Bromelain reduced pro-inflammatory mediators as a common pathway that mediate antinociceptive and anti-anxiety effects in sciatic nerve ligated Wistar rats

The involvement of pro-inflammatory mediators complicates the complex mechanism in neuropathic pain (NP). This study investigated the roles of bromelain against pro-inflammatory mediators as a mechanism that underpins its antinociceptive and anti-anxiety effects in the peripheral model of NP. Sixty-four male Wistar rats randomly divided into eight groups, were used for the study. A chronic constriction injury model of peripheral neuropathy was used to induce NP. Tail-immersion and von Frey filaments tests were used to assess hyperalgesia while open field and elevated plus mazes were used to assess anxiety-like behaviour. NF-кB, iNOS, nitrate, and pro-inflammatory cytokines were investigated in the plasma, sciatic nerve, and brain tissues using ELISA, spectrophotometer, and immunohistochemistry techniques after twenty-one days of treatment. Bromelain significantly (p < 0.05) improved the cardinal signs of NP and inhibited anxiety-like behaviours in ligated Wistar rats. It mitigated the increases in cerebral cortex interleukin (IL) -1β, IL-6, and PGE2 levels. Bromelain reduced NF-кB, IL-1β, IL-6, TNF-α, PGE2, and nitrate concentrations as well as the expression of iNOS in the sciatic nerve. Hence, the antinociceptive and anxiolytic effects of bromelain in the sciatic nerve ligation model of NP is in part due to its ability to reduce nitrosative and inflammatory activities.

Thermal hyperalgesia. A tail immersion test was used to assessed thermal hyperalgesia before and after ligating (3rd, 7th, 14th, and 21st days) the rats. Briefly, the distal portions of the tails of the rats were immersed in a water bath that was maintained at 55 °C ± 0.5 °C temperature. The time taken for the rats to flick or withdraw their tails from the hot water was measured chronologically with a stopwatch (seconds) and reported as the latency. A cut off time of ten (10) seconds was adopted to avoid thermal damages to the tails of the rats.
Mechanical allodynia test. Sensitivity to the mechanical stimulus was assessed using von Frey filaments as described in our previous study 24 . Briefly, animals were placed in the transparent Perspex cages with a wire mesh floor and allowed to rest for 15 min. Von Frey filaments grading 1.4 g, 2, 4 g, 6 g, 8 g, 10 g, 15 g, 26 g, 60 g, and 100 g bending forces, were applied individually to the plantar surface of each ligated hind paws of rats in ascending orders. Paw withdrawal latency was defined by the mechanical force (in gram) that results in three consecutive trial withdrawals, licking, or flinching of the hind limbs. Von Frey filaments that produced uplifting of the whole hind limb without sudden withdrawal, licking or flinching were taken as a positive response.
Development and progression of the mechanical allodynia were assessed before and after ligating (3rd, 7th, 14th, and 21st days) the rats.
Open field test. Locomotive activities and anxiety-like behaviours were investigated by introducing the rat into a novel open field arena on the eleventh (11th) days of treatments with either normal saline, gabapentin, or bromelain. The open-field arena (24″ by 24″) was divided into sixteen (16) equal squares with side boundary of considerable height (16″) to prevent the rats from escaping. An open-field test was conducted at night. Rats were introduced at the central square of the open field arena, and activities were recorded with a camera for 5 min. Vertical exploration, rearing, frequency of defecation, number of stretching, and duration spent at the central square were obtained from the video recordings by trained research assistants to avoid bias.
Elevated plus maze test. Anxiety-like behaviour was further investigated using elevated plus maze apparatus on the sixteenth day of post-treatment with normal saline, gabapentin, or bromelain. Activities of rats in the elevated plus-maze were recorded with a camera. Rearing, percentage of time spent in the open and close arms, and frequency of time the rats visited the open and closed arms were assessed by trained research assistants from the recorded videos.
