CBS-Induced H2S Generation in Hippocampus Inhibits EA-Induced Analgesia

Hydrogen sulfide (H2S) is an important mediator participating in both physiological and pathological systems and related to the inflammatory process. Acupuncture has a therapeutic effect on inflammatory pain. However, whether H2S generated in the central nervous system (CNS) is a mediator of electroacupuncture (EA) treatment for inflammatory pain is unknown. We injected complete Freund's adjuvant (CFA) to induce inflammatory pain and applied EA treatment as an interventional strategy for pain relief. The results presented here show that S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-β-synthetase (CBS), may reverse the therapeutic effect of EA. CBS-induced H2S generation might get involved in the mechanism of EA-induced analgesia in the hippocampus on chronic inflammatory pain.


Introduction
Hydrogen sulfide (H 2 S) is the most recently accepted endogenously produced gasotransmitter that plays an important role in human health and physiology. Emerging evidence indicates that physiological concentrations of H 2 S might have an anti-inflammatory effect, while higher concentrations can exert proinflammatory effects [1]. H 2 S is synthesized by three enzymes: cystathionine c-lyase (CSE), cystathionine β-synthetase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [2]. e expression of CBS, CSE, and 3-MST shows tissue-specific dominance. Both CBS and 3-MST, as the primary physiological source of H 2 S in the central nervous system, predominantly are localized in the brain; CSE abundantly exists at the vascular or nonvascular smooth muscle in the mammalian cardiovascular and respiratory system; therefore, H 2 S-producing enzymes are generally ubiquitously expressed in mammalian tissues and impact a wide range of cellular processes [3,4]. Homocysteine and cysteine are substrates for H 2 S production, and H 2 S-synthesizing enzymes are responsible for converting these substrates to H 2 S; in the presence of cysteine, especially homocysteine, CBS catalyzes the production of H 2 S [5].
H 2 S and its synthesizing enzymes got involved in a variety of conditions, including pain and inflammation [6]. It has been indicated that H 2 S has an effect on pro-and antiinflammation, and it is a mediator of peripheral and neurogenic inflammation [1]. e same as inflammation, H 2 S participating in modulating visceral pain is also controversial [6]. H 2 S-generating enzyme CBS is also involved in several pain conditions. CBS has been found with an increased expression in the dorsal root ganglion (DRGs) of the IBS model induced by neonatal colonic inflammation (NCI) [7]. Inhibition of the CBS-H 2 S signaling pathway markedly attenuated heterotypical intermittent stress-induced visceral hyperalgesia; particularly, aminooxyacetic acid (AOAA), one of the CBS inhibitors, suppressed voltage-gated sodium channel currents of the colon-specific DRG neurons and reversed the enhanced expression of NaV1.7 and NaV1.8 subtypes [8].
ese findings indicated that CBS plays a crucial role in visceral hyperalgesia. Furthermore, CBS expression can be also upregulated in neuropathic pain and inflammatory pain. Chronic construction injury (CCI) surgery caused significantly increased expression of CBS in the ipsilateral ventral and dorsal horn of L4-6 spinal cord segments [9]. Inhibiting CBS by intrathecal injection of AOAA attenuated mechanical and thermal hypersensitivity, and AOAA injecting suppressed the expression of Na V1.7 and Na V1.8 in DRG neurons [10]. Additionally, application of AOAA was observed to alleviate CFA-induced hyperalgesia on the temporomandibular joint and reduce CBSinduced H2S production in trigeminal ganglion by reversing the enhancement of neural hyperexcitability and increasing the voltage-gated potassium currents [11]. ese findings suggest that H 2 S and CBS could play a pivotal role in a variety of pain conditions. Acupuncture has been applied in various pain conditions, and the neurobiological mechanism has been the important focus to be investigated [12,13]. When the needle is connected with an electrode, it is called as electroacupuncture (EA). EA is also very popular in the basic research and clinic practice because the stimulus is under control [14,15]. EA stimulation has been found to induce time-dependent cumulative analgesia in rats with neuropathic pain, and the mechanism was demonstrated to be related to the activation of hippocampal MEK1 [16]. Recent studies have also shown that repeated EA intervention leads to synaptic remodeling of hippocampal neurons, and in consequence, the expression of calcium/calmodulin kinase II in the hippocampal CA3 region is upregulated, which contributed to acupuncture analgesia [17]. However, whether H2S induced by CBS in the hippocampus would be able to get involved in acupuncture analgesia still remains unclear. In this study, we hypothesized that EA might modulate the H2S generation induced by CBS in the hippocampus to alleviate chronic inflammatory pain.

