Abstract Background Depression is a prevalent and complex psychiatric disorder with high incidence in patients with chronic pain. The underlying pathogenesis of chronic pain-induced depression is complicated and remains largely unclear. An integrated analysis of endogenous substance-related metabolisms would help to understand the molecular mechanism of chronic pain-induced depression. Curcumin was reported to exert various health benefits, such as anti-depression, antioxidant, antineoplastic, analgesia, and anti-inflammation. Objective The aim of this study was to analyze the biomarkers related to depression in serum and to evaluate the anti-depression properties of curcumin in a chronic pain-induced depression model of rats. Design This is a randomized, controlled experiment. Setting This study was conducted at the Experimental Animal Center, Beijing Friendship Hospital, Capital Medical University. Methods Trigeminal neuralgia (TN) was produced by injecting 4 µL, 10% cobra venom saline solution into the infraorbital nerve (ION). Curcumin was administered by gavage twice a day from post-operation day (POD) 15 to POD 42. Mechanical allodynia was assessed using von Frey filaments. Sucrose preference and forced swimming tests were performed to evaluate depression-like behaviors. The metabolomics analysis was preceded by LCMS-IT-TOF and multivariate statistical methods for sample detection and biomarker screening. Results Cobra venom intra-ION injection led to chronic mechanical allodynia, reduced sucrose preference, and prolonged immobility during forced swimming. Curcumin treatment alleviated chronic mechanical allodynia, regained sucrose preference, and reduced immobility time. Differential analysis identified 30 potential metabolites changed under TN condition. The integrated analyses further revealed two major metabolic changes by comparing the serums from sham operated rats, TN rats, and TN rats treated with curcumin: 1) ether lipid metabolism; and 2) glycerophospholipid metabolism, and suggested that curcumin may improve chronic pain-induced depression by regulating these two types of lipid metabolisms. Conclusion Ether lipid and glycerophospholipid metabolism might be two of the pathways with the most potential related to chronic pain induced-depression; and curcumin could alleviate chronic pain induced-depression by modulating these two pathways. These results provide further insights into the mechanisms of chronic pain-induced depression and may help to identify potential targets for anti-depression properties of curcumin. Keywords: curcumin, depression, chronic pain, trigeminal neuralgia, metabolomics Introduction Chronic pain patients often develop into depression,[36]^1^,[37]^2 which leads to additional emotional and cognitive deficits,[38]^3^,[39]^4 and has become a general medical problem. The involvements of anatomical, neurochemical, and psychological changes in depression induced by chronic pain have been intensively studied.[40]^5–7 However, the potential contributions of microcosmic metabolite changes under pathologic pain conditions to development of depression has not had enough attention yet. As a powerful approach to directly reflect the underlying biochemical activity and state of cells or tissues, metabolome reveals the entire metabolic profile by detecting over 1,000 molecules in various biological samples, such as blood, urine, saliva, and cerebrospinal fluid.[41]^8 By analyzing specific biomarkers, metabolic profiling can provide a complete insight of disease progression and the outcome of drug treatment by functional readout of the physiological state of a whole biologic system.[42]^9 Due to its antioxidant, anti-inflammatory,[43]^10 antimutagenic, antimicrobial,[44]^11^,[45]^12 and anticancer properties,[46]^13^,[47]^14 curcuma longa has been traditionally used in Asian countries as a medical herb. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), also called diferuloylmethane, is the main natural polyphenol found in the root of turmeric (Curcuma longa) and in others Curcuma spp. [PMID:12680238], and has been shown to target multiple signaling molecules while also demonstrating activity at the cellular level, which has helped to support curcuma longa multiple health benefits.[48]^15 Curcumin has been shown to benefit pain and depression through inhibiting monoamine oxidase, modulating the level of serotonin and monoamine, inhibiting glutamate release in the prefrontal cortex,[49]^16–18 and activating MAPK/ERK-dependent BDNF expression in the amygdala region.[50]^19 However, a detailed metabolic profiling for the anti-depression effects of curcumin has not been done yet. In our previous studies, our team developed a new trigeminal neuralgia rat model induced by cobra venom injected into the infraorbital nerve (ION); the model displays persistent pain and mimics the trigeminal neuralgia onset in human patients.