This study revealed that the administration of casein fraction of goat milk (CNF) and its hydrolysates, produced by enzymatic hydrolysis in vitro with pepsin (HDP) and trypsin (HDT), reduce the inflammatory pain in the TMJ of rats, and that the antinociceptive efficacy these compounds appear to depend, at least in part, on the involvement of TNFα, IL-1β and IL-10. In addition, our results also suggest that the effects of these compounds on inflammatory pain in the TMJ of rats do not appear to depend on the NO pathway or on the heme oxygenase pathway.
Goat milk has beneficial effects on human health, already recognized by the scientific community (Palatnik et al., 2015). Among the milk components which can positively affect organic function are the proteins (Bhat, Kumar and Bhat, 2015), with antimicrobial, anti-inflammatory, hypocholesterolemic or hypertension control effects already recorded in the literature (Esmaeilpour et al., 2016; Eriksen et al., 2008; Yamauchi, Ohinata and Yoshikawa 2003; Hernándes-ledesma, 2014).
Heme-oxygenase (HO) is the rate-limiting enzyme that catalyzes the degradation of heme to release carbon monoxide (CO), biliverdin (BVD) and free iron in mammalian cells (Alcaraz et al., 2003). HO-1 is induced by oxidative or nitrosative stress, cytokines and other mediators during inflammatory processes, probably as part of a defense system in cells exposed to stress, to provide negative feedback for cell activation and the production of mediators, that can modulate the inflammatory response (Alcaraz et al., 2003). Over the last few years, our group have demonstrated that HO/BVD/CO pathway plays antinociceptive effects during temporomandibular joint inflammatory hypernociception in rats (Chaves et al, 2018; Coura et al, 2017; Vanderlei et al, 2011; Freitas et al, 2016) However, in the present study, after the pretreatment with ZnPP IX, a specific HO-1 inhibitor, the efficacy of both pepsin (HDP) and trypsin (HDT) hydrolysates was unchanged, suggesting that HO-1 activity is not involved in the inhibitory effects of pepsin (HDP) and trypsin (HDT) hydrolysates. Given these results, we continue in an attempt to answer the mechanisms of action of both pepsin (HDP) and trypsin (HDT) hydrolysates.
A huge number of inflammatory mediators have been suggested to play a role in temporomandibular joint disorders. Among these, NO, a small free radical, has critical signaling roles in immunological, inflammatory, and nociceptive processes. In formalin-induced hypernociception in TMJ in rats, the NO inhibitor, aminoguanidine, which is a specific inhibitor of iNOS, increased the nociceptive behavior, when compared to formalin group, showing that in this model NO has anti-nociceptive effect. In order to explain the putative mechanism of action of HDP and HDT, aminoguanidine, was administered, being followed by the HDP or HDT, which did not reduce its effectiveness, suggesting that the NO pathway integrity is not required for HDP and HDT mechanism of action. These results appear paradoxical but we would consider that within inflamed joints activated cells (neutrophils, macrophages) can secrete a plethora of inflammatory mediators, then amplifying the inflammatory response. Further, NO is involved in both pathological and physiological processes, and many lines of evidence have indicated that NO plays a complex role in the modulation of pain, producing either analgesia or nociception, depending on the dose and the pain model used (Cury et al, 2011).
During TMJ disorders, besides NO, many inflammatory mediators are produced and secreted from TMJ synoviocytes and neutrophils to synovial fluid of TMJ. Among them, the proinflammatory cytokines TNF-α and IL-1β are associated with inflammation in synovial joints and connective tissue destruction. On the other hand, IL-10 acts as inhibitors to these proinflammatory cytokines (Kacena MA et al, 2001). Fortunately, communication network of pro- and anti-inflammatory cytokines maintains the homeostasis of the TMJ (Sorenson et al, 2018). In the present study, we investigated the involvement of TNF-α and IL-1β, and IL-10 on the hydrolysates (HDP and HDT) efficacy during formalin-induced hypernociception in rat TMJ.
