Oral administration of Weissella and Pediococcus sp alleviates formalin induced inflammation in rats by Cytokine Modulation

This study was designed to study the cytokine modulatory activity of three lactic acid bacteria (LAB) strains, Weisella cibaria II-1-59, Weisella confusa JMC 1093, and Pediococcus pentosaceus DSM20336 isolated from a Nigerian locally fermented food condiment; “iru” using paw oedema acute inflammatory model induced with 1% formalin in Wistar rats. Rats were distributed into six groups (A-F). Rats in Groups A were neither administered formalin nor treated with LAB, while Group B received formalin injection only. Rats in Groups C, D, and E were administered formalin and were treated orally with 2 × 107 CFU/ml of Weisella cibaria II-1-59, Weisella confusa JMC 1093, and Pediococcus pentosaceus DSM20336 respectively, while Group F received diclofenac sodium treatment following administration of formalin. The dose of LAB strain used for the oral treatment was 2 × 107 CFU/ml for all the groups while the dose of diclofenac sodium used was 150 mg/kg body weight of the rats. Paw thickness (mm) was checked at t = 0, 1, 4, 8, 24, 72, 168 and 336 h. Cytokine assay for C-reactive protein (CRP), Interleukin (IL-10) and Transforming growth factor (TGF-β) was performed on serum samples of the rats using Enzyme-linked immunosorbent assay (ELISA). Oral administration of W. cibaria II-1-59 showed the best significant decrease in the paw thickness of the rats, which was followed by P. pentosaceus DSM20336 and W. confusa JMC 1093 respectively, and was shown to be statistically significant at P<0.05. There was also a significant decrease (below standard 2000 pg/ml) in the secretion of pro-inflammatory biomarker (CRP) in all LAB treated groups at 1 hour, while there was an increase in the serum levels of anti-inflammatory cytokines IL-10 and TGF- β in Groups C-E rats which was maximally increased in W. confusa JMC 1093 treated rats. This study suggests that W. cibaria II-1-59, W. confusa JMC 1093 and P. pentosaceus DSM20336 possess anti-inflammatory potentials. Keywords: Inflammation, Cytokines, Weisella, Formalin, Oedema, Pediococcus

Antinociceptive and Anti-Inflammatory Effects of Recombinant Crotamine in Mouse Models of Pain

Crotamine, a toxin found in the venom of the South American rattlesnake Crotalus durissus terrificus, has been reported to have antinociceptive effects. We purified recombinant crotamine expressed in Escherichia coli and investigated its antinociceptive and anti-inflammatory effects using the hot-plate test, acetic-acid-induced writhing method, and formalin test in mice. Recombinant crotamine was administered intraperitoneally (0.04–1.2 mg kg−1) or intraplantarly (0.9–7.5 μg 10 μL−1) before the tests. The paw volume was measured with a plethysmometer. To evaluate the antagonistic and anti-inflammatory effects of naloxone, subcutaneous naloxone (4 mg kg−1) or intraplantar naloxone (5 μg 10 μL−1) was administered before recombinant crotamine. For tumor necrosis factor (TNF)-α assays, blood was drawn 3 h after formalin injection and measured using enzyme-linked immunosorbent assay. Intraperitoneal and intraplantar recombinant crotamine had antinociceptive and anti-inflammatory effects, neither of which were affected by pre-treatment with naloxone. The mean serum TNF-α levels were significantly lower in the intraperitoneal recombinant crotamine (0.4 and 1.2 mg kg−1) or intraplantar (2.5 and 7.5 μg 10 μL−1) recombinant crotamine groups than in the saline group and were not affected by naloxone pre-treatment. In conclusion, recombinant crotamine possesses significant antinociceptive and anti-inflammatory effects that do not appear to be related to the opioid receptor. The antinociceptive and anti-inflammatory effects of intraperitoneal or intraplantar recombinant crotamine are related to TNF-α.

Anti-Inflammatory Activity of Artemisia vulgaris Leaves, Originating from Three Different Altitudes of Nepal

This study aimed to evaluate and compare the in vivo chronic anti-inflammatory efficacy, from the ethyl acetate and ethanolic extracts of Artemisia vulgaris leaves, grown at three different altitudes in Nepal, by formalin-induced paw edema in Swiss albino mice. Edema was induced on the mice paw by administering 0.2% of formalin injection. Indomethacin was used as a standard drug at the concentration of 5 mg/kg of body weight. Ethyl acetate and ethanolic leaves extract, at the concentration of 200 mg/kg and 400 mg/kg, were used as test drugs. Standard drug and all the extracts were administered 30 min before formalin injection. The paw thickness was measured at 0, 1, 2, 3, 24, 48, and 72 hours after formalin injection, using a Vernier caliper. It was observed that both ethyl acetate and ethanolic extract from all the altitudes exhibited significant inhibition of paw edema ( p < 0.05 ) induced by formalin. Maximum activity was shown by 400 mg/kg of the plant leaf extract taken from the temperate zone, with 54.05% of paw edema inhibition, and it is almost similar to the inhibition of standard drug (56.75%). Moreover, the ethanolic extract was found to be more effective than ethyl acetate extract in all the plant samples. The results suggested that the anti-inflammatory effect of A. vulgaris leaves increases with an increase in altitudes and this plant can be used as a useful source of medicine to treat chronic inflammation.