[51]^20 In this study, we performed a metabolomics assay using HPLC coupled with IT-TOF mass spectrometry and characterized the metabolic profiles underlying depression evoked by trigeminal neuralgia, which was induced by injecting cobra venom into the infraorbital nerve (ION), as reported in our previous study.[52]^20 Furthermore, we studied if curcumin can be used to treat depression induced by chronic pain, and its regulation to biomarkers and metabolic profiles, which were specifically changed under neuropathic conditions. Methods Animals Male SD rats (200–220 g) were purchased from The Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College and used in this study. All animal procedures were approved by the Ethical Committee of Beijing Friendship Hospital, Capital Medical University (Beijing, China), and were performed in accordance with the guidelines of the International Association for the Study of Pain. Rats were housed under a 12-hour light/dark cycle and at a temperature of 22–24°C. All rats were given food and water ad libitum. Twenty-five rats were randomly divided into three groups: sham group (n=8), TN+vehicle group (n=8), and TN+curcumin group (n=9). Curcumin or vehicle was orally administered by gavage twice a day (morning and evening) from POD 15 to 42. Drug Preparation Lyophilized cobra venom (Formosan cobra; Sigma, St. Louis, MO) was dissolved in 0.9% sterile saline to make 10% solution.[53]^20 Curcumin (CAS: 458–37-7, Sigma Aldrich, purity ≥99.5%) 0.3% was made in peanut oil and administered to rats in the TN+curcumin group (45 mg/kg, 1.5 mL) based on earlier literature.[54]^21–23 Blood Sample Preparation Orbital blood (1.5 mL) was collected from all rats weekly in the morning for 6 weeks except weeks 3 and 5, and centrifuged at 4,000×g for 15 minutes to obtain serum, which was sterilized without hemolysis and stored at −80°C. To prepare the samples for chromatography and mass spectrometry, 200 µL thawed serum was added into 600 µL methanol and mixed for 30 minutes at 4°C followed by high-speed centrifugation at 12,000 rpm and 10 minutes incubation at 4°C to remove solid debris. Finally, the supernatant was filtered through a microfiltration membrane (0.22 μm) to obtain the injection samples. Surgical Procedure Models of TN were produced according to the method described in our previous studies.[55]^20^,[56]^22 Briefly, after the rats were anesthetized by sodium pentobarbital (40 mg/kg, i.p.), a 1-cm radical incision was made above the left superciliary arch to expose the infraorbital nerve (ION). For rats assigned to the TN group, 4 µL cobra venom solution (10% in saline) was injected into the ION. The incision was closed in layers. Rats in the sham group received 4 µL of saline injection instead. Behavioral Testing Mechanical Sensitivity Test Mechanical allodynia was used to verify trigeminal neuralgia induced by cobra venom intra-ION injection. The rats were placed in a plastic chamber (20 cm × 25 cm × 15 cm) and acclimated for 15 minutes. Their mechanical withdrawal thresholds (MWT) were measured via von Frey filaments (Stoelting, Chicago, IL) and calculated through up–down method as described previously.[57]^20 Each filament was placed perpendicularly on the facial skin innervated by left ION, which is near the center of the vibrissa pad. A positive response was defined by brutally turning the head away, scratching the left side of the face, or attacking the filaments. Sucrose Preference Test Before the test, all rats received water and 1% sucrose solution alternately for 48 hours and fast for 24 hours. On the testing day rats were allowed to acclimate in the test room for 20 minutes and provided by a bottle of water and a bottle of 1% sucrose solution for 30 minutes, followed by another 30 minutes after swapping the two bottles. Sucrose preference was calculated by dividing the volume of consumed sucrose solution by total liquid consumption. Forced Swim Test In the forced swim test, immobility time was collected to evaluate depression-like behaviors. After 20 minutes acclimation in the test room rats were placed into a standard clear Porsolt chamber filled with water (25°C and 25-cm depth) for 15 minutes, and then removed from the chamber, dried, and returned to their home cages. Twenty-four hours later, the rats were placed into the same Porsolt chamber for 5 minutes. Immobility, defined as no hind paw movement for over 1 seconds during swimming,[58]^24 was recorded and analyzed in the second session by two independent researchers who were blinded to the rats before operation, and weekly after operation for 6 weeks, except the 3^rd and 5^th week. Chromatography and Mass Spectrometry Assay of Serum Samples Chromatography and mass spectrometry assay were performed on a LCMS-IT-TOF system (Shimadzu, Japan). Blood serum was separated on an XBridge^® C18 Column (3.5 μm, 2.1×100 mm, Waters). The temperature of the column was set to 40°C. The mobile phase flow rate was 0.30 mL/min and consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Serum