Antinociceptive Effect of Tetrandrine on LPS-Induced Hyperalgesia via the Inhibition of IKKβ Phosphorylation and the COX-2/PGE2 Pathway in Mice

Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid that is isolated from the Stephania Tetrandra. It is known to possess anti-inflammatory and immunomodulatory effects. We have shown that TET can effectively suppress the production of bacterial lipopolysaccharide (LPS)-induced inflammatory mediators, including cyclooxygenases (COXs), in macrophages. However, whether TET has an antinociceptive effect on LPS-induced hyperalgesia is unknown. In the present study, we investigated the potential antinociceptive effects of TET and the mechanisms by which it elicits its effects on LPS-induced hyperalgesia. LPS effectively evoked hyperalgesia and induced the production of PGE2 in the sera, brain tissues, and cultured astroglia. TET pretreatment attenuated all of these effects. LPS also activated inhibitor of κB (IκB) kinase β (IKKβ) and its downstream components in the IκB/nuclear factor (NF)-κB signaling pathway, including COX-2; the increase in expression levels of these components was significantly abolished by TET. Furthermore, in primary astroglia, knockdown of IKKβ, but not IKKα, reversed the effects of TET on the LPS-induced increase in IκB phosphorylation, P65 phosphorylation, and COX-2. Our results suggest that TET can effectively exert antinociceptive effects on LPS-induced hyperalgesia in mice by inhibiting IKKβ phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE2 and COX-2, via the IKKβ/IκB/NF-κB pathway.


Introduction
Inflammatory mediators, such as prostaglandins (PGs), PG synthases, and cyclooxygenases (COXs), can cause abnormal neuronal activity, which leads to pain hypersensitivity [1]. Over the past decade, many studies have focused on the roles of these mediators in the regulation of hypersensitivity that is induced by environmental stimuli and pro-inflammatory factors, such as bacterial lipopolysaccharide (LPS) [2].
In the central nervous system (CNS), treatment of astrocytes and microglia with low concentrations of LPS can produce PGE 2 via Toll-like receptor 4-dependent manners [3,4]. PGE 2 can directly trigger pain-sensitive neurons to induce nociception [5,6]. At the same time, PGE 2 receptors that are located in peripheral tissues can scatter to the end of nociceptive nerve endings, thus sensitizing the CNS to the existence of nociceptive stimulation [7]. The COXs are rate-limiting enzymes that catalyze the synthesis of PGs. There are two distinct isoforms: COX-1 and COX-2. Although the existence of COX-3 has been reported, its roles and effects in humans are still unclear [8][9]. COX-1 is constitutively expressed to regulate normal physiological conditions, whereas COX-2 is initiated in response to inflammatory signals, such as cytokines and LPS. Moreover, in inflammatory pain conditions, COX-2 itself can act as a nociceptive stimulator to directly cause pain. COX-2 is regulated by nuclear factor (NF)-kB, which is a well-known transcription factor that is involved in inflammation or injury. Recent reports revealed that NF-kB is also implicated in hyperalgesia [10][11], which is regulated by a series of adaptors. Under normal conditions, NF-kB is inactive, and it is bound to inhibitor kB (IkB) via its subunits, P65 and P50, in the cytoplasm. Upon IkB kinase (IKK) activation, IkB is phosphorylated, thus resulting in its ubiquitination and subsequent degradation by the 26S proteasome. NF-kB then translocates into the nucleus to regulate the transcription of genes that code for inflammatory cytokines and nociceptive substances [12,13].
Tetrandrine (TET) is an important bisbenzylisoquinoline alkaloid that is isolated from Stephania Tetrandra (Fig. 1A). It is traditionally used in China and Korea to treat patients with arthritis. Previous studies have shown that it possesses antiarrhythmic [14], anti-hypertensive [15], cardio-protective [16], antitumorigenic [17], and anti-inflammatory effects [18]. We have demonstrated that TET exhibits anti-inflammatory and hepatoprotective effects in mice [19,20], and it inhibits IL-6 and TNF-a production in macrophages. However, whether it is involved in the inflammatory processes of nociception is unknown. In this study, we tested the role of TET on LPS-induced hyperalgesia in mice and investigated the potential mechanisms by which TET elicits its effects.

