Biomedical Research
ISSN: 0970-938X (Print) | 0976-1683 (Electronic)

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Warming acupuncture affects TTX-R sodium current of DRG and PGE2 of KOA rats

Yong-Liang Zhang1,2, Jia-Hong Gang1, Hua-Min Wang1 and Yi-Qun Mi1*

1Department of Acupuncture and Massage, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, PR China

2Second Hospital of Hebei Medical University, Shijiazhuang, PR China

*Corresponding Author:
Yi-Qun Mi
Department of Acupuncture and Massage
Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, PR China

Accepted date: January 02, 2016

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Abstract

Background: This study aimed to elucidate mechanism of analgesic effect of warming acupuncture on Knee Osteoarthritis (KOA).

Methods: A total of 40 SD rats were randomly divided into control group, KOA group, Pharmacotherapy (PT) group and Warming Acupuncture (WA) group. Rats in PT and WA groups received intra-articular injection of papain into bilateral knees. In PT group, rats were intragastrically treated with meloxicam; in WA group, rats received warming acupuncture. Then, Tetrodotoxin Resistance (TTX-R) sodium channel current of Dorsal Root Ganglion (DRG) and serum PGE2 were measured.

Results: TTX-R sodium channel current was significantly different among groups (P<0.01). When compared with control group, TTX-R sodium channel current increased significantly in KOA group (P<0.01). When compared with KOA group, TTX-R sodium channel current reduced markedly in PT group and WA group (P<0.01). It was suggested that WA and PT were able to reduce TTX-R sodium channel current to exert certain analgesic effects. In addition, WA and PT also reduced peripheral serum PGE2 to a certain extent in rats with KOA.

Conclusions: Warming acupuncture may reduce TTX-R sodium channel current of DRG and decrease inflammatory PGE2 to improve inflammatory hyperalgesia, exerting analgesic effect.

Keywords

Warm puncture, Osteoarthritis, Tetrodotoxin-insensitive sodium channel current, Rat dorsal root ganglion.

Introduction

Knee Osteoarthritis (KOA) is a degenerative disease characterized by knee cartilage degradation, reactive sclerosis and hyperplasia of the subchondral bone. Clinically, symptoms of KOA include joint pain, stiffness, swelling and limitation of joint motion. Severe KOA may cause joint deformity and even loss of joint function. Traditionally, KOA is treated with nonsteroidal anti-inflammatory drugs. Dorsal Root Ganglion (DRG) is a primary sensory neuron. On the basis that skin is innervated by spinal nerves L2-4, it was speculated that DRG of spinal nerves L2-4 were associated with pathogenesis of KOA. Studies have confirmed that acupuncture may exert analgesic effects on KOA in clinical practice, but the specific mechanism is still poorly understood. This study was undertaken to investigate potential mechanism underlying analgesic effects of acupuncture on rats with early KOA.

Knee osteoarthritis belongs to the category of “heumatism” of “polioplus” in traditional Chinese medicine. The etiology and pathogenesis of polioplus is descripted in Neijing, indicating that the basic of the disease is liver and kidney deficiency, and its reality is that wind, cold, and wet occurs together. Therefore therapy about reversing the insufficiency and decreasing the wind, cold, and wet showed good curative effect [1].

Warming acupuncture belongs to moxibustion, it is the embodiment of the “if cold, warm it”. When adding a moxibustion on needle, the heat reaches the disease part along the needle while not act directly on the skin, which makes the thermal penetrates acupoints and joint to promote blood circulation, remove blood stasis, warm menstruation and relief pain. A study has proved that the effect of warm acupuncture is better than simple acupuncture [1]. Modern researches [1-5] have confirmed that warming acupuncture can repair function of articular cartilage. Such repair effect of warming acupuncture is associated with related growth factors, such as transforming growth factor β, bone protection factor, basic fibroblast growth factor, insulin-like growth factor 1, etc. All these factors have similar effect to promote cartilage cell proliferation and repair cartilage. A number of studies have demonstrated that warming acupuncture can increase the growth factor expression level [6-8], therefore plays an important role in promoting the repair of articular cartilage.

Materials and Methods

Animals and grouping

This study has been approved by the Ethic Committee of The Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University (2014-10-2J). And international guidelines for animal welfare were followed. A total of 40 male SD rats (specific pathogen free) weighing 250 ± 50 g and aging 3 months were randomly divided into control group, KOA group, WA group and PT group (n=10 per group).

