Synergism in LPS-induced RAW 264.7 macrophage cellsCell culture

RAW 264.7 macrophage cells were purchased from ATCC (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Sigma-Aldrich, MO, USA). The cells were seeded in 24-well plates at the density of 200,000 cells/well in DMEM supplemented with FBS and penicillin-streptomycin and incubated at 37 °C in a 5% CO2 atmosphere for 24 h.

Cytotoxicity profiling

The cells were treated with different concentrations of curcumin (1.25, 2.5, 5, 10, 20 μM) and piperine (12.5, 25, 50, 100, 200 μM), and the plates were incubated at 37°C in a 5% CO2 atmosphere for 24 h. The culture media was removed, and the cells were incubated with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL) for 2 h. Then, the MTT solution was removed, and dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 570 nm using a microplate reader.

NO assay

The NO production in cultured cells was measured using the Griess reaction. The cells were pre-treated with curcumin (0.625, 1.25, 2.5 and 5 μM), piperine (3.125, 6.25, 12.5 and 25 μM) and their combination (0.625 + 3.125, 1.25 + 6.25, 2.5 + 12.5 and 5 + 25 μM Cur + Pip) for 2 h and then challenged with 1 μg/mL LPS for 22 h. Then, 100 μL of cell culture media was transferred to a 96-well plate, followed by adding 50 μL of 1% (w/v) sulfanilamide and incubation in the dark for 5 min. The media was further incubated with 50 μL of 2.5% (w/v) N-1-Napthylenediamine dihydrochloride for another 5 min in the dark. The absorbance was measured at 520 nm.

Median-effect analysis

The median effect analysis described by Chou-Talalay was employed to determine the type of interaction between curcumin and piperine [31]. The dose–effect relationship between compounds was derived using the median effect equation:

$$}_}} /}_}} = \, \left[ }/}_}} } \right]^}} ,$$

where Fa, fraction effect by compound at the concentration C (Fa values ranging from 0 to 1 represent 0 to 100% inhibition of NO production); Fu, fraction unaffected (Fu = 1 – Fa); C, concentration of test compound; Cm, concentration required to produce x% effect; m, sigmoidiciy coefficient of the dose–response curve. Then, the combination index (CI) was determined using the following formula:

$$} = \, \left[ } \right]_} /\left[ }_}} } \right]_} + \, \left[ } \right]_} /\left[ }_}} } \right]_} ,$$

where [C]1 and [C2], compound 1 and 2 concentrations in combination that produce x% effect; [Cx]1 and [Cx]2, compound 1 and 2 concentrations alone that produce x% effect. The interaction between the compounds was further visualized in the fraction affected-combination index (Fa-CI) plot and isobologram. The interaction is identified as an additive, synergistic, or antagonistic if the CI values are 1, < 1, or > 1, respectively.

ELISA

The cell culture media was further analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the effect of compounds on LPS-induced proinflammatory cytokine production. IL-6 and TNF-α expression levels in cell culture media were analyzed using a commercial ELISA kit (BioLegend), according to the manufacturer’s instructions.

Safety evaluation in SH-SY5Y neuronal cellsCell culture

The SH-SY5Y neuroblastoma cells were purchased from ATCC (MD, USA). The cells were maintained in DMEM/F-12 media supplemented with 10% FBS and 1% penicillin-streptomycin and incubated at 37 °C in a 5% CO2 atmosphere.

Cell viability assay

Cells were seeded in 96-well plates at 50,000 cells/well density and incubated at 37°C in a 5% CO2 atmosphere for 24 h. The cells were exposed to EC50, EC75, and EC90 concentrations of curcumin, piperine alone, and their combination (obtained in RAW 264.7 macrophage cell line) for 24 h. Then the cell viability was measured using the MTT assay.