Animals
BALB/C mice (6-8 weeks old, 20-22 g) were obtained from the Laboratory Animal Center of Chongqing Medical University (Chongqing, China). All mice received humane care, and all studies were performed with approval from the Animal Care and Use Committee of Chongqing Medical University (approval #SCXK20070001). The mice were maintained in a SPF-grade facility under controlled conditions (22uC, 55% humidity, and 12 h day/night rhythm) and fed standard laboratory chow. After each experiment, mice were sacrificed under anesthesia with isoflurane and decapitated to ameliorate any suffering.

Materials and Drug Preparations
TET (C 38 H 42 O 6 N 2 , molecular weight: 622.8 g/mol) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and its purity was determined by HPLC, as previously described [19,20]. LPS (Escherichia coli, 0111:B4), morphine hydrochloride, and indomethacin were purchased from Sigma (MO, USA). All drugs were freshly prepared on the day of experiments. TET powder was dissolved in 0.01 M hydrochloric acid, and the pH was adjusted to 5.5 with 0.01 M NaOH. The LPS stock solution was reconstituted to a final concentration of 10 mg/ml. Indomethacin solutions for intraperitoneal injections were prepared fresh in 0.01 M sodium carbonate, pH 7.2, at a final concentration of 0.2 mg/ml.
The hot-plate and acetic acid-induced abdominal constriction (writhing) tests were performed to measure the hyperalgesic responses to LPS in the presence or absence of TET, indomethacin, or morphine. The hot plates (RB200, Chengdu TME, China) were maintained at 5561uC, and induction time was determined by measuring the latency of paw licking every 2 h until 8 h after treatment. Due to its long-term effects, acetic acid was administered only once for each individual mouse at different time points (0, 2, 4, 6, and 8 h) and concentrations. The acetic acid-saline solution (0.1 ml/10 g of 0.7% acetic acid-saline) was intraperitoneally injected, and the frequency of abdominal constrictions was counted for 20 min. Writhe was defined as the contraction of abdominal muscles, which were accompanied by the extension of forelimbs and elongation of the body.

Treatment and culture of astroglia
To prepare mouse cerebral astrocytes, cerebral cortices from P1 neonatal BALB/C mice were mechanically dissociated in astrocyte culture medium (Dulbecco's Modified Eagle Medium [DMEM] with 10% fetal bovine serum [FBS] and antibiotics). After filtering through a 70 mm cell strainer, the cells were seeded in cell culture flasks. To obtain astroglia, confluent cultures were shaken at 250 rpm overnight at 37uC. The purity of astrocytes was checked by immunostaining for GFAP (Abcam, MA, USA), and the threshold was set at .95% [21,22]. When the primary cells reached 80-90% confluency, they were digested by 0.25% trypsin and plated in 12-well tissue-culture plates at a density of 1.0-1.5610 5 cells/well. When the cells in the 2 nd passage were close to confluence, the culture medium was replaced with FBS-free DMEM. Cell treatments were performed according to following groups: control group (only the FBS-free DMEM); LPS-stimulated group (1 mg/ml); low, moderate, and high TET-treated groups (1 mg/ml LPS with TET [1610 28 mol/l, 1610 27 mol/l, and 1610 26 mol/l, respectively]). Treatments lasted for 6 hours, after which the cells and supernatants were harvested for various experiments.

Enzyme Immunoassay (EIA)
PGE 2 levels in purified plasma, brain homogenates, and cell culture supernatants were evaluated using a commercial EIA kit (Cayman, Michigan, USA), according to the manufacturer's protocol. Samples were added to a plate that was pre-coated with goat anti-mouse IgG antibodies. PGE 2 monoclonal antibodies were then added to each well, and the plates were incubated for 18 h at 4uC. Afterwards, Ellman's Reagent substrate was added to each well. The optical density of each sample was read at 412 nm. The standard curve was plotted, and the final concentrations of PGE 2 in the samples were calculated using the equations that were obtained from the curve.

