Pain, along with redness, warmth, swelling are the characteristic symptoms of inflammation. Inflammation is an evolutionary conserved and protective reaction of the body against factors that are threatening its normal functioning and homeostasis. Acute inflammation is a high-grade type of inflammation, triggered either by the presence of pathogenic microorganisms or cellular damages caused by noxious stimuli. Pathogen-associated molecular patterns (PAMPs) are activated in case of infections from pathogens, whereas damage-associated molecular patterns (DAMPs) are activated in case of cellular damage. These molecules are recognized from immune cells such as macrophages and dendritic cells (DC), which in turn are activated to produce inflammatory enzymes, such as cyclooxygenase 2 (COX-2) producing prostaglandin E2 (PGE2), and cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL-1β) (17). Recruited neutrophils are further promoting the inflammatory state. All the inflammatory mediators are playing a key role in acute inflammatory pain, through activating the nociceptors (17–19).

After a reasonable period of time and under normal conditions, when threat is eliminated and healing of the tissue has been completed, the resolution of inflammation will be achieved and relative symptoms including pain, will stop. Various factors, including environmental and genetic parameters, can contribute to preserved chronic inflammation, usually activated by DAMPs (sterile type). Persistent, non-resolving inflammation can lead gradually to detrimental effects in tissues and organs' normal structure and functioning. Pain can be one of the symptoms where the underlying pathology is a low-grade, progressive inflammatory state (3, 17, 18). Chronic conditions like osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, are characterized by pathological and persistent pain. This type of pain results from sensitization of nociceptors to the inflammatory mediators' stimuli in the foci of inflammation, causing a shift from their high-threshold to low-threshold activation.

In the absence of cure, inhibition of inflammation is the gold standard approach in management of chronic inflammatory conditions, in order to achieve prolonged remission state, and consequently relief of pain. Non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, opioids, immunobiological molecules are being used in chronic inflammatory pain. Clinicians' choice is based on pathogenesis of the inflammatory condition itself, as well as on outweighing the effectiveness of the protocols designed and the risks for side effects. However, there is still a need for more effective and safe agents that can complement the current therapies (8).

Luteolin, shows pleiotropic actions in various pathways involved in chronic inflammation, and due to its safe profile can be considered as a promising adjuvant therapeutic choice in inhibition of inflammation and related pain.

Luteolin has shown important effects in regulation of inflammation, in both in vitro and in vivo studies. Its actions are mostly exerted by inhibiting several biochemical pathways and inflammatory mediators, which are involved in several chronic diseases where prolonged inflammation is the common denominator of pathogenesis.

The anti-inflammatory activities of luteolin were comprehensively reviewed in literature in two review papers (9, 10). The reader can refer to these reviews for very detailed information on preclinical evidence about the anti-inflammatory effects of luteolin in various in vitro cell lines and in vivo animal models. Authors described extensively the regulatory effects of luteolin on inflammatory mediators such as cytokines IL-6, IL-1β, TNF-α, enzyme COX-2 and prostaglandins PGEs. Moreover, luteolin can inhibit the increased expression of inducible nitric oxide synthase (iNOS) and metalloproteinases (MMPs) in chronic inflammatory conditions (9, 10).

Nitric oxide (NO) has various roles under normal conditions. It promotes vasodilation, works as a neurotransmitter, and regulator of immune response. However, in chronic inflammation, an inducible form of NO is synthesized by synthase iNOS, leading to nitric oxide overexpression, even 1,000 times more than its physiological production. In this case, nitric oxide acts as an inflammatory mediator (20, 21).

MMPs are a family of metalloproteinases, and their primary action is to degrade extracellular matrix proteins. Although they have been attributed with many physiological roles in various essential functions, such as cell proliferation, tissue repair, wound healing, and others, they are also involved in inflammatory processes. In particular, increased expression of various types of MMPs have been implicated in tissue remodeling and destruction in chronic inflammatory conditions (22, 23).