Biochemical parameters. After twenty-one days of post-treatment, the rats were euthanised, fore-brain (Ipsilateral cerebral cortex excluding the olfactory bulb, but including frontal and parietal lobes, and their deep brain structures such as the hippocampus, amygdala), and sciatic nerve (from the point of ligature to the point near the entrance into the Lumber vertebral) were collected and homogenised. Sucrose (0.32 M) was used for fore-brain homogenate while Tris buffer was used to homogenise the sciatic nerve. Homogenates were centrifuged at 16,000 RPM for fifteen minutes, and supernatants were collected. The supernatants were used for the estimation of biochemical parameters through ELISA and spectrophotometry techniques. The blood samples of rats were collected using cardiac puncture technique. The blood samples were transferred to lithium heparinised bottles and centrifuged at the speed of 3000RPM for ten minutes. The plasma was separated with micropipettes and used for the analysis of plasma nitrate concentration.
Estimation of glutamate level. Glutamate concentration was measured in the sciatic nerve by the use of a glutamate assay kit (Abcam, Cambridge, USA) following the procedure described by the manufacturer. In brief, the reaction mix sample and background reaction mix were prepared according to the manufacturing instructions. Reaction mix (100 µL) was added into each standard (six standard samples were prepared according to the manufacturer's direction) and sample wells. Likewise, 100 µL of background reaction mix was added to the background sample wells. The mixture was incubated at 37 °C for 30 min in a dark environment. The absorbance was read at 450 nm on a microplate reader. The corrected absorbance of the standard was used to generate the standard curve from which the concentrations of the samples' glutamate concentrations were extrapolated.
Calculation: Estimation of nitrate concentration. Nitric oxide was estimated indirectly by determining the concentration of both nitrite and nitrate in the sciatic nerve homogenate and plasma supernatants using the Griess diazotisation reaction method. A nitrate kit procured from Molecular Probe Inc., USA was used for this biochemical analysis. The supernatants were first deproteinised via the addition of ZnSO 4 (15 g/l) and then centrifuged at 10000 g for 5 min under room temperature. For estimation of nitrite (NO 2 -), 100 µm of Griess reagent (50 µm of 1% sulfanilamide in 5% HCl and 50 µm of 0.1% naphthyl-ethylenediamine dihydrochloride) was added to 300 µm of the sample followed by 2.6 ml of deionised water. The mixtures were incubated for 30 min at room temperature, and absorbance was measured at 548 nm. For the estimation of nitrate (NO 3 − ), activated The concentration of glutamate = Sa Sv XDf  www.nature.com/scientificreports/ cadmium granules (2.5-3 g) were added to the deproteinised supernatants. The mixtures were stirred and incubated for 90 min so that nitrate in the supernatants were reduced to nitrite, and estimation of total nitrite was carried out as described above. The calibration curve used to determine the concentration of nitrite was generated by preparing different concentrations of sodium nitrite in distilled water which ranged from 0-500 µM/l. The absorbance of each sample was read at 548 nm, and the standard curve of nitrite concentration was plotted against the absorbance.
Histological study. Structural integrity of the sciatic nerve was investigated using haematoxylin and eosin (H & E) stain as described by Muthuraman et al. 26 . The sciatic nerve was fixed in 10% formalin solution and blocked. It was further sectioned longitudinally into 5 μm before staining with H & E and observed under a light microscope.
