Menhaden fish oil attenuates postpartum depression in rat model via inhibition of NLRP3-inflammasome driven inflammatory pathway

Background and aim: Postpartum depression (PPD) is a familiar problem which is associated with about 10–20% of women after child delivery. Fish oil (FO) has a therapeutic potentials to many diseases including mood disorders. However, there is paucity of data on the effects of FO supplementation on PPD rat model. Hence, this study aimed at investigating the potentials of FO in ameliorating depressive-like behaviors in PPD rat by evaluating the involvement of NLRP3-inflammasome. Experimental procedure: Thirty six virgin adult female rats (n = 6) were randomly divided into six groups; Group 1–3 were normal control (NC), Sham (SHAM) and ovariectomized group (OVX) respectively whereas group 4–6 were PPD rats forced-fed once daily with distilled water (PPD), fish oil (PPD + FO; 9 g/kg) and Fluoxetine (PPD + FLX; 15 mg/kg) respectively from postpartum day 1 and continued for 10 consecutive days. Rats behaviors were evaluated on postpartum day 10 through open field test (OFT) and forced swimming test (FST), followed by biochemical analysis of NLRP3 inflammasome proteins pathway in their brain and determination of neutrophil to lymphocyte ratio (NLR). Results: PPD-induced rats exhibited high immobility and low swimming time in FST with increased inflammatory status; NLR, IL-1β and NFкB/NLRP3/caspase-1 activity in their hippocampus. However, administration of FO or fluoxetine reversed the aforementioned abnormalities. Conclusion: In conclusion, 10 days supplementation with FO ameliorated the depressive-like behaviors in PPD rats by targeting the NFкB/NLRP3/caspase-1/IL-1β activity. This has shed light on the potential of NLRP3 as a therapeutic target in treatment of PPD in rats. Female albino Wistar rats with an average weight of 180–200 g were used in this study. The rats were supplied by Takrif Bistari Enterprise, Taman Kembangsari, Seri Kembangan, Selangor, Malaysia and were kept at Animal Behaviour Room, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia. The rats were housed individually per cage under 12 h/12 h day/light cycle with free access to water and standard rat chow, and they acclimatized for 2 weeks before the experiment began. All the experimental procedures were conducted following Animal handling and ethical guidelines approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia (UPM/IACUC/AUP-R097/2015). The rats were randomly allocated into 6 groups (Fig. 1) containing six animals per group. Normal control (NC), Sham-operated (SHAM), Ovariectomized (OVX), PPD rats fed with distilled water (PPD), PPD rats fed with fish oil (PPD + FO) 9 g/kg and PPD rats fed with antidepressant fluoxetine (PPD + FLX) 15 mg/kg. The doses of FO and fluoxetine selected for this study were based on previous research, where PPD rats were supplemented with FO for 15 days.8 The ovariectomized rats were intraperitoneally injected with hormones, estradiol benzoate and progesterone for 23 days; mimicking the gestation period of rodents, thus inducing postpartum state (Fig. 1)19 The sudden decrease of oestrogen and progesterone hormones leads to mood changes known as postpartum blues that will eventually end up as a PPD.20 All treatments commenced on PPD day 1 and ended onPPD day 10. Forced swimming test and open field test (OFT) were both carried-out on PPD days 2 and 10 respectively to screen for depressive-like behaviours in the rats during the 10 days fish oil supplementation.19,21 All the rats were euthanized on PPD day 10 through decapitation and their prefrontal cortex (PFC) and hippocampus were harvested for evaluation of NFкB, NLRP3, caspase-1 and IL-1β concentrations. Timeline and treatment of experimental rats groups, OVX = ovariectomy, FO = fish oil, FLX = fluoxetine, PP = Postpartum. The induction of an animal model of PPD was done using HSPW protocol.8,18,19 It began with ovariectomy followed by 23 days of ovarian hormones injection and subsequent withdrawal of the hormonal injections to mimic the postpartum condition in the rats. At the beginning of the experiment, rats were ovariectomized bilaterally using an aseptic technique. The ovariectomy was conducted under anaesthesia (ketamine 80 mg/kg and xylazine 10 mg/kg; im). The ovariectomized rats were observed daily for seven days post-surgery to full recovery. Iodine was applied unto the wounds, while their cages were cleaned daily to prevent infections and contamination. Each rat was maintained in a separate cage during the observation period. Sham-operated rats were just sutured back as their ovaries were left intact.8,18 One week after the ovariectomy, hormone regimens were started. The rats were injected with ovarian hormones estradiol benzoate and progesterone daily for 23 days (Table 1). From day 1–16, 2.5 μg/rat of Estradiol benzoate and 4 mg/rat of Progesterone were administered. However, from day 17–23 only 50μg/rat Estradiol benzoate was injected to mimic the normal gestation period of rats. The doses used were enough to induce postpartum behaviour as earlier reported.8,18 Maternal nesting test behaviour was conducted on day 22 of the hormone injection to ascertain a pregnancy-like state of the PPD rats’ model. After the last injection of hormones on day 22, paper towels were provided to each cage as nesting material for the rats. The rats were expected to make a nest with the paper towels as preparation for having their offspring as if they were pregnant. 