Chitosan Oligosaccharide Reduces Propofol Requirements and Propofol-Related Side Effects

Propofol is one of the main sedatives but its negative side effects limit its clinical application. Chitosan oligosaccharide (COS), a kind of natural product with anti-pain and anti-inflammatory activities, may be a potential adjuvant to propofol use. A total of 94 patients receiving surgeries were evenly and randomly assigned to two groups: 10 mg/kg COS oral administration and/or placebo oral administration before being injected with propofol. The target-controlled infusion of propofol was adjusted to maintain the values of the bispectral index at 50. All patients’ pain was evaluated on a four-point scale and side effects were investigated. To explore the molecular mechanism for the functions of COS in propofol use, a mouse pain model was established. The activities of Nav1.7 were analyzed in dorsal root ganglia (DRG) cells. The results showed that the patients receiving COS pretreatment were likely to require less propofol than the patients pretreated with placebo for maintaining an anesthetic situation (p < 0.05). The degrees of injection pain were lower in a COS-pretreated group than in a propofol-pretreated group. The side effects were also more reduced in a COS-treated group than in a placebo-pretreated group. COS reduced the activity of Nav1.7 and its inhibitory function was lost when Nav1.7 was silenced (p > 0.05). COS improved propofol performance by affecting Nav1.7 activity. Thus, COS is a potential adjuvant to propofol use in surgical anesthesia.


Introduction
Propofol (2,6-diisopropylphenol), as a sedative agent, has been used widely in the induction of surgical anesthesia [1]. However, propofol-induced side effects become apparent [2], including hypotension and respiratory depression [3]. Propofol-induced injection pain is a major issue for propofol as an anesthetic in surgery [4,5]. Various alternative and folk remedies have also been used effectively for many years [6][7][8]. Remifentanil preventing propofol-induced injection pain has been proved effective. However, the combination therapy will be affected by the time interval between remifentanil and propofol injection, as well as the dosage of remifentanil [4]. Lidocaine is often used before being injected with propofol. Lidocaine pretreatment or mixed with propofol has also been used successfully for preventing propofol-induced pain [9]. Although the effectiveness is obvious, the side effects of the medicine are also palpable [10,11].
Thus, it is critical to explore a new agent for preventing or treating pain disorders. Chitosan oligosaccharide (COS) is a polysaccharide mainly obtained from crustacean shells and consists of Figure 1. The effects of chitosan oligosaccharide (COS) on propofol requirements. All the selected subjects were evenly assigned to two groups before being injected with propofol: 10 mg/kg COS oral administration and 10 mg/kg placebo oral administration. After five min, propofol was started with step increases of 0.5 μg/mL/2.5 min until the patient lost consciousness. Propofol target-controlled infusion (TCI) was adjusted to maintain the values of bispectral index (BIS) at 50.

The Incidence of Propofol-Induced Injection Pain in the Subjects Undergoing Surgery
Propofol induces high-incidence pain during intravenous injection. However, few non-pharmacological methods have been applied to control propofol-induced injection pain. COS may be a potential natural product to control the pain. The effects of COS on propofol-induced injection pain were measured. As Table 1 shows, the incidence of propofol-induced pain at a four-point scale in the subjects undergoing surgery was higher in PG than in CG (p < 0.05). Furthermore, there was no toxic symptom of COS in all subjects. The results suggest that COS may inhibit the propofol-induced injection pain and can be a potential adjuvant to propofol use.

COS Pretreatment Reduces the Side Effects of Propofol
Besides propofol-induced injection pain, propofol can cause some other side effects. For instance, propofol use induces sedation and may have a significant effect on the pattern of upper airway obstruction [38]. Hypotension has been reported to be a common adverse effect caused by propofol, but there is no reliable method to determine which patients have the risk for propofol-induced hypotension [39]. Therefore, it is necessary to find a new method to control these side effects caused by propofol. Based on this idea, the effects of COS on these side effects were measured. Table 2 shows the most common side effects, which were found in both groups. The patients had lower inadequate ventilation in CG than in PG (p < 0.05). Similarly, the patients had a lower incidence of tachycardia and hypotension in CG than in PG (p < 0.05). Other side effects showed the similar incidences between two groups. However, there is no statistical significance of differences for bradypnea (p > 0.05), and no nausea or vomiting was found in both groups after seven-day surgery, although the symptoms were widely reported in propofol use [40,41]. The effects of chitosan oligosaccharide (COS) on propofol requirements. All the selected subjects were evenly assigned to two groups before being injected with propofol: 10 mg/kg COS oral administration and 10 mg/kg placebo oral administration. After five min, propofol was started with step increases of 0.5 µg/mL/2.5 min until the patient lost consciousness. Propofol target-controlled infusion (TCI) was adjusted to maintain the values of bispectral index (BIS) at 50.