The inflammatory response requires the orchestrated activation of different but interacting signaling pathways. Among of those, nuclear factor kappa B (NF-κΒ), Janus kinase - signal transducer and activator of transcription (JAK-STAT), and inflammasome NOD-like receptor (NLR family) pyrin domain containing 3 (NLRP3) play important roles in expression of pro-inflammatory genes. NF-κB is considered a master transcription factor in both acute and chronic inflammation, involved in expression of pro-inflammatory cytokines, chemokines, adhesion molecules, iNOS, and metalloproteinases (MMPs) (24). JAK-STAT is another signaling pathway whose activation is implicated in auto-immune and inflammatory disorders. It is used as a signal transduction pathway from cytokines to promote the inflammatory response and it relates to NF-κΒ signaling pathway activation (25). NLRP3 is the most studied inflammasome, a multiprotein complex whose oligomerization activated by PAMPs or DAMPs, leads to maturation of the potent immune modulators IL-1β and IL-8 (26). Research studies showed that luteolin can beneficially regulate these pathways during the inflammatory process (10, 27–29), as summarized in Figure 5.

Based on its multi-targeted anti-inflammatory actions, luteolin appears to be a very promising natural agent for inhibiting abnormal inflammatory responses in chronic inflammatory conditions. In Table 3, we provide a summary of recent in vitro and in vivo studies, reflecting this flavonoid's potential effects on conditions characterized by significant pain during flares of the inflammatory diseases: Rheumatoid Arthritis, Osteoarthritis, and Inflammatory Bowel Disease.

Antioxidant properties of luteolin could also contribute to inhibition of chronic inflammation. Oxidative stress and inflammation are closely related. In fact, evidence shows that there is a bidirectional relationship between excessive free radicals' production and inflammation. Chronic inflammatory conditions are characterized by oxidative stress, and vice versa. Excessive production of free radicals is implicated in the pathogenetic triggers of chronic inflammation: It leads gradually to accumulation of damages to fundamental components of cells, such as DNA, lipids and proteins, and induces activation of inflammatory pathways, such as the NF-κΒ (39, 40).

Flavonoids are potent antioxidant agents (41). Luteolin has shown important antioxidant actions, acting directly, via its ability to scavenge free radicals, but also by inducing endogenous antioxidant cell defenses. In various in vitro and in vivo experiments, it was found that luteolin promotes increased production of antioxidant enzymes (9). One of the main pathways involved in this pathway is suggested to be the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway (Figure 5) (42).

More detailed information on Luteolin's antioxidant capacity will be discussed in the next section about Neuropathic pain, where oxidative stress seems to play a primary role in pathogenesis.

Neuropathic pain is the type of pain that results from damage or disease of the somatosensory nervous system. Its origin can be either peripheral (e.g., peripheral nerve injury pain, chemotherapy induced, cancer pain, diabetes peripheral neuropathy, alcoholic neuropathy) or central (e.g., spinal cord or brain injury, stroke, neurodegenerative disease). Though the pathogenetic mechanisms have not been fully elucidated, there is growing evidence that nitrosative and oxidative stress, along with neuroinflammation, have crucial roles in neuropathic pain.

Oxidative stress and nitrosative stress represent the loss of redox balance in the cells, due to high levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) respectively. ROS, produced by nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) enzymes, and RNS, produced by iNOS enzymes, are generated constantly during normal metabolic cellular processes. When in low concentrations they have various protective roles, e.g., contributing to a normal immune response, but they are potentially detrimental when highly increased. ROS and RNS can easily react with other molecules, causing important oxidative damages to components which are fundamental for cells' healthy functioning and survival (lipid peroxidation, protein and DNA oxidation) (43–45).

Cells normally have endogenous defenses to maintain concentrations of these reactive species under control (redox balance), including a series of antioxidant enzymes, such as superoxide dismutases (SODs), catalase (CAT), glutathione (GSH), heme oxygenase 1 (HO-1). The transcription factor Nrf2 is considered the governor for the expression of more than 200 antioxidants enzymes. However, this antioxidative ability can be compromised as a result of disease or injury, due to excessive production of ROS and NOS (45, 46).