sample injected (5 μL) was trapped using 100% mobile phase A for 13 minutes at a flow rate of 5 μL/min before being placed in-line with the analytical column and subjected to the gradient profile which was set as follows: 5% B for 3 minutes, 3–50% B for 3–5 minutes, 50% B for 3 minutes, 50–70% B for 8.0 to 10.0 minutes, 70% B for 10 minutes, 70–95% B for 20.0 to 22.0 minutes, 95% B for 3 minutes, 95–5% B for 25.0–27.0 minutes, and 5% B for 8 minutes. The eluent was directly introduced to the mass spectrometer. To ensure the stability and repeatability of the LCMS systems, a QC was run after every 10 samples were analyzed, and followed by a blank. The Q-Exactive mass spectrometer parameters were as follows: electrospray voltage was 1.57 kV, Orbitrap precursor spectra (AGC 3×106) were collected from 100–1,000 m/z; the gas temperature was 200°C; the gas flow was 0.8 L/min; and charge state screening was with a dynamic exclusion time of 30 seconds to discriminate against previously analyzed ions. The method was validated for selectivity, sensitivity, linearity, accuracy and precision, recovery, matrix effect, dilution integrity, and stability. Multivariate Data Analysis and Statistical Analysis of Metabolic Profiling Sample data were extracted using Profiling Solution software for peak detection and alignment. The full scan mode was set in the range of 100–1,000 m/z and data collection as the initial and final retention times. Resultant data matrices was analyzed by software SIMCA-P11.0 (Umetrics, Umea, Sweden) for multivariate data analysis, such as principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Prior to PCA, all data obtained from the matrix were mean centered and scaled to a Pareto variance. As an unsupervised pattern recognition approach, PCA was usually used for reducing the matrix dimension and demonstrating the intrinsic simples clustering degree. On this basis, PLS-DA was used to identify the variables responsible for separation, and correlating with the variable influence on projection (VIP) parameter select the most significant variables contributing to discriminate metabolomics profiles between sham and TN + vehicle groups and between TN + vehicle and TN + curcumin groups. In a PLS-DA model, the VIP is a weighted sum of squares of the PLS weight, indicating the importance of the variable to the entire model. The variables with VIP values ≥1.5 were considered as significant differences and were selected for further data analysis. Then, the compounds corresponding to the significantly changed variables between groups (P-value<0.05) were screened as biomarkers.[59]^25 All potential biomarkers were annotated by the Human Metabolome Database (HMDB) and METLIN database. Finally, the compounds and their KEGG data numbers were imported to Metaboanalyst 3.0 for further pathway and enrichment analysis. Behavioral data were analyzed using SPSS 19.0 Software. Statistical significance was determined (P<0.05) using repeated measures two-way ANOVA. Results Curcumin Alleviated Mechanical Allodynia After Trigeminal Neuralgia Intra-ION injection of cobra venom but not saline produced a fast-onset (within 1 week) and long-lasting (over 6 weeks) mechanical allodynia, which is one of the symptoms of trigeminal neuralgia in rats. From POD 15–42, curcumin (45 mg/kg in 1.5 mL) was administrated twice a day by gavage (10:00 am and 18:00 pm) ([60]Figure 1A). TN rats treated by curcumin exhibited a significant higher mechanical withdrawal threshold than TN rats that only received vehicle treatment (F(2,22)=17.25, P<0.0001) ([61]Figure 1B), suggesting long-term curcumin treatment significantly alleviated mechanical allodynia observed in intra-ION injection of cobra venom induced trigeminal neuralgia. Figure 1. [62]Figure 1 [63]Open in a new tab Curcumin alleviated mechanical allodynia induced by cobra venom intra-ION injection. (A) Schematic for the timeline of surgery (cobra venom or sterile saline injection) and curcumin administrations. (B) Mechanical allodynia induced by cobra venom intra-ION injection was attenuated by curcumin treatment. Two-way ANOVA, *P<0.05 vs the sham group, #P<0.05 vs the cobra venom group, n=8 or 9. Curcumin Alleviated Trigeminal Neuralgia-Induced Depression Forced swimming test and sucrose preference test were used to evaluate rat depression-like behaviors in this study. From POD 28, TN rats exhibited significantly less sucrose consumption and longer immobility time during swimming than rats in the sham group, indicating that TN induced depression-like behaviors, which on the other hand were alleviated after curcumin treatment demonstrated by regained sucrose consumption (F(2,22)=19.85, P<0.0001) and decreased immobility time during swimming (F(2,22)=66.58, P<0.0001) ([64]Figure 2A-[65]B). Figure 2. [66]Figure 2 [67]Open in a new tab The effects of curcumin treatment on depression behaviors observed in rats with chronic pain. (A) Sucrose preferences were progressively