The Incidence of Propofol-Induced Injection Pain in the Subjects Undergoing Surgery
Propofol induces high-incidence pain during intravenous injection.
However, few non-pharmacological methods have been applied to control propofol-induced injection pain. COS may be a potential natural product to control the pain. The effects of COS on propofol-induced injection pain were measured. As Table 1 shows, the incidence of propofol-induced pain at a four-point scale in the subjects undergoing surgery was higher in PG than in CG (p < 0.05). Furthermore, there was no toxic symptom of COS in all subjects. The results suggest that COS may inhibit the propofol-induced injection pain and can be a potential adjuvant to propofol use.

COS Pretreatment Reduces the Side Effects of Propofol
Besides propofol-induced injection pain, propofol can cause some other side effects. For instance, propofol use induces sedation and may have a significant effect on the pattern of upper airway obstruction [38]. Hypotension has been reported to be a common adverse effect caused by propofol, but there is no reliable method to determine which patients have the risk for propofol-induced hypotension [39]. Therefore, it is necessary to find a new method to control these side effects caused by propofol. Based on this idea, the effects of COS on these side effects were measured. Table 2 shows the most common side effects, which were found in both groups. The patients had lower inadequate ventilation in CG than in PG (p < 0.05). Similarly, the patients had a lower incidence of tachycardia and hypotension in CG than in PG (p < 0.05). Other side effects showed the similar incidences between two groups. However, there is no statistical significance of differences for bradypnea (p > 0.05), and no nausea or vomiting was found in both groups after seven-day surgery, although the symptoms were widely reported in propofol use [40,41].

Analysis of Mechanic Hyperalgesia
Intraplantar injection of 0.9% NaCl solution did not induce mechanical hyperalgesia and is regarded as a control group ( Figure 2) Intraplantar injection of CFA increased mechanical hyperalgesia of a mouse model by reducing its thresholds for pain ( Figure 2). Propofol and COS treatment decreased CFA-induced hyperalgesia ( Figure 2). The combination treatment of COS and propofol attenuated the hyperalgesia more than propofol used alone (p < 0.05). However, Nav1.7-silenced groups attenuated hyperalgesia significantly though COS and/or propofol no longer attenuated hyperalgesia ( Figure 2). There is no statistical significance of differences among the Nav1.7-silenced groups treated or untreated by COS and/or propofol (p > 0.05).

Analysis of Mechanic Hyperalgesia
Intraplantar injection of 0.9% NaCl solution did not induce mechanical hyperalgesia and is regarded as a control group ( Figure 2) Intraplantar injection of CFA increased mechanical hyperalgesia of a mouse model by reducing its thresholds for pain ( Figure 2). Propofol and COS treatment decreased CFA-induced hyperalgesia ( Figure 2). The combination treatment of COS and propofol attenuated the hyperalgesia more than propofol used alone (p < 0.05). However, Nav1.7-silenced groups attenuated hyperalgesia significantly though COS and/or propofol no longer attenuated hyperalgesia ( Figure 2). There is no statistical significance of differences among the Nav1.7-silenced groups treated or untreated by COS and/or propofol (p > 0.05). The threshold in an inflammatory pain model. There were 32 mouse pain models evenly assigned into four groups: PG group (received 10 mg/kg propofol treatment), PCOSG group (received both 10 mg/kg COS and propofol treatment), PIG group (Nav1.7-silenced model mouse received 10 mg/kg propofol treatment) and PCOSIG group (Nav1.7-silenced model mouse received both 10 mg/kg COS and propofol treatment). All data were presented as mean ± S.D. and n = 8 in each group. There is statistical significance of differences if p < 0.05.

Analysis of Thermal Hyperalgesia
Thermal hyperalgesia was found in CFA-induced mouse pain models but not in the mice only treated with 0.9% NaCl solution ( Figure 3A). COS reduced thermal hyperalgesia by increasing its latency ( Figure 3A). COS attenuated the mechanic hyperalgesia caused by propofol (p < 0.05). COS pretreatment resulted in insensitivity to the pain in a mouse model (p < 0.05). Comparatively, Nav1.7 silence also attenuated mechanic hyperalgesia significantly but COS no longer attenuated mechanic hyperalgesia ( Figure 3A). There is no statistical significance of differences among Nav1.7-silenced groups treated or untreated by COS (p > 0.05).
For thermal pain, there is statistical significance of differences for the jumping times between COS-treated and non-treated groups (p < 0.05, Figure 3B), suggesting that COS has better effects on thermal hyperalgesia than propofol used alone (p < 0.05). Notably, Nav1.7 silence reduced jumping times significantly but propofol and/or COS was not able to reduce jumping times ( Figure 3B). There was no statistical significance of differences between the groups treated by propofol and the combination therapy of propofol and COS when Nav1.7 was silenced (p > 0.05).
For a cold-plate test, Nav1.7 silence could not reduce the rearing times, and COS and propofol could not maintain reduction of rearing times on the cold plate ( Figure 3C). There is no statistical Figure 2. The threshold in an inflammatory pain model. There were 32 mouse pain models evenly assigned into four groups: PG group (received 10 mg/kg propofol treatment), PCOSG group (received both 10 mg/kg COS and propofol treatment), PIG group (Nav1.7-silenced model mouse received 10 mg/kg propofol treatment) and PCOSIG group (Nav1.7-silenced model mouse received both 10 mg/kg COS and propofol treatment). All data were presented as mean ± S.D. and n = 8 in each group. There is statistical significance of differences if p < 0.05.