Establishment of KOA model in rats

The KOA model was established by intra-articular injection of 1.6% papain into bilateral knee joint cavities according to a previously report [9] at three time points (1, 4 and 7 days, 0.1 ml per joint). On the basis that super fatigue may cause KOA, rats were allowed to run on a roller for 30 min daily for 2 weeks. Then, stable KOA was observed in rats.

Treatments

Different treatments were administered in 4 groups: 1) control group: rats were intragastrically injected with normal saline as equal volume as in PT group and fixed on a table without other treatments. 2) KOA group: rats were fixed on a table, but did not receive any treatment; 3) WA group: warming acupuncture was done at bilateral anterior knee acupoints. Localization of anterior knee acupoint was anterior knee of hind limb. Acupuncture of anterior knee acupoint was able to exert analgesic effects and regulate hind limb functions. 4) PT group: meloxicam (7.5 mg/tablet; Shanghai Boehringer Ingelheim Pharmaceutical Co., Ltd. H20020217) was grounded and dissolved in normal saline into a 0.5 mg/ml solution. Rats were intragastrically injected with 3.75 mg/kg meloxicam once a day and also fixed as in WA group. After treatment for 20 days, rats were sacrificed, serum and DRG were collected, and ELISA was performed to detect PGE2 with rat PGE2 ELISA kit (RD company). The whole-cell patch-clamp technique was employed for detection of TTX-R sodium channel current.

Detection of serum PGE2 by ELISA

The serum was thawed to room temperature and ELISA kits were allowed to warm to room temperature. There were 14 standard wells and 2 non-specific wells, and samples were added to the remaining wells. First, 150 μl of reagent or sample was added to each well, followed by addition 50 μl of primary antibody solution (except for non-specific wells). The plates were sealed and incubated with constant shaking at 500 ± 50 rpm for 1 h. Then, 50 μl of PGE2 binding solution was added to each well, and the plate was sealed, followed by incubation with a constant shaking for 2 h. After washing with wash buffer for 4 times, 200 μl of substrate solution was added to each well, followed by incubation in dark for 30 min. Then, 100 μl of stop solution was added to each well, and absorbance was measured within 30 min at 450 nm. Wavelength at 540-570 nm was used if wavelength adjustment was possible; measurement was done at 450 nm, 540 nm and 570 nm if wavelength adjustment was impossible, and the results of absorbance at 450 nm minus that at 540 nm or 570 nm was used as a final absorbance.

Patch-clamp technique

The neurons were clamped at-70 mV and stimulated with step voltage at-70~40 mV (step=10 mV; duration=30 ms). TEA at 2 mM and CdCl2 at 100 μM were added independently to block potassium current and inward calcium current, and the total sodium current was measured. Then, TTX at 250 nM was added to block TTX-S sodium current, and TTX-R sodium current was measured. The peak of sodium current at depolarization was subtracted and final TTX-R sodium current was expressed as current/membrane capacitance (pA/pF). Origin 8.0 software was used to delineate I-V curve of TTX-R sodium current, current at corresponding voltage was recorded, and averages were obtained. The difference in I-V curve was compared.

Statistical analysis

SPSS version 18.0 was employed for statistical analysis, and data were expressed as means ± standard deviation (x̄ ± s). Comparisons of means among groups were done with Student- Newman-Keuls or least significant difference t. A value of P<0.05 was considered statistically significant.

Results

Comparisons of serum PGE2 in rats

Rats were intraperitoneally anesthetized with 5 mg/ml at 4 mg/100 g body weight, and blood (5-10 ml) was collected into a centrifuge tube, followed by centrifugation at 3000 r/min for 3 min. Serum was harvested for detection of PGE2. Results of normality test and homogeneity of variance test showed that data displayed nearly normal distribution and had homogeneity of variance. Thus, t-test was employed. Results showed that peripheral serum PGE2 was significantly different among groups (P<0.01). Compared to KOA group, serum PGE2 reduced significantly in the other groups (P<0.01), but there was no significant difference between Warming Acupuncture (WA) group and Pharmacotherapy (PT) group (P>0.05). In addition, significant difference in serum PGE2 was also observed between WA group and control group (P<0.01).