Analgesic Effect of Combined Therapy with the Japanese Herbal Medicine “Yokukansan” and Electroacupuncture in Rats with Acute Inflammatory Pain

Background: Japanese herbal medicine, called Kampo medicine, and acupuncture are mainly used in Japanese traditional medicine. In this experiment, the analgesic effect of Yokukansan (YKS) alone and a combination of YKS and electroacupuncture (EA) on inflammatory pain induced by formalin injection were examined. Methods: Animals were divided into four groups: a control group, formalin injection group (formalin), YKS-treated formalin group (YKS), and YKS- and EA-treated formalin group (YKS + EA). The duration of pain-related behaviors and extracellular signal-regulated protein kinase (ERK) activation in the spinal cord after formalin injection in the right hind paw were determined. Results: The duration of pain-related behaviors was dramatically prolonged in the late phase (10–60 min) in the formalin group. The YKS treatment tended to reduce (p = 0.08), whereas YKS + EA significantly suppressed the pain-related behaviors (p < 0.01). Immunohistochemical and Western blot analyses revealed that the number of phosphorylated ERK1/2 (pERK1/2)-positive cells and the pERK expression level, which were increased by formalin injection, were significantly inhibited by YKS (p < 0.05) and YKS + EA (p < 0.01). Conclusions: The YKS + EA combination therapy elicited an analgesic effect on formalin-induced acute inflammatory pain.

Modulation of Septo-Hippocampal Neural Responses in Anesthetized and Behaving Rats by Septal AMPA Receptor Mechanisms

This study explored the effects of septal glutamatergic transmission on septal-hippocampal theta activity via intraseptal microinjection of antagonist at AMPA receptors (AMPAR). The current results showed that microinjection of AMPAR antagonist, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione, 20 μg/μl, 0.5 μl), evoked a decrease in the frequency of theta activity evoked by various means in anesthetized and behaving rat. Theta wave activity was induced on: (a) intraseptal microinjection of carbachol, an agonist at cholinergic receptors, (b) reticular stimulation, (c) exploration in novel open field (OF), and (d) hind paw (HP) injection of the algogen, formalin. The effect on frequency in the formalin test was observed in an early period on injection of formalin, which was novel to the animal, but not in the later more sustained phase of the formalin test. The effect of NBQX, being seen in both anesthetized and behaving animals, suggests that the modulation of theta wave frequency, including in novelty, is a function of AMPAR in MS. The effect of the antagonist on theta power was less apparent, being observed only in anesthetized animals. In addition to theta power and frequency, intraseptal NBQX also attenuated suppression of CA1 population spike (PS) induced by intraseptal carbachol, thus suggesting that septal glutamate neurotransmission is involved in the spectrum of MS-mediated network responses. Indeed, in the context of behavior, formalin injection induced an increase in the level of septal glutamate, while NBQX attenuated nociceptive behaviors. Notably, MS is involved in the modulation of formalin nociception. These findings suggest that AMPA receptors are a key modulator of septal physiological function.

Activation of spinal dorsal horn astrocytes by noxious stimuli involves descending noradrenergic signaling

Astrocytes are critical regulators of neuronal function in the central nervous system (CNS). We have previously shown that astrocytes in the spinal dorsal horn (SDH) have increased intracellular Ca2+ levels following intraplantar injection of the noxious irritant, formalin. However, the underlying mechanisms remain unknown. We investigated these mechanisms by focusing on the role of descending noradrenergic (NAergic) signaling because our recent study revealed the essential role of the astrocytic Ca2+ responses evoked by intraplantar capsaicin. Using in vivo SDH imaging, we found that the Ca2+ level increase in SDH astrocytes induced by intraplantar formalin injection was suppressed by ablation of SDH-projecting locus coeruleus (LC)-NAergic neurons. Furthermore, the formalin-induced Ca2+ response was dramatically decreased by the loss of α1A-adrenaline receptors (ARs) in astrocytes located in the superficial laminae of the SDH. Moreover, similar inhibition was observed in mice pretreated intrathecally with an α1A-AR-specific antagonist. Therefore, activation of α1A-ARs via descending LC-NAergic signals may be a common mechanism underlying astrocytic Ca2+ responses in the SDH evoked by noxious stimuli, including chemical irritants.