Small-interfering RNA (siRNA) transfection
The SignalSilence siRNAs of IKKa, IKKb, and control siRNA (unconjugated) were obtained from Santa Cruz Biotechnology (CA, USA). Cells were plated onto a 6-well plate at a density of 1.6610 5 cells/well. Once they were 60-80% confluent, cells were washed with PBS, and the pre-mixed siRNA transfection solution (including siRNA duplex solution and the dilution reagent) was added directly to the culture medium. Cells were then incubated for 24 h, and the culture medium was changed for another 24 h. The reactions were stopped, and lysis buffer was added to extract proteins from the cells for further experiments.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from brain tissues and cultured cells using Trizol reagent, (Invitrogen, USA) according to the manufacturer's guidelines, followed by further purification using the RNeasy Mini Kits (Qiagen, USA). Purified RNA was reverse-

Statistical Analysis
All data were expressed as mean6standard deviation (M6SD) from at least four independent experiments. Results were analyzed by Student's t test or analysis of variance (ANOVA). P#0.05 was considered to be statistically significant.

LPS induced hyperalgesia in mice via time-and dosedependent manners
The administration of LPS (50, 100, or 200 mg/kg, i.p.) evoked dose-dependent hyperalgesia, as evaluated by the hot-plate and acetic acid-induced abdominal constriction tests (Fig 1). In the hotplate test, hyperalgesia was assessed by the action of hind-paw licking. A decrease in the latency time of paw licking was observed at 2 h after LPS treatment, and maximal reduction was observed at 6 h (from 17.0 to 9.7, 7.1 and 4.1 seconds at LPS of 50, 100, 200 mg/kg, respectively). This was maintained until 8 h after LPS treatment (Fig 1B). Acetic acid-induced abdominal constriction numbers increased with LPS treatment, and the analogous initial and peak time points were determined ( Fig 1C). Based on these results, we chose the 6-h time point for further experiments. In addition, mice that were exposed to the highest concentration of LPS (200 mg/kg) exhibited obvious hyperalgesia. However, this was accompanied by adverse reactions, including cachexia, diarrhea, and sustained tumbling, all of which may potentially influence the evaluation of the algesic effect. Alternatively, a lower dose of LPS (100 mg/kg) induced obvious hyperalgesia without the adverse effects. Therefore, this concentration of LPS was used for further experiments.

Analgesic effects of TET on LPS-induced hyperalgesia in mice
Different doses of TET were administered to mice 30 min prior to LPS treatment. LPS-induced hyperalgesia was significantly repressed, as indicated by the elongated threshold time in hot-plate tests and the decrease in the number of writhing in acetic acidinduced abdominal constriction tests. The percentages of protection at 6 h by TET concentrations of 15, 30, and 45 mg/kg were 11.7%, 27.8%, and 59.6%, respectively, in the hot-plate test (Fig. 1D) and 9.7%, 16.8%, and 49.6%, respectively, in the acidinduced abdominal constriction test (Fig. 1E). Indomethacin (5 mg/kg) and morphine (10 mg/kg) were used as the positive controls, due to their known antinociceptive effects [23,24]. The percentages of protection by morphine and indomethacin were 81.2% (hot-plate test) and 62.7% (acid-induced abdominal constriction test), respectively. These results indicate that TET may possess both peripheral and central antinociceptive properties on LPS-induced hyperalgesia in mice.

TET repressed PGE 2 production in LPS-induced hyperalgesia in mice and cultured astroglia
To explore the antinociceptive mechanism of TET, PGE 2 production was measured by EIA in the sera and brain tissues of LPS-induced hyperalgesic mice and in the supernatants of cultured astroglia. In vivo, LPS significantly increased PGE 2 levels, which were markedly suppressed by TET pretreatment in dosedependent manners ( Fig. 2A, 2B). To exclude the pathophysiological conditions in vivo that may potentially affect the intrinsic reactions, we cultured astroglia cells to verify the mechanism in vitro. The toxicity of TET on astroglia was evaluated by the MTT assay, and we confirmed that TET concentrations from 1610 28 mol/l to 1610 26 mol/l did not significantly repress cell viability (Fig. 2C). Therefore, these concentrations of TET were used in further experiments. Treatment of astroglia with 1 mg/ml LPS significantly increased PGE 2 levels. Similarly, TET cotreatment attenuated PGE 2 levels in a concentration-dependent manner. The repressive peak was at the TET concentration of 1610 26 mol/l (Fig. 2D). These results suggest that the antinociceptive effect of TET on LPS-induced hyperalgesia in mice may be partially mediated through downregulation of the PGE 2 signaling pathway.