Immunohistochemistry. Expression of inducible nitric oxide synthase (iNOS) was assessed using the immunohistochemistry technique by modifying the procedure described by Emokpae and his colleagues 27 . Briefly, rats were anaesthetised with ketamine hydrochloric (100 mg/kg, i.p.) and perfused transcardially with phosphate buffer (pH 7.4) followed by 10% formalin. The sciatic nerve was isolated and post-fixed in the same 10% formalin for 18 h. It was then blocked with paraffin wax and further sectioned into 5 μm transversely and laid on a gelatin-coated glass slide. The sections were deparaffinised, hydrated, and an antigen retrieval procedure was carried out. Tissue sections were incubated with primary antibody rabbit-iNOS (1:500, Abcam) for 20-30 min at room temperature (25 °C) according to the manufacturer's instructions. The sections were then incubated for 20-30 min with secondary antibodies at room temperature (1:500, Jackson ImmunoResearch). Few drops of ready to use 3, 3′-diaminobenzidine (DAB) reagent were added to each tissue sections and allowed to incubate for 6-10 min at room temperature (25 °C) before washing with PBS 5-7 times and then with distilled water. The slides were incubated with hematoxylin for 30-60 s, rinsed with distilled water, and allowed to drain before mounting with appropriate mountant. Images were acquired using Leica ICC50 E Digital Camera (Ger-  www.nature.com/scientificreports/ many) connected to a computer and light microscope, and the expression of immunopositive cells was analysed using Image J software (NIH, Bethesda, MD, USA).
Statistical analysis. Data were expressed as mean ± SEM. Two-way analysis of variance (ANOVA) was used to analyse behavioural pain tests while one-way ANOVA was used for the analysis of behavioural emotion tests, behavioural tests, and biochemical parameters. Bonferroni post hoc multiple comparison test was used for comparison among groups. The entire statistical analysis was conducted using GraphPad Prism software version 5. Means with p < 0.05 were considered as significantly different from each other.

Pain behavioural tests (thermal and mechanical hyperalgesia). Bromelain significantly reversed
the thermal hyperalgesia developed by the CCI-induced NP rats. Both doses of bromelain increased the threshold sensitivity of the rats to a thermal stimulus. Pre-treating rats with bromelain prevented the development of thermal hyperalgesia in ligated rats as there was a significant difference (p < 0.05) between the pre-treated bromelain group and the unligated control group (6.05 ± 0.11 s. vs 2.95 ± 0.15 s.). The results of this study ( Fig. 2A) show that pre-treated bromelain groups yielded a better antinociceptive effect as it was significantly different from the post-treated bromelain group. Mechanical allodynia was observed in all the sciatic nerve ligated Wistar rats, but bromelain improved the allodynia (55.75 ± 4.25 g vs 5.25 ± 0.53 g) as showed in Fig. 2B. Likewise, pretreating animals with bromelain did not yield a better anti-allodynia effect compared with the post-treated bromelain group. The effect of bromelain on allodynia was not significantly different from that of gabapentin.
Open field test. Exploratory and anxiety-like behaviour were indicated in Fig. 3A-G. CCI resulted in reduced exploratory behaviour in rats, as shown in Fig. 3A,B. There were reductions in locomotive activities in both the vertical and horizontal explorations in ligated rats (p < 0.05) compared with unligated control. Pre-treatment with bromelain significantly increased (p < 0.05) the number of lines crossed (48.00 ± 1.65 vs 29.80 ± 1.65) in rats exposed to open field arena but not the rearing frequency (8.50 ± 0.53 vs 7.13 ± 0.40). Chronic constriction injury increased the number of stretches and the rate of defecation observed in rats. Stretching and the number of faecal boli produced in sciatic ligated rats was reduced by treatment with bromelain or gabapentin, as shown in Fig. 3C,D. www.nature.com/scientificreports/ Furthermore, CCI decreased the grooming duration in rats, but there was a significant difference between the unligated control and the ligated control groups. Both pre-treated (48.79 ± 3.77 s. vs 32.04 ± 2.57 s.) and post-treated (65.70 ± 3.35 s. vs 32.04 ± 2.57 s.) bromelain groups significantly increased the grooming duration as shown in Fig. 3 E. Rats treated with bromelain made their first moved as quickly as possible with less confusion. There were decreases in both centre time duration, and frequency in rats induces with CCI, as shown in Fig. 3F,G. The ligated control rats did not visit the centre box of the open field arena frequently. However, pre-treatment with bromelain increased the centre time duration and frequency. Pre-treatment with a high dose of bromelain significantly increased the centre frequency but not the duration. Figure  Pro-inflammatory mediators. CCI increased the level of pro-inflammatory mediators (IL-1β, IL-6, TNF-α, and PGE 2 ) in the sciatic nerve of rats, as shown in Fig. 5A-D. Administration of bromelain significantly (p < 0.05) inhibited IL-1β, IL-6, TNF-α, and PGE 2 concentrations. It was observed that gabapentin slight reductions in the concentrations of IL-1β, TNF-α, and PGE 2 but not IL-6. There were no significant differences between the bromelain treated group and the unligated control group. Bromelain significantly reduced the concentration of PGE 2 in the sciatic nerve (22.89 ± 0.33 pg/ml/mg protein vs 32.23 ± 0.48 pg/ml/mg protein) compared with the unligated control group.