Quercetin action on pain modulation/ Ação da quercetina sobre a modulação da dor

Background: Quercetin is a flavonoid widely found in plant kingdom and target of studies in pharmacological area due to its potent antinociceptive effect compared to analgesics used in conventional therapies. The aim was to evaluate its antinociceptive activity and antinociception mechanism. Methods: For this, 40 Norvegicus Wistar rats were used, divided into 4 groups: Q50 (treated with quercetin 50 mg/Kg), Q100 (treated with quercetin 100 mg/Kg), Q500 (treated with quercetin 500 mg/Kg) and Positive control (PC) without quercetin treatment), who were submitted through the pain induction methods by tail immersion and formalin in the first step to assess antinociceptive action and in the second step, tail immersion method receiving antagonists from opioid, cholinergic and nitric oxide - L-arginine to evaluate the action mechanism. Results: Quercetin antinociceptive activity was verified at the dose of 50 mg/kg and 100 mg/kg in tail immersion test after formalin injection, with better performance at the dose of 50 mg/kg. There were no statistically significant results in paw opening and capsaicin tests. Quercetin demonstrated a possible influence on opioid and cholinergic pathway, which was not observed on the nitric acid - L-arginine pathway in view of parameters tested. Conclusion: Quercetin performed the best antinociceptive activity at a dose 50 mg/kg and there was a possible influence on opioid and cholinergic pathways.

Role of the NO-cGMP-K+ channels pathway in the peripheral antinociception induced by α-bisabolol

The aim of this study was to examine if the peripheral antinociception of α-bisabolol involve the participation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) synthesis followed by K+ channel opening in the formalin test. Wistar rats were injected in the dorsal surface of the right hind paw with formalin (1%). Rats received a subcutaneous injection into the dorsal surface of the paw of vehicles or increasing doses of α-bisabolol (100-300 µg/paw). To determine whether the peripheral antinociception induced by α-bisabolol was mediated by either the opioid receptors or the NO-cGMP-K+ channels pathway, the effect of pretreatment (10 min before formalin injection) with the appropriate vehicles, naloxone, naltrexone, L-NAME, ODQ, glibenclamide, glipizide, apamin, charybdotoxin, tetraethylammonium or 4-aminopyridine on the antinociceptive effects induced by local peripheral α-bisabolol (300 µg/paw) were assessed. α-bisabolol produced antinociception during both phases of the formalin test. α-bisabolol antinociception was blocked by L-NAME, ODQ, and all the K+ channels blockers. The peripheral antinociceptive effect produced by α-bisabolol was not blocked by the opioid receptor inhibitors. α-bisabolol was able to active the NO-cGMP-K+ channels pathway in order to produce its antinoceptive effect. The participation of opioid receptors in the peripheral local antinociception induced by α-bisabolol is excluded.

Mechanistic insights into the role of the chemokine CCL2/CCR2 axis in dorsal root ganglia to peripheral inflammation and pain hypersensitivity

Background Pain is reported as the leading cause of disability in the common forms of inflammatory arthritis conditions. Acting as a key player in nociceptive processing, neuroinflammation, and neuron-glia communication, the chemokine CCL2/CCR2 axis holds great promise for controlling chronic painful arthritis. Here, we investigated how the CCL2/CCR2 system in the dorsal root ganglion (DRG) contributes to the peripheral inflammatory pain sensitization. Methods Repeated intrathecal (i.t.) administration of the CCR2 antagonist, INCB3344 was tested for its ability to reverse the nociceptive-related behaviors in the tonic formalin and complete Freund’s adjuvant (CFA) inflammatory models. We further determined by qPCR the expression of CCL2/CCR2, SP and CGRP in DRG neurons from CFA-treated rats. Using DRG explants, acutely dissociated primary sensory neurons and calcium mobilization assay, we also assessed the release of CCL2 and sensitization of nociceptors. Finally, we examined by immunohistochemistry following nerve ligation the axonal transport of CCL2, SP, and CGRP from the sciatic nerve of CFA-treated rats. Results We first found that CFA-induced paw edema provoked an increase in CCL2/CCR2 and SP expression in ipsilateral DRGs, which was decreased after INCB3344 treatment. This upregulation in pronociceptive neuromodulators was accompanied by an enhanced nociceptive neuron excitability on days 3 and 10 post-CFA, as revealed by the CCR2-dependent increase in intracellular calcium mobilization following CCL2 stimulation. In DRG explants, we further demonstrated that the release of CCL2 was increased following peripheral inflammation. Finally, the excitation of nociceptors following peripheral inflammation stimulated the anterograde transport of SP at their peripheral nerve terminals. Importantly, blockade of CCR2 reduced sensory neuron excitability by limiting the calcium mobilization and subsequently decreased peripheral transport of SP towards the periphery. Finally, pharmacological inhibition of CCR2 reversed the pronociceptive action of CCL2 in rats receiving formalin injection and significantly reduced the neurogenic inflammation as well as the stimuli-evoked and movement-evoked nociceptive behaviors in CFA-treated rats. Conclusions Our results provide significant mechanistic insights into the role of CCL2/CCR2 within the DRG in the development of peripheral inflammation, nociceptor sensitization, and pain hypersensitivity. We further unveil the therapeutic potential of targeting CCR2 for the treatment of painful inflammatory disorders.