Animals.
Male BALB/c mice (8-9 weeks, 20-23 g) were purchased from Chengdu Da Shuo Experimental Animals Co. Ltd. All mice were adapted to the standard laboratory conditions (24 ± 2°C room temperature and 65 ± 5% humidity on 12/12 h light-dark cycles) with drinking water and food available ad libitum. e experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine. After adaptive domestication for one week, mice were randomly divided into different groups based on random numbers generated by SPSS software.

EA Treatment.
An electroacupuncture apparatus (HANS-200A acupoint nerve stimulator, Nanjing Jisheng Medical Co., Ltd) was used for EA treatment. EA stimulation was provided at the first day after CFA injection and subsequently lasted for one week. Mice were shaved to expose ST36, located at the posterolateral part of the knee about 2 mm below the fibular head [20], and two stainless steel needles (0.18 mm in diameter, 13 mm in length) were inserted directly at ST36 of both hind limbs about 3 mm deep. e stimulated parameters of EA included 0 Hz, 2 Hz, 100 Hz, and 2/100 Hz (0.2 mA intensity), lasting for 30 min during one session. During treatment, all mice were restrained on a specific device [21].

Surgical Procedures and Microinjection.
Stereotaxic surgery was performed 7 days prior to experiments. Mice were anesthetized with isoflurane and fixed on a stereotaxic platform. Coordinates of A-P: 2.5 mm relative to the bregma, lateral: ±2.5 mm relative to the midline, and depth: 2 mm from the duramater were used for implanting guide cannulas (RWD, China) into the bilateral hippocampi [22]. After recovery from surgery, HA (9 nmol/1 μl [23]), AOAA (9 nmol/1 μl [24,25]), or SAM (10 pmol/1 μl [26]) was bilaterally microinjected into the hippocampi. DAO and CAT inhibitors followed the same procedure. A 5 μl microsyringe was held by a micromanipulater on the stereotaxic apparatus (RWD, China), and injection was performed at a rate of 0.5 μl/min. e volume of each microinjection was 1 μl. Afterwards, the needle was kept at the injection place for another 5 minutes after each injection to allow for sufficient diffusion. No adverse effect was observed during each injection.

ermal Withdrawal Latency (TWL).
Mice were placed in behavior boxes on a glass platform before the behavior test [27]. ermal hyperalgesia was assessed by measuring the TWL with a Plantar Test Apparatus (10% light intensity, 10 sec baseline latency, and 20 sec cutoff time. Hargreaves method, PL-200, Tai Meng, China). After 30-min of acclimatization, a mobile radiant heat source was focused on the plantar surface of the right hind paw. Each mouse was tested three times with intervals of 5 min. Pain threshold was tested as the baseline at day 1 before CFA injection and day 1, 3, 5, and 7 after CFA injection. In order to eliminate observational bias, all behavioral data were collected and recorded by the same investigator who was not responsible for drug injection or EA treatment.

Statistical
Analysis. All data were analyzed and graphed using Graphpad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as data � MEANS ± SEM (standard error of means). Two-way repeated-measure ANOVA followed by the Tukey-Kramer test was performed to compare the differences between groups. A value of P < 0.05 was considered statistically significant.

Low-Frequency EA Stimulation Alleviated Chronic Inflammatory Pain.
To investigate the efficacy of EA in chronic inflammatory pain, we injected CFA in the right plantar surface of the hind paw and measured responses to the thermal stimuli in separate groups at a different time point. We chose the ST36 acupuncture point, which is one of the most frequently used acupoints for pain relief [28,29] (Figure 1(a)). After one day, different groups received acupuncture treatments with different stimulated parameters at ST36, and then we measured changes of TWL on mice (Figures 1(b)-1(e)). On the first day after CFA injection, we found that the baseline value of TWL was significantly decreased (Figures 1(b)-1(e)).
On the 3rd day after acupuncture treatment, EA at low frequency (2 Hz) had an analgesic effect on thermal hyperalgesia compared with EA at high-frequency stimulation(100 Hz) and alternating frequency(2/100 Hz) on mice with inflammatory pain (P < 0.05) (Figures 1(c)-1(e)). In addition, acupuncture without electrostimulation also failed to alleviate pain sensation (P > 0.05) (Figure 1(b)). e result suggests that lowfrequency EA has a therapeutic effect on CFA-induced inflammatory pain, but acupuncture and high or alternating frequency EA stimulation failed to alleviate pain.