Elevated plus maze test.
Furthermore, it was observed that CCI significantly increased the concentration of IL-1β, IL-6, and PGE 2 in the cortex but not TNF-α, as shown in Fig. 6A-D. High dose of bromelain significantly reduced the brain levels www.nature.com/scientificreports/ of IL-1β, IL-6, and PGE 2 . However, the low dose only reduced the concentrations of IL-6 and PGE 2 . Likewise, gabapentin did not produce any significant effect on brain IL-6 compared with the ligated control group.

Nuclear factor kappa light chain enhancer B-cell inhibitor (NFкB). CCI significantly increased
the level of the NFкB in the sciatic nerve. Treatment with the two doses of bromelain significantly reversed this effect (15.92 ± 0.24 µg/ml/mg protein vs 20.28 ± 0.33 µg/ml/mg protein). Furthermore, the pre-treated bromelain group showed significantly reduced NFкB concentration compared with the gabapentin administered group (Fig. 7).

Nitrate and glutamate concentration level. CCI increased the concentrations of nitrate and glutamate
in both the plasma and sciatic nerves compared with the unligated control. Increased nitrate and glutamate concentration were ameliorated by the administration of different doses of bromelain, as shown in Fig. 8A-C. The effect of bromelain on nitrate and glutamate concentration was comparable with those of the rats treated with gabapentin.
Immunohistological study. CCI induced axonal degeneration of the sciatic nerve (Fig. 9C). There were reductions in the numbers of myelinated neurons. Most of the myelinated neurons were swollen and deranged. CCI also increased the expression of iNOS in the sciatic nerves, and there were disorganisations of the structural integrity of the axons. Treatments with bromelain reduced the expression of iNOS, increased axonal myelination, and reduced axonal swelling (Fig. 9E-H). The group treated with a high dose of bromelain (Fig. 9G,H) exhibited an improved sciatic nerve structural integrity which was comparable with that of the unligated control group (Fig. 9A,B). There were also increases in myelination of neurons, reduced neuronal swellings, and expression of iNOS in the group treated with gabapentin (Fig. 9D). www.nature.com/scientificreports/

Discussion
In this study, bromelain ameliorated hyperalgesia and allodynic signs in the CCI-induced peripheral neuropathy in male Wistar rats. This followed the trend of our previous research 24 . There is evidence of anxiety-like and depression-like comorbidity signs in rats with CCI-induced peripheral neuropathy. Anxiety and depression are common comorbid emotional deficits that are associated with neuropathic patients that adversely affect the patient's quality of life 28,29 . The use of open field and elevated maze plus in the analysis of anxiety-like and depressive behaviour in rodents has been well documented 30 . The study revealed that only a high dose of bromelain was effective in reversing the anxiety-like and depressive-like behaviours. It was observed that pre-treatment with bromelain yielded better anti-comorbidity effect, which suggests that bromelain could be used primarily as both therapeutic and prophylaxis agent. Precipitous increases in the concentration of IL-1β, IL-6, and TNF-α induces hyperalgesia via enhanced neuroexcitation 4,7,31,32 , and disinhibition 33 . Likewise, steep increases in central pro-inflammatory cytokines have been implicated as the neurobiological molecules that mediate the emotional comorbid of NP 14,15 . It is evident from this study that CCI promotes the elevation of both peripheral and central inflammatory mediators. Treatment with bromelain reversed the increases in the concentrations of the inflammatory mediators. It is therefore proposed that the antinociceptive effect of bromelain is mediated by its anti-inflammatory properties. Elimination of elevated pro-inflammatory cytokines by intrathecal injection of anti-inflammatory cytokines 10,11,34,35 , knockout cytokine receptors 9 or anti-inflammatory agents 36 have been shown to relieve NP effectively. Observations from the results showed that bromelain produced antinociceptive effects via inhibition of TNF-α, IL-1β, and IL-6 in the peripheral neurons and IL-1β, and IL-6 in the central neurons.
