Koumine enhances spinal cord 3α-hydroxysteroid oxidoreductase expression and activity in a rat model of neuropathic pain

Neuropathic pain is pain resulting from an injury or disease of the somatosensory system [1]. A wide variety of insults to the peripheral and central nervous systems, including cerebrovascular accident, chemotherapy, nutritional deficiencies, surgery, systemic diseases, and trauma, can result in neuropathic pain. Neuropathic pain can cause abnormal pain sensations, including allodynia, hyperalgesia, dysesthesia, and spontaneous pain, which are difficult to treat. Current pharmacologic therapy for neuropathic pain consists mainly of nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, anticonvulsants, antidepressants, and topical remedies. Unfortunately, the treatments available for neuropathic pain are far from satisfactory: nearly two-thirds of patients experiencing neuropathic pain receive insufficient relief [2]. Therefore, novel analgesics may contribute to the development of effective treatment strategies against neuropathic pain.

Gelsemium is a genus of the family Loganiaceae; it comprises 3 species: (1) Gelsemium elegans Benth. (Fig. 1), native to Asia; (2) Gelsemium sempervirens Ait.; and (3) Gelsemium rankinii Small., native to North America [3, 4]. An increasing body of evidence indicates that alkaloidal extracts from G. elegans Benth. elicit numerous biological effects, including analgesic, antidepressant, anxiolytic, and antitumor effects [5‐9]. G. elegans Benth. has long been used in Chinese folk medicine to alleviate pain, inflammation, and cancer [9]. Consistently, alkaloids of G. elegans Benth. are thought to have analgesic properties and exhibit pharmaceutical potential [10, 11]. The most abundant alkaloid in G. elegans Benth. is koumine (molecular formula, C₂₀H₂₂N₂O; molecular weight, 306.30; CAS registry number, 1358-76-5) (Fig. 1). According to our previous behavioral observations in animals, koumine reverses chronic constriction injury (CCI) to the sciatic nerve and thermal hyperalgesia induced by lumbar 5 (L5) spinal nerve ligation (SNL) in a dose-dependent manner. Furthermore, mechanical allodynia in rats is reduced by koumine in a dose-dependent manner [12]. Koumine differs substantially from the currently available analgesics, since it belongs to a class of chemicals known as indole alkaloids. Moreover, it lacks the adverse effects associated with most analgesic agents [6, 11]. Therefore, we hypothesized that the analgesic profile and underlying mechanism by which koumine induces analgesia are unique.

Allopregnanolone, also known as 3α, 5α-tetrahydroprogesterone (3α, 5α-THP), is one of the most important neuroactive steroids. Upregulation of allopregnanolone was shown to induce significant analgesia, implying that allopregnanolone in the spinal cord (SC) may be an important key modulator of neuropathic pain. Interestingly, our previous work has demonstrated that increased allopregnanolone levels in the SC mediated the analgesic effect of koumine on neuropathic pain [12]. Although allopregnanolone has been found to be upregulated in the SC of rats with CCI following koumine treatment, little is known about the cellular and molecular mechanisms underlying its antinociceptive actions. Since allopregnanolone biosynthesis is dependent on the activity of 3α-hydroxysteroid oxidoreductase (3α-HSOR), we performed molecular time-course experiments to analyze 3α-HSOR’s cellular distribution, gene expression, and bioactivity in the lumbar SC following koumine treatment of CCI-induced pain symptoms. The aim of this study was to investigate the relationship between the analgesic effect of koumine on neuropathic pain and 3α-HSOR in SC after peripheral nerve injury in rats to clarify koumine’s analgesic mechanism of action.

Gelsemium elegans Benth. has diverse biological effects; however, its clinical use is hampered by its toxicity. The crude alkaloid Gelsemium extract exhibits a high level of toxicity with an LD₅₀ of 15 and 4 mg kg⁻¹ bw following intragastric and intraperitoneal (i.p.) administration, respectively, in rats [13]. Several research groups are currently trying to derive alkaloid monomers with high potency and low toxicity from G. elegans Benth. In a previous study, we were able to obtain several different alkaloid monomers using the pH-zone–refining counter-current chromatography technique [14], which enabled us to perform pharmacodynamic screening. Our preliminary findings suggested that the most toxic alkaloid derived from G. elegans Benth., gelsenicine, exerted analgesic activity during inflammatory and neuropathic pain [5]. Recently, Zhang et al. reported that gelsemine, another G. elegans Benth.-derived main alkaloid, displayed potent and specific antinociceptive properties in chronic pain [15]. Furthermore, we previously demonstrated that koumine exhibited potent analgesic effects with a relatively low toxicity compared to that of other G. elegans Benth. alkaloid extracts [12, 16]. Koumine’s acute toxicity was previously investigated in both mice and rats. The LD₅₀ of s.c.-administered koumine to mice was 99.0 mg kg⁻¹ bw [12]. Acute lethality generally occurred within 10 min of s.c. injection in mice, with labored respiration and brief coordinated clonic convulsions occurring immediately before death [12]. Interestingly, koumine’s toxicity is much lower than that of the crude G. elegans extract. The LD₅₀ of intragastrically administered koumine was 300 mg kg⁻¹ bw in rats (unpublished data), a level much higher than that estimated for the crude alkaloidal extract (15 mg kg⁻¹ bw) using the same administration route [13]. Moreover, in a previous study, the spontaneous travel velocity was not affected after administration of koumine in mice as determined by the spontaneous motor activity test [17], and no adverse effects were observed in the rats treated with a range of koumine doses (0.28–7 mg kg⁻¹ bw) in the current study. The relatively wide therapeutic index of koumine suggests that it may be a promising agent in clinical applications. In this study, the TWL and MWT behavioral data indicated that koumine may ameliorate sciatic nerve CCI-induced chronic pain. Repeated administration of koumine reversed thermal hyperalgesia and mechanical allodynia in a dose-dependent manner in our CCI model. Additionally, our previous work has provided evidence of koumine’s analgesic effect in a number of animal models of pain, including SNL and diabetic-induced neuropathic pain [12, 18], indicating a role for koumine in the treatment of peripheral neuropathic pain.

