species.
Vitexin protects against isoflurane-induced caspase-3 activation. (A) Gel images and (B) quantitative analysis of caspase-3 protein expression in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin by western blot analysis. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group.
Vitexin protects against the isoflurane-induced increase in BACE levels. (A) Gel images and (B) quantitative analysis of BACE protein expression in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin using western blot analysis. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group. BACE, β-secretase 1.
Vitexin protects against the isoflurane-induced increase in cytosolic calcium levels. The fura-2 ratio in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group.
Vitexin protects against the isoflurane-induced increase in TRPV1 expression. (A) Gel images and (B) quantitative analysis of TRPV1 protein expression in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and100 µM vitexin using western blot analysis. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group. TRPV1, transient receptor potential cation channel subfamily V member 1.
Vitexin protects against isoflurane-induced NR2B expression. (A) Gel images and (B) quantitative analysis of NR2B protein expression in PC12 cells treated without (control) or with 2% isoflurane plus 0, 1, 10 and 100 µM vitexin using western blot analysis. ##P<0.01 vs. control group; **P<0.01 vs. the 0 µM vitexin-treated group. NR2B, glutamate ionotropic receptor NMDA type subunit 2B.