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
]. Various alternative and folk remedies have also been used effectively for many years [6
]. 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
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 2-amino-2-deoxy-d
-glucan combined with glycoside linkages. COS is made from chitin, which is a homopolymer of 1-4 linked 2-acetamido-2-deoxy-β-d
-glucopyranose. COS will be formed when chitin is deacetylated >50%. COS can be applied in many primary industries, including microbial control in agriculture, maintenance of overall fruit and vegetable quality [12
] and nutritional dietary additive [13
]. Chitosan has many medical and pharmaceutical uses with anti-inflammation and antioxidant activities and fewer side effects [14
]. The analgesic effect of COS on pain has been proved due to its absorption of proton ions [16
]. Thus, COS may be a potential adjuvant to propofol use. To understand the functions of COS, it is necessary to explore the molecular mechanism for the role of COS in propofol therapy.
Voltage-gated sodium channels (Navs) are important indicators of the development of mammalian hyperalgesia [17
]. Navs are localized in a mammalian central nervous system [18
] and DRG (dorsal root ganglia) [20
]. Navs participate in the pain caused by inflammatory responses [22
]. Carrageenan and complete Freund’s adjuvant (CFA) have been used widely to produce mechanical and thermal hyperalgesia in an inflammatory animal model [23
]. Thus, these models provide convenient tools in exploring the molecular mechanism of a pain cause. There are many members of Navs with different functions. Three main voltage-gated sodium channels, Nav1.7, Nav1.8, and Nav1.9, are preferentially expressed in dorsal root ganglia (DRG) cells [27
]. These channels are involved in different pain. Nav1.9 and Nav1.8 play important roles in the development of cold pain [28
]. Previous work showed that little change could be found for inflammation-induced hypersensitivity in the mice lacking Nav1.8 or Nav1.9 [28
]. Comparatively, a great reduction in hypersensitivity could be found in Nav1.7 knockout mice [29
]. Furthermore, Nav1.7 is essential for burn-induced heat hypersensitivity [30
]. An alpha-subunit gene, SCN9A, encodes the Nav1.7 sodium channel [31
]. An earlier study indicated that SCN9A is essential for human nociception [33
]. Sodium channel Nav1.7 is associated with the reduction of neuropathic pain, which is caused by chronic constriction injury of the sciatic nerve in animal models. Behavior tests indicated that the thresholds for thermal and mechanical hyperalgesia were greatly reduced in neuropathic pain models. Meanwhile, the levels of Nav1.7 were significantly increased in DRG cells [34
]. In contrast, loss-of-function mutations of Nav1.7 caused congenital insensitivity to pain [35
]. Intrathecal injection of Navl.7 shRNA reduced the levels of Nav1.7 and inactivated astrocytes and microglia of DRG. Nav1.7 can improve the pain tolerance in an animal model [36
]. Given the key role of Nav1.7 in human pain, the effects of dual therapy on Nav1.7 were investigated.
To uncover the more specific functions of the combined therapy of COS and propofol, the present study was performed to examine the effects of a combined therapy on the level of Nav1.7 in a mouse pain model.
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
] and neural disorders [46
], 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)(Figure 2
and Figure 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 (Figure 6
, Figure 7
and Figure 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.
4. Materials and Methods
4.1. 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
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.
4.3. 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.
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). Four-week-old C57BL/6 male mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). A total of 32 mice (20–25 g) were anesthetized with 2% isoflurane (Cat. No. CDS019936, Sigma, St. Louis, MO, USA) and injected with complete Freund’s adjuvant (CFA, Cat. No. F5881, 10 μL 0.5 mg/mL heat-killed M. tuberculosis) (Sigma, St. Louis, MO, USA) in the plantar of one hind paw to cause inflammatory pain symptoms. Meanwhile, another hind paw was injected with 10 μL 0.9% NaCl as a control. Animal behaviors were observed after one-day pain induction.
4.5. Nav1.7 Gene Silencing
pTZU6+1 vector was from Chongqing Medical University (Chongqing, China). shRNA for Nav1.7 gene silencing was constructed by using the primers: sense, 5′-ACCTCGACCTCAGAGCTTCGTTCACTTTGGAGTGAACGAAGCTCTGAGGTCTT-3’; antisense, 5’-CAAAAAGACCTCAGAGCTTCGTTCACTCCAAAGTGAACGAAGCTCTGAGGTCG-3′. Restriction sites, SalI and XbaI, were added on either end of the oligos and linked with pTZU6+1, and pTZU6+1-Nav1.7 were reconstructed. The reconstructed plasmids were injected into mice via tail veins. Eight hours after injection, propofol injection was performed and animal behaviors of mechanical and thermal hyperalgesia were analyzed.
4.6. 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).
4.7. 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).
4.8. Western Blot
According to a previous report, CFA infection increases the expression of Nav1.7 [49
]. Nav1.7 can be upregulated in L4/5 DRG neurons in a certain evoking situation [50
]. Therefore, L4-5 DRG samples from different groups were obtained. Protein was isolated using a plasma membrane protein isolation kit (Cat. No. ab65400, Abcam Trading (Shanghai) Company Ltd., Shanghai, China). Rabbit anti-mouse monoclonal Nav1.7 antibody (Cat. No. 62758, dilution 1:5000, Abcam Trading (Shanghai) Company Ltd., Shanghai, China) was used as the first antibody. Polyclonal Goat Anti-Rabbit IgG H&L (Cat. No. ab6721, dilution 1:3000, Abcam Trading (Shanghai) Company Ltd., Shanghai, China) was used as a secondary antibody. A rabbit anti-mouse β-actin polyclonal antibody (1:2000 dilution; Cat. No. 4967, Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. All protein bands were visualized by using an enhanced chemiluminescence substrate (Sangon Biotech, Co., Ltd., Shanghai, China). The image intensity of protein bands was quantified by using NIH ImageJ software (Bethesda, MD, USA).
4.9. 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 following test solution was prepared (mM): 100 NaCl, 50 choline chloride, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 15 glucose. The pH value was adjusted to 7.0 with tetraethylammonium hydroxide (Sigma, St. Louis, MO, USA). The pipette solution consists of the following components (mM): 140 CsF, 10 NaCl, 1 ethyleneglycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid, 10 HEPES and pH value was adjusted to 7.0 with CsOH.
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.
4.10. 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
), where ymax
is the maximal amplitude, IC50 is the concentration at which y
= 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
= 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.