Hippocampal Injection of CBS Inhibitors Attenuated ermal Hypersensitivity.
To study the relationship between CBS-induced H 2 S in the brain and thermal hypersensitivity, the CBS inhibitor HA (9 nmol) [23] or AOAA (9 nmol) [24,25] was bilaterally microinjected into the hippocampus. Moreover, the CBS-specific agonist SAM (10 pmol) [26] or saline was also bilaterally injected into the hippocampus one day after CFA injection, and CAT and DAO inhibitors [37] followed the same procedure (Figure 3(a)). Results showed that HA and AOAA microinjection significantly increased the TWL and attenuated thermal hypersensitivity (Figures 3(b) and 1(c)). However, saline, SAM, IC2A, or L-aspartate injection failed to alleviate thermal pain behavior (Figures 3(d) and 3(e)). erefore, suppressing H 2 S induced by CBS in the hippocampus might relieve CFA-induced chronic inflammatory pain.

SAM Reversed the erapeutic Effect of Low-Frequency EA.
To determine whether CBS influences EA therapeutic effect on chronic inflammatory pain, EA intervention(2 Hz, 0.2 mA) was performed immediately after SAM injection or microinjection. However, EA stimulation failed to alleviate thermal pain behavior after SAM injection (Figures 4(a) and 4(b)). ese results indicated that activating CBS reversed EA therapeutic effect on inflammatory pain, and CBS-induced H 2 S release might inhibit EA therapeutic effect.