Repeated s.c. administration of koumine was not associated with adverse effects commonly associated with opioids, such as physical and psychological dependence [19]. Interestingly, LaBuda and Little [20] revealed that agents such as gabapentin and morphine completely or partially reversed tactile allodynia in L5 SNL, another animal model of painful peripheral neuropathy. Conversely, indomethacin had no effect. Koumine also exhibited positive analgesic effects in the L5 SNL model [12]. Taken together, these findings suggest that koumine’s analgesic mechanism of action differs from that of NSAIDs and opioids. Interestingly, we found that koumine exhibited its analgesic effect by upregulating allopregnanolone, one of the most potent neurosteroids in the SC [12].

The pathophysiology underlying neuropathic pain is complicated, with many aspects remaining unclear. A striking finding in the past three decades was the discovery that neurons are capable of synthesizing neurosteroids independently of the classical mechanism involving endocrine glands [21, 22]. The involvement of endogenous neurosteroids is well established in the area of chronic pain modulation [21‐25]. Inflammatory and neuropathic pain states are associated with the upregulated synthesis of endogenous neurosteroids in the SC, such as allopregnanolone and pregnenolone [21, 23, 26]. The administration of exogenous allopregnanolone has been shown to prevent and suppress oxaliplatin-evoked painful neuropathy [25]. Allopregnanolone is an allosteric modulator that controls several important neurophysiological mechanisms through its actions on the γ-aminobutyric acid A (GABA_A) channel [27]. Indeed, neurosteroids modulate pain perception by potentiating the effects at GABA_A channels and inhibiting T-type calcium channels located in peripheral sensory neurons [23, 28‐30]. Furthermore, neurosteroid synthesis has been reported in the SC, partly explaining the vital role of SC in the control of pain processing. The SC contains key enzymes related to steroid synthesis, including cytochrome P450 side chain cleavage enzymes, 3α-reductase, and 3α-HSOR [22, 23, 31]. Therefore, the key cellular and molecular components involved in neurosteroid biosynthesis in the SC may provide potential targets that may be useful for the development of novel analgesics for the treatment of persevering neuropathic pain. Consequently, an increasing number of scientists are focusing their research toward the discovery of novel neurosteroid-directed analgesics [29, 30, 32‐34].

3α-HSOR belongs to the aldo–keto reductase superfamily. It is crucial for the synthesis of neuroactive 3α-reduced steroids, such as allopregnanolone and tetrahydrodeoxycorticosterone, in a reversible manner. The dorsal horn of the SC, a pivotal structure that controls pain and nociception, exhibits intense immunostaining of 3α-HSOR [22, 35]. Furthermore, neurons and glial cells synthesize various neurosteroids. Our double immunolabeling experiments showed 3α-HSOR immunostaining in SC neurons, microglia, and astrocytes. This prompted us to investigate the relationship between koumine and 3α-HSOR and koumine’s effects on allopregnanolone upregulation in the SC. The present study draws on data generated by different experimental techniques that clearly show that koumine increases the cellular immunoreactivity, gene expression, and bioactivity of 3α-HSOR in SC during chronic pain. Importantly, our time-course experiments revealed that the koumine-induced upregulation of 3α-HSOR cellular immunoreactivity and gene expression correlated with the development of neuropathic pain symptoms.