Analysis of Thermal Hyperalgesia
Thermal hyperalgesia was found in CFA-induced mouse pain models but not in the mice only treated with 0.9% NaCl solution ( Figure 3A). COS reduced thermal hyperalgesia by increasing its latency ( Figure 3A). COS attenuated the mechanic hyperalgesia caused by propofol (p < 0.05). COS pretreatment resulted in insensitivity to the pain in a mouse model (p < 0.05). Comparatively, Nav1.7 silence also attenuated mechanic hyperalgesia significantly but COS no longer attenuated mechanic hyperalgesia ( Figure 3A). There is no statistical significance of differences among Nav1.7-silenced groups treated or untreated by COS (p > 0.05).
For thermal pain, there is statistical significance of differences for the jumping times between COS-treated and non-treated groups (p < 0.05, Figure 3B), suggesting that COS has better effects on thermal hyperalgesia than propofol used alone (p < 0.05). Notably, Nav1.7 silence reduced jumping times significantly but propofol and/or COS was not able to reduce jumping times ( Figure 3B). There was no statistical significance of differences between the groups treated by propofol and the combination therapy of propofol and COS when Nav1.7 was silenced (p > 0.05).
For a cold-plate test, Nav1.7 silence could not reduce the rearing times, and COS and propofol could not maintain reduction of rearing times on the cold plate ( Figure 3C). There is no statistical significance of differences among Nav1.7-silenced groups treated or untreated by COS and/or propofol (p > 0.05), suggesting that Nav1.7 is also not associated with cold pain.
Marine Drugs 2016, 4, 234 5 of 15 significance of differences among Nav1.7-silenced groups treated or untreated by COS and/or propofol (p > 0.05), suggesting that Nav1.7 is also not associated with cold pain.

The Protein Level of Voltage-Gated Sodium Channels (Nav)1.7 in Dorsal Root Ganglia (DRG) Neurons
The protein level of Nav1.7 was analyzed by Western blot. The results showed that Nav1.7 was at a low level when the mice were injected with 0.9% NaCl solution (Figures 4). CFA increased the protein level of Nav1.7 (p < 0.01) and there was statistical significance of differences between control and model groups (Figures 4). There was no change in protein level when the mice were treated with propofol and COS ( Figure 4) (p > 0.05), suggesting that propofol or COS cannot affect the protein level of Nav1.7.  The protein level of Nav1.7 was analyzed by Western blot. The results showed that Nav1.7 was at a low level when the mice were injected with 0.9% NaCl solution ( Figure 4). CFA increased the protein level of Nav1.7 (p < 0.01) and there was statistical significance of differences between control and model groups ( Figure 4). There was no change in protein level when the mice were treated with propofol and COS ( Figure 4) (p > 0.05), suggesting that propofol or COS cannot affect the protein level of Nav1.7.
Marine Drugs 2016, 4, 234 5 of 15 significance of differences among Nav1.7-silenced groups treated or untreated by COS and/or propofol (p > 0.05), suggesting that Nav1.7 is also not associated with cold pain.

The Protein Level of Voltage-Gated Sodium Channels (Nav)1.7 in Dorsal Root Ganglia (DRG) Neurons
The protein level of Nav1.7 was analyzed by Western blot. The results showed that Nav1.7 was at a low level when the mice were injected with 0.9% NaCl solution (Figures 4). CFA increased the protein level of Nav1.7 (p < 0.01) and there was statistical significance of differences between control and model groups (