Discussion
Our data demonstrated that low-frequency EA stimulation notably alleviated the thermal pain behavior, and injection of CBS inhibitors might have a therapeutic effect on thermal pain. As an essential synthetase of H 2 S, CBS is mostly responsible for the production of H 2 S in brain tissue and is highly expressed in the hippocampus [38,39]. erefore, H 2 S generation in the hippocampus might participate in the CFA-induced chronic inflammatory pain. Our data further showed that SAM, a CBS agonist, could reverse EA therapeutic effect on chronic inflammatory pain. Taken together, the downregulation of CBS-induced H 2 S generation might be one of the mechanisms of EA treatment for chronic inflammatory pain.
H 2 S is the third gasotransmitter discovered after NO, CO. It is considered as an important signaling molecule in the regulation of pathophysiological processes, including pain and inflammation [34,40,41]. However, the precise role of H 2 S in inflammation is not clear. It was supposed to have pro-or anti-inflammatory effects under different conditions. In general, H 2 S at physiological concentrations might have an anti-inflammatory effect, while increased concentrations of H 2 S can exert proinflammatory effects [1]. Intraplantar administration of NaHS, the donor of H 2 S, evoked mechanical hyperalgesia [42]. It was found that H 2 S might activate Cav3.2 T-type Ca2+ channels that led to sensitization of nociceptive processing and hyperalgesia [43]. H 2 S synthetase CSE and CBS also play a pivotal role in pain and inflammation. Pretreatment with the CSE inhibitor propargylglycine significantly reduced hind paw edema and decreased granulocyte infiltration into the tissue in response to an injection of carrageenan [44]. In the visceral pain rat model, the expression of CBS in the spinal cord was notably increased [45]. Some investigations also indicated that CBS was upregulated by TLR4 in NCI rats and mediated by the NF-κB signaling pathway, thus contributing to visceral hypersensitivity [46]. In addition, the expression of CBS also has an effect on inflammatory pain. It was found that the expression of CBS in the rat dorsal root ganglia (DRG) was significantly upregulated after intraplantar administration of CFA, and the mechanical hyperalgesia was expectedly attenuated by intraperitoneal injection of CBS inhibitors in a dose-dependent manner [34]. ese investigations suggest that H 2 S and its enzymes in the peripheral nervous system are regulated by nociception. However, analgesic effect of H 2 S and its enzymes on the brain are yet to be investigated in future studies.
CBS is responsible for most of the H 2 S production in the brain, and it is highly expressed in the hippocampus. Multiple evidences indicated that the hippocampal formation responded to external nociceptive stimuli [47][48][49]. Previously, investigations showed that SAM resulted in an up to eight or ten-fold increase of the enzymatic activity of CBS [50][51][52], and CBS inhibitors HA and AOAA suppressed H 2 S production in brain homogenates which can be enhanced by the CBS activator [39]. In addition, several investigations found that intraperitoneal injection of CBS inhibitors could decrease CBS expression and activity in brain tissue. For instance, Liu and his colleagues found that intraperitoneal injection of HA inhibited CBS protein expression as examined by western blot analysis and immunohistochemical analysis [53]. AOAA (i.p) also reduced CBS activity in brain tissue [54].
us, in order to study the relationship between CBS-induced H 2 S and inflammatory pain, HA or AOAA was intraperitoneally injected into mice. Results has shown that injecting HA or AOAA led to relieve pain by inhibiting CBS. It is in consistent with the previous research [34]. However, analgesic effect of CBS-induced H 2 S on the brain is yet to be investigated in future studies. To further investigate whether CBS located in the hippocampus would affect pain sensation, HA or AOAA was bilaterally microinjected into the hippocampus. We found that inhibiting H 2 S generation in the hippocampus alleviated Evidence-Based Complementary and Alternative Medicine inflammatory pain and activated H 2 S generation by microinjected SAM had no effect on inflammatory pain. However, how CBS in the hippocampus participates in pain sensation is yet to be investigated. According to previous studies, CBS converted homocysteine to cysteine [55]. CBS inhibitors could promote accumulation of L-homocysteine and L-cysteine. It was reported that reducing L-cysteine and L-homocysteine induced time and dose-dependent peripheral hyperalgesia which could be modulated by 3bOH, a potent T-type Ca2+ channel blocker [56]. erefore, homocysteine and cysteine might participate in inflammatory pain process, but it also needs to be further investigated.
Acupuncture has a therapeutic effect on inflammatory pain, and EA therapy has a cumulative effect [16,57,58]. e frequency of EA is one of the crucial factors to significantly influence its therapeutic effects. EA with different frequencies is regarded as distinct therapeutic methods and have been used in different diseases. High-frequency (100 Hz) stimulation was more effective as compared with the low frequency (2 Hz) in amelioration of muscle spasticity, and it might be mediated by dynorphin generating from the central nervous system [59]. Low-frequency EA released β-endorphin and enkephalins, while high-frequency EA released the dynorphins to suppress pain sensation [60]. However, low-frequency EA produced the significant rapid decrease in ankle edema after ankle sprain which is an important feature of inflammation [61]. Furthermore, low-frequency EA (10 Hz) significantly reduced CFA-induced hind paw edema to suppress inflammation by activating the hypothalamus-pituitary-adrenal axis (HPA); however, 100 Hz EA had no effect on it [62]. Some investigations demonstrated that low-frequency EA could regulate the expression level of genes in the nucleus arcuate region, and low-frequency EA at ST36 in rats markedly relieved pain [63,64]. Low-frequency EA could also suppress spinal long-term potential (LTP) to alleviate pain sensation in colitis rats [65]. Additionally, low-frequency stimulation, rather than high frequency, invoked endogenous mechanisms for the release of 5-hydroxytryptamine (5-HT) in the spinal cord, which activated 5-HT receptors to reduce hyperalgesia in rats with joint inflammation [66]. In accordance with these results, we found that low-frequency EA delivered to ST36 could alleviate pain. It has been reported that EA applied to ST36 could influence hemodynamic Evidence-Based Complementary and Alternative Medicine signaling in the hippocampus [42]. Additionally, the previous report indicated that EA protected against hypoxic ischemic brain damage in immature rats via decreasing H 2 S generation in brain tissue [53]. Also, EA treatment decreased CBS expression level and increased HO-1 and HIF-1α expression levels in perinatal rat cortex cells [67]. us, EA protected against hypoxic damage via the hydrogen sulfide/ CBS-CO/HO-1-HIF-1α system. To further determine the role of H 2 S in EA stimulation, EA treatment was performed after injection of the CBS agonist SAM on a CFA-induced inflammatory mouse model, and we found that the EA therapeutic effect was suppressed by SAM. H 2 S was expected to play an important role in modulating EA effect on inflammatory pain. Although the H 2 S/CBS system participates in the inflammatory pain and it is related to EA treatment, the mechanisms of how EA modulates H 2 S release need to be confirmed in our future work. Previous research has shown that intracellular Ca 2+ can be regulated by the cAMP/ PKA pathway, and H 2 S can regulate [Ca 2+ ] i through the cAMP/ PKA system [68]. EA pretreatment could produce an antiarrhythmic effect by regulating the L-type Ca 2+ channel [69]. Additionally, AOAA, an inhibitor of CBS, could also suppress the potentiation of ATP-induced intracellular calcium signals in DRG neurons to markedly attenuate pain hypersensitivity [70]. Some investigators also showed that EA pretreatment could inhibit Ca 2+ influx to alleviate the LPS-induced inflammation in rats [71]. erefore, we speculated that acupuncture regulation of H 2 S production might be related to calcium channel opening.
is needs to be proved in our future work.

Conclusions
Current data indicated that the CBS-specific agonist SAM may reverse the therapeutic effect of EA. Downregulation of CBS might be a mediator of EA-induced analgesia on chronic inflammatory pain.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Evidence-Based Complementary and Alternative Medicine 7