Apoptosis and necrosis assay

Hoechst 33342 and Propidium Iodide (PI) staining were used to visualize the morphology and characteristics of apoptotic and necrotic cells, respectively. Cells were seeded in 24-well plates at the density of 200,000 cells/well and treated with EC90 concentrations of curcumin, piperine, and their combination for 24 h. Then the cells were washed with PBS and stained with Hoechst 33342 and PI solutions for 15 min. The cell morphology was observed under a fluorescence microscope (Olympus IX51 inverted microscope, Tokyo, Japan), and the images obtained were further processed using Image-J (NIH, MD, USA).

Animals

Male ICR mice aged 5–8 weeks (Nomura International, Bangkok, Thailand) were used for all experiments. Mice were acclimatized in the animal facility for at least 1 week before the experiment. The mice were housed 4–5 mice per cage and maintained on 12 h light/dark conditions, with a humidity of 40–60% and a temperature of 23 ± 1 °C with food and water ad libitum. In the experiments, animals were randomly selected for a given group. The protocols and procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences, Chulalongkorn University (Protocol No. 20–03-003).

Compound preparation and administration

Curcumin (> 95.0%) was obtained from Shaanxi Kanglai Ecology Agriculture Co., Ltd., Xi’an, 110 China. Piperine (> 97.0%) was obtained from Sigma, St. Louis, MO, USA. Mice were randomly allocated into five groups for each compound. The behavioral tests were carried out at 09.00 -17.00 in a quiet room during the daytime. On the day of the experiment, mice were allowed to acclimatize 1–2 h to the laboratory room. For curcumin treatment in the formalin test, each group of mice received carboxymethyl cellulose (CMC, 0.5%, in normal saline) and curcumin at 10, 30, 100, 300 mg/kg body weight orally. For piperine treatment in the formalin test, 3, 10, 30, and 100 mg/kg doses of piperine were selected. In a thermal nociceptive test by tail-flick, 3, 10, 30, 100 mg/kg of curcumin and 1, 3, 10, 30 mg/kg of piperine were administered orally. For the cold plate test, curcumin and piperine at 3, 10, 30, 100 mg/kg doses were used. The dose ranges of curcumin and piperine alone were selected according to previous studies [14, 32]. Furthermore, the coadministration of curcumin and piperine to the mice was performed using at least four doses of the combination in a fixed ratio (1:1) of ED50 of each treatment alone: 1/2, 1/4, 1/8, and 1/16 × (curcumin ED50 + piperine ED50). All drugs were suspended in 0.5% CMC and administered orally in a constant volume of 10 ml/kg bodyweight.

Assessment of pain-like behaviorsFormalin test

The subplantar surface of the left hind paw was subcutaneously administered with 10 µL of 5% formalin diluted in normal saline one hour after compound administrations. Duration of licking behaviors as a representative of pain-like behaviors was recorded for 40 min as previously described [33]. The licking behaviors were categorized to phase I (0–5 min) and phase II (10–40 min) for analysis. The percentage antinociceptive efficacy of the test compounds was calculated using the following formula:

$$\% } = 100 - \left[ }_}}} /}_}}} } \right)} \times }00} \right]$$

Dtreatment is the duration of licking behaviors of mice receiving either monotherapy of curcumin, piperine, or their combination, whereas Dcontrol represents the duration of licking behaviors of mice receiving 0.5% CMC.

Biochemical analysis of paw tissues and spinal cord

After behavioral measures, mice were euthanized by CO2 aspiration, and ipsilateral paw and spinal cord tissues were extracted. Isolated tissues were weighed and mixed with ice-cold PBS (20%, w/v), centrifuged at 10,000 rpm, 4°C for 10 min. Supernatants were collected and stored at -80°C until used for ELISA. IL-6 and TNF-α expression levels in tissue supernatants were analyzed using a commercial ELISA kit (BioLegend, San Diego, CA, USA), according to the manufacturer’s instructions.