COS Reduces the Activity of Nav1.7
To investigate the effects of COS on propofol performance for blocking Nav1.7 activities, the electrophysiological properties of Nav1.7 were compared by using whole-cell patch-clamp recordings. As shown in Figure 5A, propofol blocked Nav1.7 activities in a concentration-dependent manner and COS improved propofol blocking the channels ( Figure 5B). Resting channels were measured at a holding potential of −120 mV by test pulses to 0 mV applied at 0.1 Hz. The IC50 values for propofol were 231 ± 12 µM (Hill coefficient 1.8 ± 0.4, n = 10) and the values of the combination of COS and propofol were 165 ± 18 µM (Hill coefficient 1.1 ± 0.2, n = 10). Figure 5C showed that there was statistical significance of differences for the blocking potencies of resting Na + channels between the propofol and combined groups (p = 0.02, unpaired t-test). Figure 5D showed that COS enhanced the tonic block of inactivated Na + channels when compared to the group only treated with propofol (propofol, IC50 value 188 ± 10 µM; Hill coefficient 1.6 ± 0.2, n = 10; propofol and COS, IC50 value 121 ± 8 µM; Hill coefficient 1.3 ± 0.1, n = 10; p = 0.02, unpaired t-test). To investigate the effects of COS on propofol performance for blocking Nav1.7 activities, the electrophysiological properties of Nav1.7 were compared by using whole-cell patch-clamp recordings. As shown in Figure 5A, propofol blocked Nav1.7 activities in a concentration-dependent manner and COS improved propofol blocking the channels ( Figure 5B). Resting channels were measured at a holding potential of −120 mV by test pulses to 0 mV applied at 0.1 Hz. The IC50 values for propofol were 231 ± 12 μM (Hill coefficient 1.8 ± 0.4, n = 10) and the values of the combination of COS and propofol were 165 ± 18 μM (Hill coefficient 1.1 ± 0.2, n = 10). Figure 5C showed that there was statistical significance of differences for the blocking potencies of resting Na + channels between the propofol and combined groups (p = 0.02, unpaired t-test). Figure 5D showed that COS enhanced the tonic block of inactivated Na + channels when compared to the group only treated with propofol (propofol, IC50 value 188 ± 10 μM; Hill coefficient 1.6 ± 0.2, n = 10; propofol and COS, IC50 value 121 ± 8 μM; Hill coefficient 1.3 ± 0.1, n = 10; p = 0.02, unpaired t-test). were held at a holding potential of −120 mV and test pulses were stepped to 0 mV and applied at 0.1 Hz; (C), a tonic block of resting Nav1.7 channels by propofol and/or the combination of COS and propofol. Resting channels were measured at a holding potential of −120 mV; (D), a tonic block of inactivated Na+ channels by propofol and/or the combination of COS and propofol. Inactivated channels were induced by a 10 s pre-pulse to −70 mV followed by a 100 ms pulse at −120 mV and a test pulse to 0 mV. Peak amplitudes of Nav1.7 currents were normalized with respect to the peak amplitude in control solution and plotted against the concentration of propofol or a combination of propofol and COS.

COS Also Promotes Propofol-Produced Stabilization of Fast and Slow Inactivation
Fast inactivation was caused by 50 ms pre-pulses ranging from −120 to 0 mV in a five-mV step ( Figure 6A), and the remaining fraction of channels was measured with a 20 ms pre-pulse to 0 mV. were held at a holding potential of −120 mV and test pulses were stepped to 0 mV and applied at 0.1 Hz; (C), a tonic block of resting Nav1.7 channels by propofol and/or the combination of COS and propofol. Resting channels were measured at a holding potential of −120 mV; (D), a tonic block of inactivated Na+ channels by propofol and/or the combination of COS and propofol. Inactivated channels were induced by a 10 s pre-pulse to −70 mV followed by a 100 ms pulse at −120 mV and a test pulse to 0 mV. Peak amplitudes of Nav1.7 currents were normalized with respect to the peak amplitude in control solution and plotted against the concentration of propofol or a combination of propofol and COS.

COS Also Promotes Propofol-Produced Stabilization of Fast and Slow Inactivation
Fast inactivation was caused by 50 ms pre-pulses ranging from −120 to 0 mV in a five-mV step ( Figure 6A), and the remaining fraction of channels was measured with a 20 ms pre-pulse to 0 mV. Figure 6B showed that 100 µM propofol caused a ten-mV hyperpolarization shift of steady-state fast inactivation from V 1/2 of −75 ± 2 mV (n = 10) in control to V 1/2 of −85 ± 5 mV (n = 10) (p < 0.01). COS stabilized the fast inactivation and caused a ten-mV hyperpolarization shift of the steady-state fast inactivation of propofol (propofol: V 1/2 of −85 ± 5 mV; propofol and COS: V 1/2 of −95 ± 6 mV; n = 10) (p < 0.05). There is statistical significance of differences when compared with the combination treatment of COS and propofol (p < 0.05). Figure 6B showed that 100 μM propofol caused a ten-mV hyperpolarization shift of steady-state fast inactivation from V1/2 of −75 ± 2 mV (n = 10) in control to V1/2 of −85 ± 5 mV (n = 10) (p < 0.01). COS stabilized the fast inactivation and caused a ten-mV hyperpolarization shift of the steady-state fast inactivation of propofol (propofol: V1/2 of −85 ± 5 mV; propofol and COS: V1/2 of −95 ± 6 mV; n = 10) (p < 0.05). There is statistical significance of differences when compared with the combination treatment of COS and propofol (p < 0.05). Slow inactivation was caused by 10 s pre-pulses ranging from −120 to −10 mV in ten-mV step, followed by a 100 ms pulse at −120 mV, which allows recovery from fast inactivation, and followed by a test pulse to −10 mV. Propofol at 100 μM induced a small shift of the voltage dependency of slow inactivation of Nav1.7 (control: V1/2 of −20 ± 1 mV; propofol: V1/2 of −65 ± 2 mV n = 10; Figure 6C). In contrast, combination treatment caused the shift of slow inactivation when compared with only propofol used (propofol: V1/2 of −65 ± 2 mV, combined: V1/2 of −90 ± 4 mV, n = 10; Figure 6C). Neither propofol nor a combination of propofol and COS caused an apparent shift of the voltage-dependency of activation (data not shown).