24 h later, the cages were examined for the presence of nest-like shape which indicates a positive result for maternal nesting behaviour test. Rats with positive results for maternal nesting test behaviour were considered a success in the induction of hormone-simulated pregnancy-like state and were withdrawn from the hormones injection to finalise PDD induction. The postpartum depressed rats were allocated into PPD experimental groups administered with distilled water (PPD), fish oil (PPD + FO) and fluoxetine (PPD + FLX) respectively. Hormone regime for induction of hormone-simulated pregnancy model (postpartum depression model induction).8 The FO supplementation protocol was conducted according to previous reports from the same laboratory with slight modifications.8 Briefly, the FO was administered to the rats through oral gavage, the feeding regimen lasted for 10 days postpartum (Fig. 1). The dose of the FO (Menhaden fish oil, Sigma F8020) administered was calculated daily using the formula (body weight (g) x 0.009)/density (g/ml) for dose 9 g/kg. While its composition includes, 30% omega-3 fatty acids with 10–15% Eicosapentaenoic acid and 8–15% Docosahexaenoic acid which has been proven to be safe in rats.8,22,23 Tail vein blood was taken from the rats for evaluation of neutrophil/lymphocyte ratio (NLR) on day 23 of hormone-simulated pregnancy. A thin blood smear was prepared and stained with Leishman’s stain. Observations were made using a light microscope at 40x magnification to calculate the number of neutrophils and lymphocytes. The NLR is a good indicator of distress condition in rats.24 Reports revealed that there would be an increased level of neutrophils and decreased level of lymphocytes in depressed subjects.25 To evaluate the antidepressant-like effects of 10 days FO supplementation on PPD rats’ model, FST was conducted. The protocol for FST was previously described,8 the rats were forced to swim and both their passive and active behaviours were recorded using a video camera for later evaluation. In passive behaviour, their time of immobility was recorded, while in active behaviour both swimming and climbing times were recorded and subsequently calculated. The OFT was conducted to evaluate the locomotor activities of the rats following treatments given. The behaviour of rats crossing the squares on the floor of a square box measuring 75 cm length x 75 cm breadth and 42 cm high were observed. The floor of the box was further subdivided by black lines into 25 smaller squares of equal dimensions, 15 cm square each. Each rat was allowed an hour for habituation in the behavioural room, prior to the test. The test session for each rat lasted for 5 min and was recorded using a video for later use8,18 On PPD day 10, rats were euthanized using a guillotine, their brains were collected as described previously.8,18 Briefly, the brain was dissected out carefully and washed with cold 1x phosphate-buffered saline (PBS) (BR0014G, Oxoid Ltd, UK), 1 tablet dissolved in 100 mL deionized water. The rats prefrontal cortex and hippocampus were carefully harvested, weighted and homogenized using Polytron PT-MR 1600 E (Kinematica AG, Switzerland)with cold PBS (100 mg wet tissue in 1 mL of 1 x PBS) for 3 min. The homogenates were freeze-thawed for 2 cycles to further break down the cells and were centrifuged for 5 min at 5000×g, 4 °C. The supernatants were pipetted and used for the analysis of NFкB, NLRP3, caspase-1 and IL-1β levels, all the biomarkers were measured using their respective ELISA kits according to the user manuals; pro-inflammatory cytokines interleukin-1 Beta (IL-1β) (Rat Il-1β/IL-IF2, E-EL-0012, Elabscience, USA), inflammatory transcription factor, Nuclear Factor Kappa B (NF-kB) (Rat NF-кB, E-EL-0673, Elabscience, USA), inflammasome complex (caspase-1 (Rat CASP1, E-EL-0371, Elabscience, USA) and NACHT, LRR and PYD Domains- Containing Protein 3 (Rat NLRP3, E-EL-1463, Elabscience, USA) which is associated with the production of IL-1β. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s posthoc multiple comparisons using statistical software SPSS IBM version 23. All the results were expressed as mean ± SEM. Significance difference was accepted when the p-value is equal or less than 0.05 (p < 0.05).