Our research group demonstrated that intra-TMJ injection of formalin, which is a noxious stimulus commonly used to investigate the efficacy of analgesic and anti-inflammatory compounds in pre-clinical behavioral trials, was associated with increased levels of TNF-α in the TMJ tissue, trigeminal ganglion, and subnucleus caudalis compared with saline group (Alves et al, 2018). High TNF-α levels were found in symptomatic TMJs when compared with normal ones (Nordahl, Alstergren and Kopp, 2000; Emshoff et al, 2000). Further, some authors demonstrated that therapy with TNFα inhibitors have improved the outcomes of treatment for rheumatoid arthritis (RA) (Sikorska et al, 2019). Our current data revealed that both HDP and HDT reduced TNFα immunostaining in synovial cells of the TMJ synovial membrane, in the peripheral nerve, as well as, in the primary afferent neuron cell body, and in glial cells in the trigeminal ganglion, suggesting antinociceptive and anti-inflammatory activity involving the peripheral nervous system.
Two studies have shown that the most representative pro-inflammatory cytokines in the synovial fluid of patients with TMD are TNF-α IL-1β (Kellesarian et al, 2016; Nordahl, Alstergren and Kopp, 2000). In this regard, similar to that observed for TNF-α, our research group demonstrated that the intra-TMJ injection of formalin significantly increased the release of IL-1β in periarticular tissues, in relation to sham groups (Rivanor et al, 2018). Cytokines from glial cells may play important roles in glial-neuron communication, which contributes to pain hypersensitivity. In fact, after inflammation or nerve damage, inflammatory mediators released from nerve terminals activate glial cells, which release a diversity of mediators (such as cytokines, including IL-1β) that in turn affect neuronal activity (Ren and Dubner, 2008) (Guo et al ,2007). Notably, it was demonstrated that satellite glial cell activation in the trigeminal ganglionic modulates neuronal excitability via IL-1β post-inflammation (Takeda et al, 2007). Also, some authors suggested how IL-1β contributes to TMJ inflammatory hypernociception, showing evidence that glial IL-1β upregulated neuronal Nav1.7 expression in the trigeminal ganglionic via the crosstalk between signaling pathways of the glial IL-1β/ COX-2/PGE2 and the neuronal EP2/PKA/CREB/Nav1.7 (Zhang et al, 2018). Our present data show that both hydrolysates (HDP and HDT) reduced IL-1β immunostaining in synovial membrane, and in trigeminal ganglion, when compared to formalin group. Pharmaceutical blockade of IL-1 activity has been used to successfully treat diseases of the joints, bones and muscles, such as rheumatoid arthritis, erosive osteoarthritis and traumatic joint injuries (Dinarello, Simon and Van der Meer, 2012). Bearing in mind that the specific targeting of pathological mediators, including the IL-1 cytokine family, can significantly reduce inflammation and prevent further TMJ degeneration (Sorenson et al, 2017), the above results suggest a possible role of cytokines in the pathogenesis of TMD.
Fortunately, the inflammatory response is a well-orchestrated and strictly regulated event, so in the inflammatory milieu, anti-inflammatory cytokines also are secreted, such as IL-10, which may inhibits the synthesis of IL-1β and TNF-α, providing a negative feedback for cell activation and the production of inflammatory mediators, which could modulate, at least partially, the inflammatory pain process. Our research group demonstrated that intra-TMJ injection of formalin significantly decrease the levels of IL-10 in the trigeminal ganglion and subnucleus caudalis, when compared to saline-inected group (Coura et al, 2017). In present study, we demonstrated that the administration of both HDP and HDT increased immunostaining for IL-10 in the synovial membrane and in the trigeminal ganglion. IL-10 is a powerful anti-inflammatory cytokine with a wide spectrum of biological effects, expressed by many cells of the adaptive immune system, as well as by innate immunity cells (Saraiva and Garra, 2010). It inhibits Th1 cytokines, including both IL-2 and IFN-g (Driessler et al, 2004), in addition to disabling the synthesis of monocyte/macrophage pro-inflammatory cytokines (Brandtzaeg 1996).
Taken together our data suggest that the efficacy of casein fraction and its hydrolysates of goat milk in formalin-induced TMJ inflammatory hypernociception in rats involves, at least in part, TNF-α and IL-1β inhibition, and IL-10 secretion.
The production of peptides by hydrolysis appears to be a promising technique. This study is the first to evaluate whether casein fraction of goat milk (CNF), and its hydrolysates, produced by enzymatic hydrolysis in vitro with pepsin (HDP) and trypsin (HDT), can inhibit nociceptive response in a formalin challenge rat model. Thus, considering the safety profile of goat milk, we postulated that CNF, HDP, and HDT, could offer a novel way to treat TMJ pain. We trust that our results may support in the understanding of TMJ inflammatory pain and the development of a new strategy to deal with this disorder.