COS Promotes Propofol Blocking Veratridine-Induced Persistent Sodium Current of Nav1.7
To understand the activity of the propofol and the combination of propofol and COS on the persistent Nav1.7 currents, tonic activation was created by adding 50 μM veratridine. Figure 7A shows that veratridine caused a prominent persistent current, which was stimulated by 50 ms pulses in cells at a holding potential of −120 mV. Figure 7B shows that COS promoted propofol blocking the persistent current. The calculated IC50 values of propofol were at 202 ± 27 μM, n = 8, and a combination of COS and propofol at 126 ± 47 μM (p = 0.03, unpaired t-test). Slow inactivation was caused by 10 s pre-pulses ranging from −120 to −10 mV in ten-mV step, followed by a 100 ms pulse at −120 mV, which allows recovery from fast inactivation, and followed by a test pulse to −10 mV. Propofol at 100 µM induced a small shift of the voltage dependency of slow inactivation of Nav1.7 (control: V 1/2 of −20 ± 1 mV; propofol: V 1/2 of −65 ± 2 mV n = 10; Figure 6C). In contrast, combination treatment caused the shift of slow inactivation when compared with only propofol used (propofol: V 1/2 of −65 ± 2 mV, combined: V 1/2 of −90 ± 4 mV, n = 10; Figure 6C). Neither propofol nor a combination of propofol and COS caused an apparent shift of the voltage-dependency of activation (data not shown).

COS Promotes Propofol Blocking Veratridine-Induced Persistent Sodium Current of Nav1.7
To understand the activity of the propofol and the combination of propofol and COS on the persistent Nav1.7 currents, tonic activation was created by adding 50 µM veratridine. Figure 7A shows that veratridine caused a prominent persistent current, which was stimulated by 50 ms pulses in cells at a holding potential of −120 mV. Figure 7B shows that COS promoted propofol blocking the persistent current. The calculated IC50 values of propofol were at 202 ± 27 µM, n = 8, and a combination of COS and propofol at 126 ± 47 µM (p = 0.03, unpaired t-test).

Discussion
Present findings indicated that COS greatly inhibited the incidence and severity of propofol-induced injection pain if the patients received 10 mg/kg COS via oral administration before being injected with propofol (Table 1). No toxic symptom or fewer side effects were observed in all the patients treated with COS ( Table 2). The results suggest that COS may be a potential natural adjuvant to improve propofol performance.
From pain analyses, an animal pain model was successfully established after CFA injection. The mouse model had mechanical and thermal hyperalgesia because of inflammatory pain, which was tested by a von Frey filament assay and hot/cold plate assay. Propofol is one kind of medicine mainly used for decreasing human pain. Present findings indicated that Nav1.7 was increased in CFA-induced hyperalgesia, which suggested that Nav1.7 plays a critical role in inflammatory pain. Subsequent work showed that COS and propofol reduced pain thresholds.
Injection pain is a normal unwanted adverse effect for propofol use. The side effects can be reduced when combined with COS because they can produce more analgesic efficacy [42]. Another study also used COS as an anesthesia supplement of propofol injection, which was successfully used in topical local anesthesia for surgery on a child [43]. All the results suggest that propofol and COS may have synergistic functions. However, the complementary functions remain unclear. Since many Navs play important roles in pain [44,45] and neural disorders [46,47], we want to explore the effects of combined medicine on the level of Navs. The mutant SCN9A gene-encoding Nav1.7 caused insensitivity to pain in mammals [35]. Furthermore, many pyrrolo-benzo-1,4-diazine derivatives were synthesized to inhibit the activity of Nav1.7, and showed anti-nociceptive oral efficacy in an inflammatory pain model [48].
CFA increasing the expression of Nav1.7 was also reported in an earlier study [49]. CFA increased the colocalization of protein kinase B/Akt with Nav1.7 in L4/5 DRG neurons while Akt pathway induced the upregulation of Nav1.7 [50]. Thus, the level of Nav1.7 was higher than in an animal model than in a healthy control. However, no evidence has shown that propofol and COS can reduce the level of Nav1.7 yet (Figure 4). According to a previous report, opioid receptor activation will reduce the level of Nav1.7 [51] while propofol can increase the expression of an opioid receptor [52]. Present work revealed a functional role of COS for controlling pain, which was not associated with the changes of Nav1.7 level (Figure 4). The present findings showed that the combined treatment was better than only one kind of medicine used for decreasing the mechanic and thermal pain (p < 0.05) (Figures 2 and 3).
The main aim of our work was to evaluate whether COS and propofol functionally interact with the sodium channel Nav1.7. Our data suggested that COS was a potential adjuvant to improve propofol performance, concentration-and state-dependent inhibitors of Nav1.7. Our results also suggested that propofol and COS interacted and modulated Nav1.7. Therefore, the findings showed that COS reinforced the inhibitory properties of propofol on Nav1.7 activity.

