The present study revealed that LPS significantly reduced the response time in hot plate test and increased the licking and biting time in both phases of formalin test. Both doses of CE (300,500 mg/kg) and esculetin (40 mg/kg) increased the hot plate response time compared to LPS group. Both doses of the CE and ESC decreased the licking and biting time in the second phase of formalin test, but during the first phase of formalin test only 500 mg/kg of CE showed a significant reduction in comparison with LPS group. Moreover, LPS administration significantly increased serum TNF- α and IL-1β levels. On the other hand, pre-treatment with the CE (only, 500 mg/kg) prevented LPS-induced serum production of TNF- α, but IL-1β increase was inhibited by both doses of CE, and ESC.
Previous studies have shown that systemic administration of LPS can lead to a generalized hyperalgesia . Various mechanisms including the release of pro-inflammatory cytokines and glial cell activation have been proposed to be responsible for the LPS-induced hyperalgesia [7, 8]. TNF- α and IL-1β are two essential cytokines involved in the inflammatory response as well as neuropathic pain induction. IL-1β release through p38 MAPK signaling pathway, enhances the expression of COX-2 and leads to production of PGE2 in the white and gray matter of spinal cord and DRG [6, 8] .TNF- α a cytokine which is secreted by immune and glial cells, induces MAPKs phosphorylation and NF-κB activation. In addition to be an anti-inflammatory mediator, TNF- α can act as a potentiator to increase other inflammatory cytokines . Also, in some in vitro studies on neuronal cells line and hippocampal slices, it has been reported that TNF- α can reduce the GABA inhibitory activity . Based on this important evidence, one of our main conclusions is that an increase in TNF- α following LPS leads to neuropathic pain, and this may be due to its inhibitory effect on GABAergic neurotransmission.
Here, our findings demonstrated that CE inhibits LPS-induced hyperalgesia and this effect may be achieved by inhibiting the production of two pro-inflammatory cytokines, TNF- α and IL-1β. Previous studies have demonstrated that hexadecanoic acid, sesquiterpene lactones and flavonoids - the three major CE compounds shown in GC-MS analysis- have anti-inflammatory and analgesic activities.
Medicated oils rich in n-hexadecenoic acid had rigorous use for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda . It is a phospholipase A2 (PLA2) competitive inhibitor that reduces the production of pro-inflammatory cytokines. Furthermore, it increases the activity of aromatic amino acid decarboxylase and enhances DOPA production, which improves dopaminergic neurotransmission and relives pain [15, 25].
Sesquiterpene lactones have potent anti-inflammatory activity (l). Anti-inflammatory effect of 8-deoxylactucin is mediated by two mechanisms: 1) inhibition of NF-kB activity and 2) cyclooxygenase (COX-2) inhibition . In an in vitro study, it has been shown that SLs extracted from chicory root, in a dose-dependent manner downregulated the genes expression of COX-2, inducible nitric oxide synthase (iNOS), TNF- α and IL-1β in LPS-stimulated macrophages . It was observed that lactucin and lactucopicrin, found in CI, exerted an analgesic effect in the tail-flick and hot plate tests in mice and their antinociceptive activity was similar to ibuprofen [17, 28].
The flavonoid quercetin is a well-established molecule with strong analgesic, anti-inflammatory and anti-oxidant activity. In vivo and in vitro studies revealed that these effects are associated with several mechanisms, including inhibition of NF-κB, PLA2, reduction of pro-hyperalgesic and inflammatory cytokines/mediators such as PGE2, NO, TNF- α, IL-1β and iNOS production and expression. It also exerts opioid like effects, stimulates GABAA and serotonin receptors, decrease/downregulates TLR4 and COX2, acts as inhibitor of TRPV1/NMDA receptors and also decreases NMDA receptors functionality [16, 29, 30]. Studies indicated that flavonoids can cross the blood–brain barrier (BBB) . The quercetin inhibits CNS and spinal cord glial cells activation, and consequently reduces cytokine production and oxidative stress resulting in reduced neuro-inflammation and hyperalgesia . Catechin, belonging to the flavonoid family, can alleviate the inflammation and pain response in rats by decreasing the generation of TNF- α, PGE2, and IL-1β .We now know that circulating matrix metalloprotease 9 (MMP-9) plays an important role in neuro-inflammation and oxidative stress as two leadings factors of the neuropathic pain. It has been proved that Catechin reduces MMP-9 in diabetic rats .