TET suppressed COX-2, but not COX-1, levels
Following its release from membrane phospholipids by cytosolic or secretory phospholipases, arachidonic acid is converted to PGE 2 by COX-1 and COX-2 [25]. We then investigated the expression of COX-1 and COX-2 at the mRNA and protein levels. As shown in Figure 3A, brain tissues from LPS-stimulated mice exhibit four-fold increases in COX-2 protein levels, and TET pretreatment decreases these levels of COX-2. No changes in COX-1 were observed in the presence or absence of LPS or TET. This indicates that TET can selectively suppress COX-2 expression. Similar trends in the mRNA levels of COX-1 and COX-2 were observed ( Fig. 3B and 3C). Thus, the mechanisms of action in vivo and in vitro appear to be the similar processes.

TET decreased COX-2 expression through IKKb, which further inhibited the NF-kB pathway
The NF-kB signaling pathway is consensually involved in LPSinduced cell activation and inflammation [26,27]. To investigate whether NF-kB activity is also regulated by TET, the expression of various components of the NF-kB pathway, including P65, pP65, IkBa, and pIkBa, were assessed by western blotting in cultured astroglia. As shown in Figure 4A, P65 expression is not significantly changed in the presence of LPS or TET. However, pP65 was notably up-regulated after LPS stimulus and gradually reversed by TET pretreatment. Meanwhile, pIkBa levels increased dramatically after LPS treatment and decreased with TET pretreatment. No changes in IkBa levels were observed. These results suggest that TET can inactivate the NF-kB signaling pathway through the inhibition of LPS-induced increases in IkBa phosphorylation, thus preventing the degradation of IkBa and retaining NF-kB in the cytoplasm (Fig. 4A).
The phosphorylation of IkBa is catalyzed by the IKK complex, which is comprised of the IKKa, IKKb, and IKKc subunits. Among these, IKKa and IKKb serve as the catalytic subunits to phosphorylate IkB for degradation via ubiquitination [28]. Hence, we investigated whether IKKa and IKKb are the upstream targets for TET in the NF-kB pathway. Western blotting showed that LPS increased the phosphorylation of IKKb without affecting IKKa, which was consistent with previous reports [29,30,31]. Similarly, TET pretreatment effectively inhibited IKKb phosphorylation without affecting IKKa (Fig. 4B), which suggests that TET represses IkBa activity by inhibiting IKKb.
To verify whether TET specifically targets IKKb, cells were transfected with IKKa or IKKb siRNA (si) to knock down their respective gene expression levels. As shown in Figure 5A, cells transfected with siIKKb exhibit decreases in the protein expression of phosphorylated IKKb, and pP65. LPS stimulus partially rescued these trends, although not to levels of those seen in LPStreated control siRNA-transfected cells. SiIKKb transfection also did not affect IKKa phosphorylation, and phosphorylated IKKb did not functionally affect pIKKa in the case of LPS challenge, as shown by other groups [32,33,34]. On the other hand, transfection of cells with siIKKa only decreased the expression of pIKKa without affecting the levels of pIKKb or pP65. Collectively, these results show that IKKa and IKKb played distinct roles in the pathway of LPS-induced hyperalgesia. Moreover, IKKa may not directly participate in the LPS-and TET-signaling cascade. TET treatment in cells with IKKb knockdown had no effect on the levels of pIKKb and pP65. This was partially due to the low levels of IKKb that already existed in these cells with IKKb knockdown.
Next, to further determine whether the pathway of ''LPS/ TET-IKKb-NF-kB-COX-2/PGE 2 '' practically take effect in astroglia, we investigated the COX-2 generation under the control siRNA, siIKKa, siIKKb knockdown with or without TET treatment (Fig 5B). Results showed both siIKKb and TET could decrease COX-2 generation, but not siIKKa, which more solidly supported the conclusion of TET specifically inhibited IKKb phosphorylation and subsequently downregulate COX-2/PGE 2 levels.