Nerve cells are more vulnerable to nitro-oxidative damage, because they have weaker antioxidant defenses mechanisms and higher lipid content, compared to other types of cells. Therefore, nitro-oxidative stress in neurons can be considered as a primary cause of neuropathic pain. It can lead to mitochondrial dysfunction - further increasing ROS levels - and induce pro-inflammatory cytokines production. Moreover, ROS have been involved in the enhancement of excitatory signaling of nociceptive nervous cells. They can induce excitatory N-methyl-D-aspartate (NMDA) receptors, inhibiting glutamatergic regulation, and contribute to reduction of GABA-ergic inhibitory signaling. In addition, ROS seem to contribute to activation of the ion channels TRP, which are highly expressed in nociceptors neurons and have an important role in transduction of pain (44, 47).

Apart from damage caused directly by ROS and NOS, neuroinflammation seems to be a key process in induction and maintenance of neuropathic pain. Neuroinflammation is the type of inflammation triggered by damage of neuronal tissue in the peripheral or central nervous system. When the peripheral nerve is damaged, resident immune cells (e.g., mast cells and macrophages), along with immune cells recruited from blood circulation (e.g., neutrophils and T-cells) release inflammatory mediators, such as cytokines IL-1b and TNF-a, prostaglandins, and ROS. Moreover, microglia and astrocytes in the spinal dorsal horn are activated, which in turn contribute to the release of inflammatory mediators in the central nervous system and enhancement of excitability. This cascade of inflammatory events leads to peripheral and central sensitization (44, 48).

Both inflammation and nitro-oxidative stress shall not be considered as independent processes in neuropathic pain, as they rather interact in a bidirectional relationship.

Excessive ROS and RNS induce neuronal inflammatory response and vice versa. For example, the transcriptional factor NF-κΒ is one of the most characteristic examples of an inflammatory biochemical pathway that can be activated by ROS/RNS leading to increased expression of inflammatory mediators, but also itself can result to increased nitro-oxidative species production (44, 48, 49).

Pharmacological options currently available for neuropathic pain target the pain as a symptom, rather than inhibit the causes of its pathogenesis. First line drugs include tricyclic antidepressants, serotonin-norepinephrine reuptake inhibitors, gabapentin and pregabalin. Second-line therapy includes tramadol, capsaicin patches, and lidocaine patches. Last line of choice are strong opioids. Due to their inadequate efficacy and important side effects in long-term use of these drugs, effective management of neuropathic pain seems to be quite difficult. An outcome of 30%–50% pain reduction is considered meaningful (50, 51), with a significant number of patients, even >50% in relative surveys, reporting insufficient management of their painful symptoms (52, 53).

Direct antioxidants (e.g., Vitamins C, E, coenzyme Q) have been tried but clinical outcomes failed to support their use (α-Lipoic acid seems however to have therapeutic efficacy in case of diabetic neuropathy). It is worth noting that apart from their unfavorable pharmacokinetic and pharmacodynamic profile, another possible reason for the failure of direct antioxidants is that they may interfere with the physiological (and protective) functions of ROS (44, 45).

Newer and safer approaches that could target selectively multiple pathways may be more effective, protecting from nitro-oxidative damage and at the same time inhibiting neuroinflammation.

Luteolin is a very promising agent against neuropathic pain, due to its effects on two molecular mechanisms involved in neuropathy pathogenesis: oxidative stress and inflammation. Its potent antioxidant abilities, its actions against neuroinflammation, along with its analgesic effects, justify luteolin as a possible complementary therapeutic compound in neuropathy (Figure 6).