TTX-R sodium channel current

Neurons were clamped at-70 mV, and TTX-R sodium channel current of DRG at whole-cell mode was measured in control group, KOA group, WA group and PT group (Figures 1A-1D). Results of data analysis showed that there were significant differences of TTX-R sodium channel current among four groups. Current density was calculated according to the peak of TTX-R sodium channel current. Results showed that current density displayed in normal distribution, with heterogeneity of variance. Thus, non-parametric Kruskal-Wallis H test and Mann-Whitney U test were employed. Results showed there were significant differences in TTX-R sodium channel current among groups (P<0.01). When compared with control group, density of TTX-R sodium channel current in KOA group increased significantly (P<0.01); when compared with KOA group, current density in WA group and PT group reduced significantly (P<0.01). There was no significant difference in current density between WA group and PT group (P>0.05). On the basis of voltage-dependence of TTX-R sodium current, the I-V curve was delineated. A current at a specific voltage was recorded, and averages were obtained. A I-V curve of TTX-R sodium current was V-shaped, a TTX-R sodium current was gradually activated and peaked at an amplitude of-20 mV. Thereafter, current amplitude reduced gradually, and TTX-R sodium current became inactivated. These suggested voltagedependence of TTX-R sodium current (Figure 2). Bothman equation was employed to fit a curve according to current and voltage (Figure 3). There was no significant difference in V1/2 among 4 groups (Figure 1C). The slope factor K (2.123) in WA group was comparable to those in PT group (2.078) and control group (2.32). Taken together, current-voltage dependence of TTX-R sodium current remained unchanged.

biomedres-sodium-current

Figure 1: A: TTX-R sodium current in blank control group. B: amplitude of TTX-R sodium current in KOA group increased significantly when compared with control group (P<0.05); C: TTX-R sodium current density in WA reduced markedly as compared to KOA group (P<0.05); D: TTX-R sodium current density in PT reduced markedly as compared to KOA group (P<0.05).

biomedres-similar-groups

Figure 2: The I-V curves of TTX-R sodium current were similar in 4 groups. Current was first measured at-70 mV and peaked at-20 mV (P>0.05).

biomedres-depolarizing-voltage

Figure 3: Bothman equation was used to fit an equation according to the standardized current and depolarizing voltage. Significant difference was not observed in the slope factor V1/2 among 4 groups (P>0.05).

Discussion

Mechanism of pain is complex, which generally divides into nerve pathological pain and inflammatory pain. Knee osteoarthritis pain belongs to the inflammatory pain. TTX-R sodium channel current testing on the DRG of electrophysiology was used to determine the presence of inflammatory pain. And inflammatory pain occurs with a variety of inflammatory cytokines. PGE2, among many inflammatory cytokines, can be used as clinical biomarker of early detection of joint damage. So the combination of those two was tested in the present study.

Prostaglandins are metabolites produced by arachidonic acid cyclooxygenase and their production undergoes a series of enzyme-mediated catalyses. PGE2 acts via activating a group of G protein-coupled receptors including EP1, EP2, EP3 and EP4 [10]. PGE2 has been found to play an important role in acute inflammation and may act on peripheral circulation system to induce congestion and oedema. In EP knockout mice, results showed prostaglandins including PGE2 may exert both pro-inflammatory and anti-inflammatory effects, which are dependent on the regulation of corresponding genes [11]. PGE2 may not only promote osteogenesis, but stimulate bone absorption. PGE2 at a low concentration induces osteogenesis, but PGE2 at a high concentration may cause bone absorption [12]. After warming acupuncture in rats with KOA, ELISA was performed to detect nitric oxide (NO) and PGE2 in knee synovial fluid and serum, and results showed that NO and PGE2 increased significantly in rats with KOA when compared with control rats; and warming acupuncture could reduce elevated NO and PGE2 in the knee synovial fluid and serum when compared to untreated KOA rats. This suggested that the increase of PGE2 was closely related to pathogenesis of KOA and might be an important participant in pathogenesis and pathology of KOA; and warming acupuncture might exert therapeutic effects [13].

TTX-R sodium ion channel current is mainly produced by NaV1.8 and NaV1.9 [14]. This current is formed due to inward sodium fluxing into cells. TTX-R sodium ion channel current is involved in depolarization activation of action potential and maintenance of cell excitation [15]. In addition, it also plays an important role in peripheral pain transmission [16]. The abnormal TTX-R sodium ion channel current may cause hyperalgesia, and trigger neuropathic and inflammatory pains [17,18]. DRG has both TTX-S and TTX-R sodium ion channel currents which play crucial roles in pathophysiology of different pains. TTX-R is mainly produced in sensory neurons and involved in pain transmission [19,20]. Especially, TTX-R plays important roles in the pathogenesis of chronic inflammatory and neuropathic pains [21]. DRG is a primary neuron in pain transmission and usually does not produce action potential spontaneously. With presence of inflammation, sodium channel is opened, and depolarizing sodium current is produced to form action potential, which causes frequent discharge, reduces threshold of mild pain and delays progression of inflammatory hyperalgesia [22,23]. The reduced threshold of sodium channel activation in neurons may cause frequent formation of action potential, signal transmission increase and cell excitation elevation, which further deteriorate cell swelling and cause cell apoptosis [24-26].