Consistent with these findings, our in vivo enzymatic activity assays revealed that the catalytic activity of 3α-HSOR in the SC was increased in CCI rats after 7 days of koumine treatment. Furthermore, we observed that koumine produced significant analgesia and dose-dependently reversed the mechanical pain thresholds induced by the intrathecal injection of the 3α-HSOR pharmacological inhibitor MPA at a dose of 0.5 or 1.25 mg kg⁻¹ bw. It is worth noting that the highest dose of MPA used (1.25 mg kg⁻¹ bw) had no effect on the MWT values measured in naïve CCI rats. Thus, inhibiting 3α-HSOR activity in the sensory nerve circuit of the SC could reverse the analgesic effect of koumine in CCI rats. Our findings demonstrate the direct role of 3α-HSOR in mediating pain modulation and koumine’s influence thereon. Therefore, 3α-HSOR may be a fundamental molecular target for koumine in the modulation of pain sensation. However, although the 3α-HSOR fluorescence density and mRNA expression were still elevated on postoperative day 14, the analgesic effect had disappeared by then (Figs. 3, 5). One possibility is that koumine may regulate the catalytic activity of 3α-HSOR. Allopregnanolone biosynthesis in the SC may thus be insufficient in the absence of koumine because of low 3α-HSOR catalytic activity. However, this hypothesis needs to be confirmed by the determination of allopregnanolone levels in the SC on postoperative day 14. Another explanation may be that a molecular target other than 3α-HSOR, and whose function is also altered by koumine, participates in the observed analgesic effect of koumine.

The endogenous biosynthesis of neurosteroids is also upregulated in the SC during inflammatory and neuropathic pain states [21, 36, 37]. We demonstrated a similar increase in allopregnanolone and pregnenolone levels in CCI rats [12]. The observed increase in allopregnanolone levels in CCI rats is consistent with the findings of Kawano et al. in SNL neuropathy [37]. Similarly, we found increased immunohistochemical staining, mRNA expression, and bioactivity of 3α-HSOR in the SC during sciatic nerve CCI-induced chronic pain. On the basis of the results of our and other studies, we consider that a state of chronic pain in animals is predominantly determined by two sets of factors. The first includes the pro-nociceptive mechanisms mediated by neurotransmitters supporting the development and maintenance of pain symptoms, such as substance P, bradykinin, prostaglandin, and histamine. The second group of factors opposes the pro-nociceptive processes, and its effects are mediated by endorphins, neurosteroids, and neuroprotective factors that possess adaptive or antinociceptive properties. The latter help the animal to cope with or accelerate the recovery from the pathological pain state. Consequently, the selective upregulation of the 3α-HSOR immunoreactivity, mRNA expression, and bioactivity and the 3α-HSOR-induced increase in spinal synthesis of endogenous allopregnanolone may represent an intrinsic adaptive response to neuropathic pain and elicit beneficial effects against diverse pathological pain symptoms. However, these natural or adaptive mechanisms are not sufficient to achieve the suppression of pain sensation in rats with CCI [21]. As observed in rats with CCI, an insufficient increase in 3α-HSOR and allopregnanolone levels fails to reverse the development of neuropathic pain. Only a sufficient increase in allopregnanolone would offer adequate protection against the development of neuropathic pain [12, 37]. Allopregnanolone that is endogenously formed in the central nervous system significantly alters nociception through paracrine and autocrine mechanisms [36, 38]. Therefore, koumine and other alkaloid extracts of G. elegans Benth. that are capable of stimulating allopregnanolone formation in neural networks may provide a novel approach for the development of analgesic therapies [39].

Our fluorescence immunohistochemistry experiments also revealed that 3α-HSOR was widely distributed in the SC dorsal horn and co-localized with neural and non-neural cells, including neurons and microglia (Fig. 4, merged photomicrograph). In recent years, there has been a growing consensus that glial cells located in the spinal dorsal horn are activated following peripheral nerve injury [40‐42]. Microglial activation induces the release of proinflammatory cytokines, such as interleukin-β, tumor necrosis factor-α, and interleukin-6, which play important roles in the development of neuropathic pain. The protective property of allopregnanolone in pain perception in relation to glial cells has been well documented [25, 34, 43]. Allopregnanolone treatment not only significantly reduced astrocyte proliferation and microglial activation, but also enhanced myelination in mice [44]. Neurosteroids, such as allopregnanolone, could reduce inflammatory cytokine levels, which were, for example, elevated following traumatic brain injury [45]. Our findings demonstrated that koumine administration increased 3α-HSOR activity, which could contribute to increased allopregnanolone levels in the SC of CCI rats. Consequently, we hypothesized that elevated allopregnanolone levels may exert analgesic effects through allosteric modulation of GABA_A and by suppressing the release of microglia activation-induced inflammatory cytokines. Further studies are warranted to determine whether koumine influences the activation of glial cells during the neuropathic pain state.

This study demonstrated that koumine could relieve neuropathic pain in rats by enhancing the mRNA expression and bioactivity of 3α-HSOR in the SC. Our study also suggested that by targeting 3α-HSOR, koumine altered 3α-HSOR-regulated allopregnanolone levels in the SC of rats. Therefore, koumine may be promising in the search for novel analgesic agents that are protective against painful neuropathy.