Luteolin's antioxidant properties are well established. It works as a direct free radical scavenger, due to the presence of the hydroxyl groups in the B ring that act as an electron-donating system. In addition, it has been shown that luteolin can induce the enhancement of endogenous antioxidant capacity. It can lead to increased expression of antioxidant enzymes, such as SOD, CAT, glutathione peroxidase (GPx), GSH, HO-1, NADPH quinone dehydrogenase 1 (NQO1). This particular action is of very special interest in the case of neuropathic pain (9, 42, 54–56). As mentioned previously, direct antioxidants may not be as effective as expected, and current research has turned its focus on agents that induce endogenous antioxidant mechanisms, rather than just neutralize directly the free radicals. Luteolin seems to achieve this action by activating the major antioxidant transcription factor Nrf2. Moreover, luteolin has been extensively researched for its neuroprotective effects. It appears that it can cross the blood-brain barrier and exert important antioxidant and anti-inflammatory effects in the brain (57–59).

In various preclinical studies, it has been shown that luteolin has a pleiotropic function against mechanisms involved in neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Multiple Sclerosis. It can prevent the activation of microglial and astrocytes, the upregulation of inflammatory mediators, such as cytokines and iNOS, and can also reduce oxidative stress. Again, the major signaling pathways on which Luteolin acts, inducing this anti-inflammatory and antioxidant profile, include the inhibition of NF-κΒ and induction of Nrf2 respectively, among others (11, 60, 61).

Even though the neuroprotective role of luteolin in the central nervous system is well established from in vitro and in vivo studies, its benefits on peripheral neuroinflammation still lack evidence. However, as one would notice from the present review, the pathways involved in inflammatory response and oxidative damage are universal, independently of the tissue damaged. Therefore, we can suspect that luteolin will most likely act beneficially also against damages of the peripheral nervous system. In fact, the limited but important in vivo studies about luteolin's effect on neuropathic pain are very encouraging (Table 4).

Luteolin could attenuate in a dose dependent manner, the mechanical and cold hyperalgesia in an animal model of neuropathic pain (62). The researchers also investigated the mechanism of its antinociceptive action. Antihyperalgesic effects of luteolin (intrathecally administered luteolin 1.5 mg or intraperitoneally administered luteolin 50 mg/kg) were inhibited by intrathecal pretreatment with the γ-aminobutyric acid A (GABAA) receptor antagonist bicuculline and μ-opioid receptor antagonist naloxone, but not by intrathecal pretreatment with either the benzodiazepine receptor antagonist flumazenil or glycine receptor antagonist strychnine.

In a diabetic neuropathy model, luteolin was able to improve the impaired nerve functions (63). Intraperitoneally daily treatment with luteolin (50 mg/kg, 100 mg/kg and 200 mg/kg) induced activation of Nrf2, increased activities of antioxidant enzymes SOD, GST, GPx and CAT, and reduced ROS production. Nerve function, as measured by increase of motor and sensory conduction velocities were also improved with luteolin treatment. Moreover, mechanical withdrawal threshold, cold, and heat withdrawal latencies were increased, indicating that luteolin can attenuate hyperalgesia and allodynia. All results were induced in a dose dependent manner, with doses of 100 mg/kg and 200 mg/kg of luteolin treatment leading to a significantly better outcome than dose of 50 mg/kg.

Administration of luteolin dose (5 mg/kg and 10 mg/kg body weight) significantly reduced neuropathic pain in another animal model. Interestingly, co-administration of luteolin and morphine (1 mg/kg), potentiated morphine analgesic effects (64).

Intraperitoneally treatment with luteolin ameliorated Lewis's lung cancer (LLC)-induced bone pain in mice in a dose-dependent manner (65) (50 mg/kg significantly improvement in pain behavior, than 1 mg/kg and 10 mg/kg). Bone cancer pain (BCP) is a severe complication in cancer patients with both inflammatory and neuropathic components. Intrathecal injection of luteolin 0.1 mg also reduced BCP in this study. Further analysis about the mechanism of action, showed that luteolin inhibited important components of neuroinflammation, such as activation of glial cells in spinal dorsal root and NLRP3 inflammasome.