Tail-flick test

The tail-flick test was selected to assess the effects of the test compounds on thermal/heat nociception. The thermal stimuli from the tail-flick apparatus (Harvard Apparatus, Massachusetts, USA) were applied to the tail of the mice at the mid-region of the dorsal surface. The heat lamp intensity was adjusted to obtain the baseline latency of 3–4 s. The duration of the stimulation until the flicking of the tail was considered tail-flick latency. The cut-off value was set to be 8 s to avoid tail tissue damage. The effects of the test compounds were assessed at time intervals of 0, 15, 30, 60, 90, 120, and 240 min post-compound administration. Percentage antinociception was presented as the percentage of the maximal possible effect of the treatment (%MPE), which was determined using the following formula:

$$\% } = }\left[ } - }} \right)}/}\left( } - }} \right)} \right]} \times }00$$

Cold plate test

The cold plate test was used to assess the effects of curcumin and piperine in monotherapy and combination therapy on cold nociception using a cold plate apparatus (Ugo Basile, VA, Italy). The apparatus was set at 2°C constant temperature, and the baseline latencies to pain-like behaviors (licking, lifting, or shaking of hind paws or jumping out from the cold surface) before compound administration was recorded in triplicate. Then the mice were orally administered with test compounds, and the cold plate latencies were measured 60 min post-compound administration. A cut-off time of 60 s was established to avoid tissue damage. Percentage antinociception was presented as the percentage of the maximal possible effect of the treatment (%MPE), which was determined using the following formula:

$$\% } = \, \left[ } - }} \right) \, / \, \left( } - }} \right)} \right] \, \times 00$$

Assessment of CNS safety profile—LABORAS automated home cage behavioral analysis

The effects of the test compounds on spontaneous locomotor activity were assessed in the LABORAS automated home cage behavioral analysis as previously described [34]. Mice were administered with the highest dose of curcumin (300 mg/kg), piperine (100 mg/kg), and their theoretical ED50 doses in the formalin test and tail-flick test (44.9 and 21.3 mg/kg, respectively). The spontaneous locomotor activity was measured at one hour post-compound administration for 30 min. The effects of individual curcumin, piperine, and the combination on spontaneous locomotor activity were presented as duration and frequency of mobile behaviors (climbing, rearing, locomotion), immobility, speed, and distance traveled. The position distribution of mice in the cage was also visualized.

Data analysisED50 analysis

The doses that produce 50% antinociceptive effects in formalin, tail-flick, and cold plate tests were further analyzed. For the individual compound and their coadministration, experimental ED50 was determined by linear regression analysis of the log dose–response curve.

Isobolographic analysis

Isobolographic analysis was performed to determine the interaction between curcumin and piperine in the formalin, tail-flick, and cold plate tests, as previously described by Tallarida [35]. The experimental ED50 and theoretical ED50 were determined. The theoretical ED50 is calculated using the following formula:

$$}_}}} = }\left( }_}1}} } \right) + \left( }} \right)\,\,\left( }_}1}} } \right)$$

where ED50 add represents theoretical ED50, ED50 D1 represents ED50 of curcumin, ED50 D2 represents ED50 of piperine, f represents fraction.

The isobologram was constructed using the ED50 data, and the theoretical ED50 of curcumin and piperine were connected using a line (additive line). Further, the experimental ED50 of the combination was also included in the isobologram presented as a point. The location of the experimental ED50 of the combination in the isobologram was used to determine the antinociceptive interaction between curcumin and piperine. If the point is below the additive line, the interaction is considered synergistic, whereas if the point lies above the additive line, the interaction is considered antagonistic. The significant difference between the theoretical and experimental ED50 of the combination was also assessed by t-test to further confirm the antinociceptive interaction. Furthermore, the interaction index was calculated using the following formula:

$$\gamma = }_ \,} \mathord}_ \,} }}}} \right. \kern-\nulldelimiterspace} }}}_}}}$$

where γ represents the interaction index, ED50 exp represents experimental ED50, and ED50 add represents theoretical ED50. The interaction index values of < 1, 1, and > 1 are used to define synergistic, additive, and antagonistic interaction, respectively.

Statistical analysis

All data are presented as means ± SEM. Data were analyzed using GraphPad Prism 9.4.1 by analysis of variance (ANOVA) followed by Bonferroni post hoc test and t-test. The significant level is p < 0.05.