Furthermore, the findings suggested that IL-1β and IL-6 are responsible for the emotional dysfunction associated with neuropathic pain. It is, thus, proposed that increases in the concentrations of IL-1β and IL-6 are the common pathways that mediate emotional dysfunction, allodynia, and hyperalgesia symptoms of NP. Bromelain reversed the observed increases in IL-1β and IL-6 concentrations in the cerebral cortex and its deep structures. Therefore, the anti-anxiodepressive-like effects of bromelain may be due to its inhibitory effect on IL-1β, IL-6, and PGE 2 . The pro-inflammatory inhibitory effects of bromelain may also account for the observed reductions in glutamate concentration. Glutamate is an excitatory neurotransmitter that mediates pain perception. Proinflammatory cytokines such as IL-1β promotes the release of glutamate in the afferent nerves 37 , which mediate central sensitisation.
The drastic reductions in PGE 2 concentrations in the sciatic nerve and brain neurons below the level observed in unligated rats may be an essential mechanism of action of bromelain in mitigating the hyperalgesic effect of neuropathic pain. This can be explained partly by an indirect mechanism via the inhibitory effect of bromelain on pro-inflammatory cytokines and partly by a direct inhibitory effect on the COX 2 enzyme. The increased concentration of PGE 2 has also been linked with neurogenic inflammatory responses 38 . Elevated concentration of IL-1β, and IL-6 results in increases in the expression of cyclo-oxygenase-2 (COX 2 ) which induces the production of PGE 2 8 . This study showed that bromelain decreases NF-кB in sciatic nerve ligated rats. Bromelain has been  www.nature.com/scientificreports/ reported to block the activation and transmigration of NF-кB 39,40 that induces production of pro-inflammatory cytokines and subsequently expression of COX 2 38,41-43 . Furthermore, CCI induced the up-regulation of the expression of nitric oxide synthase (iNOS). iNOS mediates the production of peroxynitrite nitrogen species and COX 2, both of which increase the phosphorylation of excitatory receptors that potentiates hyperalgesia and allodynia 44 . The down-regulatory effect of bromelain on iNOS expression suggests that bromelain may have acted on this pathway to mediate its antinociceptive effects. Wen et al. 45 also reported that bromelain decreases the expression of nitric oxide synthase while studying its proliferative effects on the defaecation of rats.

Conclusion
In conclusion, bromelain is a potential biochemical supplement that could provide therapeutic options for the management of NP. Its anti-allodynic, anti-hyperalgesic, and anti-anxiodepressive properties match that of gabapentin that is traditionally used in the management of NP. Pre-treatment with bromelain offers more significant antinociceptive effects in the treatment of CCI-induced NP in rats compared with gabapentin. The antinociceptive effects of bromelain mediated its amelioration of neuroinflammation via the inhibition of the pro-inflammatory mediators. This serves as an effective mechanism that underpins its mitigating effects on allodynia, hyperalgesia, and comorbidity of neuropathic pain. The superiority of the anti-inflammatory effects of bromelain compared with gabapentin maybe its crucial advantage over gabapentin.  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.