Discussion
Present findings indicated that COS greatly inhibited the incidence and severity of propofol-induced injection pain if the patients received 10 mg/kg COS via oral administration before being injected with propofol (Table 1). No toxic symptom or fewer side effects were observed in all the patients treated with COS ( Table 2). The results suggest that COS may be a potential natural adjuvant to improve propofol performance.
From pain analyses, an animal pain model was successfully established after CFA injection. The mouse model had mechanical and thermal hyperalgesia because of inflammatory pain, which was tested by a von Frey filament assay and hot/cold plate assay. Propofol is one kind of medicine mainly used for decreasing human pain. Present findings indicated that Nav1.7 was increased in CFA-induced hyperalgesia, which suggested that Nav1.7 plays a critical role in inflammatory pain. Subsequent work showed that COS and propofol reduced pain thresholds.
Injection pain is a normal unwanted adverse effect for propofol use. The side effects can be reduced when combined with COS because they can produce more analgesic efficacy [42]. Another study also used COS as an anesthesia supplement of propofol injection, which was successfully used in topical local anesthesia for surgery on a child [43]. All the results suggest that propofol and COS may have synergistic functions. However, the complementary functions remain unclear. Since many Navs play important roles in pain [44,45] and neural disorders [46,47], we want to explore the effects of combined medicine on the level of Navs. The mutant SCN9A gene-encoding Nav1.7 caused insensitivity to pain in mammals [35]. Furthermore, many pyrrolo-benzo-1,4-diazine derivatives were synthesized to inhibit the activity of Nav1.7, and showed anti-nociceptive oral efficacy in an inflammatory pain model [48].
CFA increasing the expression of Nav1.7 was also reported in an earlier study [49]. CFA increased the colocalization of protein kinase B/Akt with Nav1.7 in L4/5 DRG neurons while Akt pathway induced the upregulation of Nav1.7 [50]. Thus, the level of Nav1.7 was higher than in an animal model than in a healthy control. However, no evidence has shown that propofol and COS can reduce the level of Nav1.7 yet (Figure 4). According to a previous report, opioid receptor activation will reduce the level of Nav1.7 [51] while propofol can increase the expression of an opioid receptor [52]. Present work revealed a functional role of COS for controlling pain, which was not associated with the changes of Nav1.7 level (Figure 4). The present findings showed that the combined treatment was better than only one kind of medicine used for decreasing the mechanic and thermal pain (p < 0.05) (Figures 2 and 3).
The main aim of our work was to evaluate whether COS and propofol functionally interact with the sodium channel Nav1.7. Our data suggested that COS was a potential adjuvant to improve propofol performance, concentration-and state-dependent inhibitors of Nav1.7. Our results also suggested that propofol and COS interacted and modulated Nav1.7. Therefore, the findings showed that COS reinforced the inhibitory properties of propofol on Nav1.7 activity.
Previous work showed that steady-state plasma concentration of propofol during sedation was in the order of 22-44 µM [53]. It can intensively (97%-98%) bind plasma proteins [54]. In most cases, only the unbound fraction is able to interact with Na + channels. Therefore, a higher concentration was used in pain therapy [55]. Propofol is mainly eliminated by hepatic conjugation to inactive metabolites, which are secreted from the kidney [56]. On the other hand, the persons have a reduced clearance for propofol and may have increased levels of plasma propofol [57]. Additionally, the terminal half-life of propofol ranges from one to three days [58].
COS showed as a preventive agent by improving propofol performance in a pain model. COS improves propofol performance by suppressing pain symptoms and inhibiting Nav1.7 activity (Figures 6-8). Furthermore, COS caused an obvious hyperpolarization shift of the steady-state fast inactivation of Nav1.7 ( Figure 6). There is statistical significance of differences when compared to the combination of COS and propofol (p < 0.05, unpaired t-test). COS has no systemic adverse effects on the mouse model. Clinically relevant plasma levels of propofol will cause related effects on Nav1.7. Therapeutic levels of COS are low in the present experiment (10 mg/Kg).