Although available studies reported that warming acupuncture might exert favourable analgesic effects on inflammatory pain, the specific mechanism is still unclear. In the present study, whole-cell patch clamp technique was employed to detect TTX-R sodium channel current of DRG in control group, KOA group, WA group and PT group. Results of this study showed that warming acupuncture could significantly reduce density of TTX-R sodium channel current. This indicated that warming acupuncture reduced number of opened sodium channels and decreased inward sodium flux; depolarization compensation of action potential reduced; action potential might not be induced; refractory period of neurons prolongs and frequency of signal transmission reduced. Moreover, threshold of TTX-R sodium channel current activation reduced which increased activation potentials of NaV1.8 and NaV1.9, and then decreased discharge frequency of DRG. The reduced density of TTX-R sodium channel current may also reduce discharge frequency of neurons and improve hyperexcitability of DRG [27,28], exerting protective effects on neurons. Reflex arc of pain transmission center in sensor was blocked, which reduced stimulation of central nervous system by pain signals, and thus pain was alleviated.

PGE-2 plays an important part in the pathogenesis of knee osteoarthritis. In this study, warm acupuncture treatment affected the concentration of PGE-2 in serum and joint fluid and reduced the high excited state DRG ionization sodium channel, promoting new ion channels and nerve fiber hyperplasia and transforming the high sensitivity of the peripheral pain, thus decreasing the central nervous sensitization and playing a role in analgesia. By the in-depth study about the link of warm acupuncture affecting PGE-2 concentration in serum and joint fluid and DRG TTX-R channel current density change in rats with knee osteoarthritis, we provided reliable experimental basis for acupuncture treatment of knee osteoarthritis and guidance of the clinical curative effect. Besides, we explained the relationship between DRG TTX-R Na current with the mechanism of pain. Pain exists in knee osteoarthritis, and it is inflammatory pain. The generation mechanism of inflammatory pain is related to DRG TTX-R Na current. The detection of DRG TTX-R Na current determined the degree of inflammatory pain. The TTX-R Na current data showed that the inflammatory pain was relieved, which further confirmed the curative effect for knee osteoarthritis, and at the same time further inferred that the key for the treatment of knee osteoarthritis may be through blocking the DRG TTX-R Na current.

In WA group, TTX-R sodium channel current was still voltage dependent, suggesting that physiological voltage dependence of sodium channel remained unchanged. In the present study, density of TTX-R sodium channel current reduced in both WA group and PT group, but there was no significant difference between them. It is suggested that both warming acupuncture and pharmacotherapy exerted favourable therapeutic effects on KOA. And when reducing the number of open sodium channels, flow in the sodium ions is decreased, depolarization compensation of action potential is reduced, and the action potential cannot be triggered, refractory period of neurons cannot be extended, and signal conduction frequency is slowed. Meanwhile, the no longer decrease of TTX-R sodium channel current activation threshold causes NaV1.8 and NaV1.9 channel activation potential increase, thereby reducing the dorsal root ganglion discharge frequency. Overall TTX-R sodium channel current density is reduced, and neuron discharge frequency is reduced at the same time, which improves the high excitability of dorsal root ganglion neurons [27] and shows protective effect on neurons. Receptors of nociceptive transmission backbone of reflex arc are impeded, thus reducing the stimulation of pain signals in the central nervous system. The corresponding clinical symptoms are expressed as pain relief. However, combination use of warming acupuncture and pharmacotherapy required further studies.

Conclusions

In the present study, results indicated that TTX-R sodium channel played an important role in pathogenesis of pain secondary to KOA, and warming acupuncture was able to inhibit TTX-R sodium channel current to exert analgesic effects. In addition, blocking TTX-R sodium channel may become one of the directions in future studies on KOA.

Acknowledgements

The study was supported by Three-year Program of Traditional Chinese Medicine for Special Talents of Shanghai Health and Family Planning Committee (ZY3-PCPY-3-1018) and Traditional Chinese Medicine Fund of Traditional Chinese Medicine (2012J012A).

References