Some other CE compounds with lower percentages (shown in Table 1) may be involved in the anti-inflammatory and analgesic activities of chicory.
GC-MS identified two anthocyanin compounds in the CE, Cyanidin-3-glucoside and Delphinidin. Anthocyanins (ANCs) are phenolic compounds which belong to the subclass of flavonoids. glycosylation is essential to increase ANCs molecular stability . The ANCs or their bioactive metabolites readily penetrate the BBB and act as centrally antinociceptives to reduce CNS inflammation and scavenge free radicals [6, 32]. It has been demonstrated that ANCs attenuate inflammation-induced thermal and mechanical hyperalgesia with similar efficacy to indomethacin (6). It has been reported that cyanidin-3-glucoside possesses antioxidant and anti-inflammatory properties (33). Delphinidin can reverse mechanical, thermal and chemical hyperalgesia, as well as local inflammation (32). Also, due to its antioxidant activity, delphinidin has been proposed as a potential clinical cure to treat inflammatory pain (34). Delphinidin downregulates NF-κB signaling pathway and leads to decrease TNF- α level. Moreover, ANCs are strong COX inhibitors with inhibitory capacity even comparable to NSAIDs such as naproxen and ibuprofen, hence, abridging the inflammation and nociceptive sensitization. In addition, ANC was also demonstrated to suppress glutamate release from inflamed neurons (6).
Lupeol is a triterpenoid compound. A previous study showed that it reduces the inflammatory response through the modulation of p38 pathway. The researchers reported that lupeol inhibited LPS-induced IκBα/degradation and decreased the DNA binding activity of NF-κB in the macrophages (4). It also down-regulates TNF- α gene expression (35).
The name Octadecatrienoic acid refers to many different structural and conformational isomers. Fatty acids can modulate immune responses by acting directly on T cells. Linoleic acid exerts anti-inflammatory effect by decreasing production of the inflammatory mediators such as PGE2, IL-1β, TNF- α and nitric oxide. Also, α-linolenic acid exhibits high anti-inflammatory effect (15). Furthermore, 9,12,15-octadecatrienoic acid- the other component which was found in the CE- has been introduced as an anti-inflammatory compound (36).
Although some studies have shown that the anti-nociceptive property of esculetin may be mediated by inhibition of lipooxygenase (5-LOX) and PGE2 production, understanding the mechanisms underlying the analgesic activity of esculetin needs further studies (37). Recently, one study on the fibromyalgia, a syndrome of persistent widespread pain, showed that esculetin can reduce pain by decreasing TNF- α and IL-1β levels in fibromyalgia (38). In consistence with these results, we demonstrated that esculetin inhibits LPS-induced hyperalgesia and this effect may be due to inhibition of IL-1β, but not TNF- α production. This may be resulted from different ways to regulate IL-1β and TNF- α mRNA expression. LPS induces IL-1β mRNA expression by both protein kinase C (PKc) and calmodulin (CaM) kinase dependent pathways, while TNF- α expression depends on PKc pathway but not CaM kinase pathway, apparently . So, it seems that esculetin interferes with PKc signaling pathway.
The present study showed that hydro-alcoholic extract of chicory (500 mg/kg) was more effective than esculetin 40 mg/kg in behavioral and biochemical tests. There are many compounds with anti-inflammatory and analgesic properties in the chicory root which obviously may improve the efficacy of chicory compared to esculetin.