Marine Drugs 2016, 4, 234 9 of 15
Previous work showed that steady-state plasma concentration of propofol during sedation was in the order of 22-44 μM [53]. It can intensively (97%-98%) bind plasma proteins [54]. In most cases, only the unbound fraction is able to interact with Na + channels. Therefore, a higher concentration was used in pain therapy [55]. Propofol is mainly eliminated by hepatic conjugation to inactive metabolites, which are secreted from the kidney [56]. On the other hand, the persons have a reduced clearance for propofol and may have increased levels of plasma propofol [57]. Additionally, the terminal half-life of propofol ranges from one to three days [58].
COS showed as a preventive agent by improving propofol performance in a pain model. COS improves propofol performance by suppressing pain symptoms and inhibiting Nav1.7 activity (Figures 6-8). Furthermore, COS caused an obvious hyperpolarization shift of the steady-state fast inactivation of Nav1.7 ( Figure 6). There is statistical significance of differences when compared to the combination of COS and propofol (p < 0.05, unpaired t-test). COS has no systemic adverse effects on the mouse model. Clinically relevant plasma levels of propofol will cause related effects on Nav1.7. Therapeutic levels of COS are low in the present experiment (10 mg/Kg). One important thing should be mentioned here: −120 mV hyperpolarized potentials were artificial and did not present the membrane properties of DRGs in vivo. With a physiological resting membrane potential around −50 mV, and with an ongoing DRG activity, the data from inactivated channels can be used to evaluate the function of Na + channel blockers. A tonic block of Nav1.7 channels by propofol and COS may be a better means for pain therapy. Present findings showed that COS were potential adjuvants to induce a higher tonic block as compared to use of only propofol.
There are some limitations for the present study: (1) Most studies, if not all, examined the effect of COS in addition to propofol, and the possible effects of COS alone have not been studied. This seems to make the mechanisms of COS effects vague and mysterious. Propofol has been proved to be an important sedative. However, we are not sure whether only COS can be a kind of sedative although it has been reported to have anti-pain functions. To avoid unknown risks, the test was not performed in the patients receiving surgeries. We are influenced by the design for human experiment and the test was not performed in the animal models with only COS treatment; (2) Low-molecular-weight COS cannot be injected in most cases although it has been used widely as healthy products in China; (3) Detail molecular mechanism for the inhibitory function of COS and propofol for Nav1.7 remains unknown; (4) Nav1.7 is only one critical effector for evaluating the functions of COS, and many other Nav members should be analyzed in the future.

COS Preparation and MALDI-TOF (Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight) MS Analysis
Low-molecular-weight, water-soluble COS was purchased from GlycoBio Company (Dalian, China). The COS was marine natural products and prepared from marine resources according to a One important thing should be mentioned here: −120 mV hyperpolarized potentials were artificial and did not present the membrane properties of DRGs in vivo. With a physiological resting membrane potential around −50 mV, and with an ongoing DRG activity, the data from inactivated channels can be used to evaluate the function of Na + channel blockers. A tonic block of Nav1.7 channels by propofol and COS may be a better means for pain therapy. Present findings showed that COS were potential adjuvants to induce a higher tonic block as compared to use of only propofol.
There are some limitations for the present study: (1) Most studies, if not all, examined the effect of COS in addition to propofol, and the possible effects of COS alone have not been studied. This seems to make the mechanisms of COS effects vague and mysterious. Propofol has been proved to be an important sedative. However, we are not sure whether only COS can be a kind of sedative although it has been reported to have anti-pain functions. To avoid unknown risks, the test was not performed in the patients receiving surgeries. We are influenced by the design for human experiment and the test was not performed in the animal models with only COS treatment; (2) Low-molecular-weight COS cannot be injected in most cases although it has been used widely as healthy products in China; (3) Detail molecular mechanism for the inhibitory function of COS and propofol for Nav1.7 remains unknown; (4) Nav1.7 is only one critical effector for evaluating the functions of COS, and many other Nav members should be analyzed in the future.

COS Preparation and MALDI-TOF (Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight) MS Analysis
Low-molecular-weight, water-soluble COS was purchased from GlycoBio Company (Dalian, China). The COS was marine natural products and prepared from marine resources according to a previous report [59]. A 1 µL sample solution was mixed with 2 µL 2,5-dihydroxybenzoic acid (15 mg/mL) in 30% ethanol. Mass spectra were made on an Agilent 6530 Accurate-Mass (Santa Clara, CA, USA) in a positive ion mode. In the measurement, a nitrogen laser (Spectra-Physics, Mountain View, CA, USA) (at 337 nm, 3 ns pulse width, 3 Hz) was performed. All spectra were examined in a reflector mode by using external calibration. MALDI-TOF MS analysis of COS showed that the degree of polymerization (DP) of the main products were DP4, 5, 6 and 7 when potassium adduct ions were summed together in MALDI-TOF (Figure 8).

Participants
Before the present study, all protocols were approved by the Ethical Committee of the First Hospital of Jilin University (Changchun, China). The subjects with the physical status of American Association of Anesthesiology (ASA) I or II received surgery at our hospital from 3 May to 12 October. Including criteria was used according to previously reported [60]. Excluding criteria includes following items: (1) the patients could not express themselves clearly; (2) they took other anti-pain medicine within one day of surgery; (3) the patients refused to sign an informed consent for present experiments. Finally, a total of 188 patients were selected.