Discussion
LPS, which is a component of the cell wall of gram-negative bacteria, is known to activate a number of cellular signals in various cell types and tissues during inflammation and infection. In addition to its ability to cause endotoxic shock, LPS induces hyperalgesia in mice at lower doses [35]. A single dose of LPS that is administered centrally or peripherally can evoke a hyperalgesic reaction by decreasing mechanical nociceptive thresholds. In this study, we generated a hyperalgesic mouse model, in which BALB/ C mice were treated with LPS. The hyperalgesic effect of LPS was verified by the shortened latency time of paw licking using the canonical hot-plate test, as well as the increase in writhing numbers in the acetic acid-induced abdominal constriction test. Because the writhing model is sensitive to the antinociceptive action of non-steroidal anti-inflammatory drugs, including indomethacin [23], and the hot-plate test focuses on the pathophysiological process above the spinal cord level [24], we chose indomethacin and morphine to be the positive controls of the  writhing and hot-plate tests, respectively. Using these models, tests, and drugs, we were able to investigate the peripheral and central anti-nociceptive effects of TET.
TET dose-dependently reduced the nociceptive responses in the writhing and hot-plate tests in LPS-treated mice, thus suggesting that TET has both central and peripheral anti-nociceptive effects. Because PGE 2 is a critical pro-inflammatory and algesic factor, we measured its levels in vivo and in vitro. PGE 2 levels were significantly increased and repressed with LPS and TET treatments, respectively, in mouse sera, brain tissues, and cultured astroglia. This suggests that PGE 2 plays pivotal roles in LPS-induced hyperalgesia and TET-mediated analgesia. The COXs are key enzymes that regulate the formation of PGE 2 from arachidonic acid. LPS increased COX-2 expression in mouse brain tissues and cultured astroglia. No effects on COX-1 were seen. Consistent with the physiology of canonical pain, COX-2 acted as a key regulatory  synthase in the production of PGE 2 in our hyperalgesic mice and astroglia models. These results show that PGE 2 /COX-2 was the appropriate central pathway of hyperalgesia. Proportional decreases in central and peripheral PGE 2 /COX-2 levels by TET were also observed.
A crucial role for astroglia in mediating pain has been implicated by studies involving animal models and patients with persistent pain conditions [36]. Pro-inflammatory cytokines are produced and released by activated microglia and astrocytes in the CNS. The IKK/IkB/NF-kB signaling pathway regulates the expression of these inflammatory cytokines, including COX-2 and IL-1 [37]. Therefore, we isolated astrocytes from the brains of newborn mice and co-treated them with TET and LPS. The phosphorylation of IKKb, IkBa, P65 and COX-2 increased proportionally upon LPS stimulus, and these increases were significantly reversed by TET co-treatment, thus implicating the IKKb/IkBa/NF-kB pathway in LPS-induced hyperalgesia and TET-induced antinociception. No effects on IKKa were observed. Knockdown experiments with IKKa or IKKb siRNAs further clarified the mechanism by which TET elicits its analgesic effects, and the results show that LPS induced NF-kB pathway activation by, at least in part, triggering the phosphorylation of IKKb but not IKKa. Interestingly, TET specifically targeted IKKb phosphorylation in LPS-treated astroglia, and eventually depressed NF-Kb activation and COX-2/PGE 2 expression. These results allow us to better understand the mechanisms by which LPS and TET induce hyperalgesia and antinociception, respectively, and show that both effects were elicited via the activation or inhibition of IKKb phosphorylation and the downregulation of the NF-kB/COX-2/ PGE 2 pathway.
Although TET appears to mediate analgesia via inhibiting IKKb phosphorylation, it may also target other components of the pathway that are upstream of IKK. Additionally, the modulation of pain by peripherally derived inflammatory mediators involves factors and effector cells other than PGE 2 and astroglia, respectively. The microglia and spinal glia also participate in pain modulation [38,39]. Whether the central modulation of pain involves the actions of the other eicosanoid metabolites, nitric oxide, or pro-inflammatory mediators requires further elucidation. Therefore, more work needs to be done to reveal the exact mechanisms of hyperalgesia, as well as the main mechanisms behind the analgesic effects of TET.