Patient Grouping
All the selected subjects were evenly assigned to two groups before being injected with propofol: 10 mg/kg COS (CG) treatment and 10 mg/kg placebo (PG) treatment daily. COS and placebo were administered orally. To avoid the intervention of baseline characters for final results, demographic data were investigated including age, gender, BMI (body mass index), lifestyle and ASA. After 2 h pretreatment, the patients received 2 mg/kg/h saline treatment. After five min, propofol TCI was started with step increases of 0.5 µg/mL/2.5 min until the patient lost consciousness. Cis-atracurium was injected at 0.2 mg/kg to promote tracheal intubation. Meanwhile, propofol TCI was adjusted to maintain BIS values at 50. The pain was evaluated by clinical experts according to a four-point scale (no pain, mild pain, moderate pain and severe pain) from propofol injection to the time when the patients lost consciousness. Side effects were recorded from day 4 to 7 after the surgery. Table 3 showed that the baseline characters were similar between CG and PG groups, including age, gender, BMI, lifestyle and ASA (p < 0.05). The results suggest that the baseline clinical characters will not affect the final results of COS and propofol treatment.

Animals
To explore the molecular mechanism, an animal pain model was established. All the protocols were established according to the guidance for the use of laboratory animals (National Academy Press) and approved by the Ethical Committee of the First Hospital of Jilin University (Changchun, China).

Animal Grouping
The mice received 10 mg/kg COS treatment before 2 h propofol injection and the dosage was used according to a previous report [61]. There were 32 pain-model mice evenly assigned into four groups: PG group (received 10 mg/kg propofol treatment), PCOSG group (received both 10 mg/kg COS and propofol treatment), PIG group (Nav1.7-silenced model mouse received 10 mg/kg propofol treatment) and PCOSIG group (Nav1.7-silenced model mouse received both 10 mg/kg COS and propofol treatment).

Animal Behavior of Mechanical and Thermal Hyperalgesia
Mechanic pain sensitivity was measured immediately by testing the responding forces to the stimulation by Electronic von Frey monofilaments (Nanjing Jisheng Medical Technology Company, Nanjing, China) after propofol injections. The thermal pain was examined by an algesiometer (Shanghai AoBopharmtech, Shanghai, China). Hot-and cold-induced pains were tested by a Hot/Cold Plate Analgesia Meter (YLS-6B, Huaibei Zhenghua Biologic Apparatus Facilities Ltd. Co., Huaibei, China).

Electrophysiology Analysis of Nav1.7
Primary DRG cells were cultured in DMEM media and treated with different concentrations of propofol and/or 10 µg/mL COS for 24 h. To investigate the activities of Nav1.7, the electrophysiological properties of Nav1.7 were compared in primary DRG cells by using whole-cell patch-clamp recordings.
The membrane currents were recorded by using a patch clamp and an EPC10 amplifier (HEKA Instruments Inc., Bellmore, NY, USA). Data were obtained and stored with Patchmaster v20 × 60 software (HEKA Instruments Inc., Bellmore, NY, USA). Patch pipettes were pulled from glass capillaries (Science Products, Hofheim, Germany) by using a DMZ-Universal Puller (Zeitz, Germany) and then heat polished to give a resistance of 2.0 to 2.5 MΩ when it was filled with pipette solution. Currents were filtered at 5 kHz. The series resistance was compensated by 60%-80% to minimize voltage errors, and the capacitance artifacts were canceled using the amplifier circuitry. Linear leak subtraction based on resistance estimates from hyperpolarized pulses was applied before the pulse test.

Statistical Analysis
M Data were represented as mean ± S.D. Chi-square test was used for the comparison between two groups. The comparisons of independent groups of data were performed with the ANOVA test by using IBM SPSS Statistics 20.0 (Brea, CA, USA). Data analysis, curve fitting, and statistical analyses were also performed using the same software. IC50 values were calculated by normalizing peak current amplitudes at different concentrations to the value obtained in control solution. Data were fitted with Hill equation y = y max × (IV50n/IC50n × Cn), where y max is the maximal amplitude, IC50 is the concentration at which y/y max = 0.5, and n is the Hill coefficient. To obtain inactivation curves, peak currents evoked by a test pulse were measured, normalized, and plotted against the conditioning repulse potential. Data were fitted by the Boltzmann equation [62], y = 1/(1 + exp(Epp − h0.5)/kh), where Epp is the membrane potential of test pulse, h0.5 is the voltage at which y equals 0.5, and kh is a slope factor.

Conclusions
Taken together, propofol and chitosan oligosaccharide (COS) can synergistically reduce inflammation pain symptoms. While propofol causes some adverse effects, COS improves the propofol performance with fewer side effects by reducing inflammation and inhibiting the activity of voltage-gated sodium channel (Nav)1.7. Our data demonstrate that both substances block the Na + channel Nav1.7 and potentially contribute to pain relief. Thus, this study identified a potential adjuvant for the pain therapy with low-dose propofol.