These findings show that patients belonging to the treatment group had a significant change and a global reduction in the expression of pro-inflammatory cytokines compared to the control group (Table 2, Figure 1).

It is traditionally known that OA pathophysiology is complex and influenced by many factors that converge in a single disease model of a purely inflammatory nature. The pathogenesis of OA is therefore based on the action of secreted inflammatory molecules, such as proinflammatory cytokines, which are the most important mediators of all the processes implicated in this chronic degenerative disease. In fact, pro-inflammatory cytokines, together with nitric oxide, prostaglandin E2, and neuropeptides, are responsible for articular cartilage matrix degeneration. They are produced by the inflamed synovial tissue and upset the physiological balance between repair and degradation, ensuring catabolic processes prevail thanks to the action of lytic enzymes which are able to destroy the cartilage [30]. Although, from a clinical point of view, OA presents phases of quiescence and phases of reactivation, the biochemical mechanisms that underlie it do nothing but smolder, giving this pathology a chronic degenerative evolution due to the ineluctable inflammation that modifies the physiological functioning of joint cells, favoring chondrocyte apoptosis and the activation of macrophages.

This inflammatory process then inevitably extends to the entire joint microenvironment, leading to the formation of geodes and osteophytes and a reduction in the joint space; swelling, deformity, and joint pain thus cause a functional limitation that can determine severe forms of motor disability and can negatively impact the physical and mental wellbeing of the patients, especially in its advanced stages, which are typically related to old age.

The above description can be valid for all joints, starting with the arthritic knee. The knee joint, indeed, is certainly the most affected by OA, followed by the hip, hands, and spine. For this reason, it is also traditionally the most investigated, given that it may represent the first site of the manifestation of OA itself, even in isolated forms [31]. Although the knee is a joint subjected to a continuous and intense load, and, consequently, it is more exposed to the risk of joint degeneration in the presence of predisposing factors such as obesity and reduced muscle tone and strength, it is now also well known that the OA-related biomechanical factors in this case go strictly hand-in-hand with the biochemical ones. In a recent review by P. Dainese et al., the association between inflammation (as measured by effusion, synovitis, baker’s cysts, cytokines, and the C-reactive protein) and pain in patients with radiographic KOA was investigated [32]. Despite varying results in terms of the strength of evidence, what emerges is that both imaging tests and blood tests confirmed the inflammatory nature of KOA, which therefore results in joint pain.

As a result, the modulation of the proinflammatory cytokine profile represents a possible new target for new OA treatments [33]. The real challenge is to find increasingly selective and innovative solutions which allow for personalized therapies, amplifying the benefits and reducing the side effects of the drugs currently used to fight inflammation. In fact, non-steroidal anti-inflammatory drugs are effective but burdened by side effects such as an increase in blood pressure, variations in blood sugar, irritation to the gastric mucosa, and negative changes in the coagulation profile. This imposes severe limitations on their use and calls for the scientific community to make an effort in the search for effective but well-tolerable molecules, both topically and systemically. As far as joint injections are concerned, hyaluronic acid remains a reliable solution with long-lasting effects over time; nevertheless, regenerative medicine is opening up to new solutions such as platelet-rich plasma and stem cells, with very encouraging clinical results, and even nanomedicine, an interdisciplinary discipline which aims to manipulate particles the size of 1–100 nanometers, is studying the possibility of using new bioparticles such as exosomes, dendrimers, micelles, and lipid vesicles as transports for drug release control [34]. In the near future, this could avoid excessive infiltration practices, which are minimally invasive procedures that are poorly tolerated by some patients, especially in the presence of coagulopathies and systemic diseases that expose them to the risk of infections due to a low white blood cell count. In this sense, experimenting with natural molecules that can be taken orally and that can develop an anti-inflammatory power without significant side effects is still an open game that researchers and clinical physicians are necessarily called upon to play.

This is the reason why this research aimed to deepen the understanding of the effects of dietary supplements containing hemp seed oil and terpenes in the inflammatory cytokine expression in a population of KOA patients.

With regard to the IL-2 and TNF-α levels, these were not measurable. This can be explained by the exclusion criteria which required the absence of systemic chronic inflammatory diseases. In fact, many cytokines of the IL-2 family as well as TNF-α have been reported to be a driving force in immune cell activation and systemic inflammation [35,36,37]. The enrollment criteria predicted that patients were suffering from monolateral KOA, which can be considered a district pathology, and that they were not affected by rheumatoid arthritis (RA), metabolic inflammatory arthropathy, or any other chronic inflammatory systemic diseases. In fact, these conditions are associated with a general activation of several pro-inflammatory cytokines. In a study conducted by Kondo N et al. in 2021, the role of TNF-α in the pathogenesis of RA was demonstrated and an increased expression of TNF-α was documented in affected patients, inducing autoimmune arthritis in transgenic animals [38]. Moreover, the increased serum level of these cytokines in chronic systemic inflammatory diseases such as vasculitis [39], psoriasis [40], and long COVID-19 syndrome [41] is now clear. This explains why, in our subjects, the Bio-Plex Pro Human Cytokine 5-plex assay (IL-1β, IL-1α, IL-2, IL-6, and TNF-α) did not find measurable levels of the pro-inflammatory cytokines IL-2 and TNF-α and low levels of IL-1β.

IL-1β levels decreased between T0 and T1 in both groups, but significantly more in the treatment group. IL-1 family members are the most highly profiled in OA and have all been shown to be present in the synovial fluid and subchondral bone of OA patients [42]. IL-1β is also actively involved in the pathogenesis of cartilage loss related to OA [43]. The low levels of IL-1β induced by the local inflammatory process in KOA [44] were further reduced by hemp seed oil and terpenes, which therefore demonstrated their synergic anti-inflammatory action. Terpenes’ healing potential for OA is rapidly being documented. In a 2021 study, the therapeutic effect of limonin on OA was assessed in chondrocytes in vitro in IL-1β-induced OA and in the destabilization of the medial meniscus (DMM) mice in vivo [45]. Similarly, aucubin, a natural compound isolated from Eucommia ulmoides, inhibits IL-1β-induced chondrocyte apoptosis [46] and morusin ameliorates IL-1β-induced chondrocyte inflammation and OA via the NF-κB signal pathway [47].

Although further studies are needed to confirm the clinical effectiveness of different terpenes, there is no doubt that these molecules can now be considered as a complementary therapy to OA by interfering with IL-1β.

Although a minor increase in its levels could be glimpsed in the treatment group between T0 and T1, the IL-1α levels did not have any statistically significant modifications between the two groups at the detection times. Also, these data are in line with the available literature, since the most recent evidence suggests that the role of IL-1α in OA pathogenesis is still controversial [48]. However, it seems that an IL-1α increase could have a protective action for OA patients, though at the moment it is not possible to unequivocally establish the role of this cytokine in joint inflammatory processes [49].

The particular case of IL-6 caught our attention. Indeed, this interleukin decreased slightly between T0 and T1 in the control group, while it significantly increased in patients in the treatment group after 45 days of treatment.

This suggests an unexpected action of the IL-6 cytokine, generally known as a pro-inflammatory factor, which was recently found to have anti-inflammatory activity in its muscle-derived form, which could be induced by hemp seed oil and in particular by terpenes (Figure 1).

Interestingly, hemp seed oil and terpene treatment increased the levels of IL-6, which is a cytokine with established pro- and anti-inflammatory action [50]. In classic signaling, IL-6 induces intracellular signaling pathways after binding to its membrane-bound receptor (IL-6R) expressed on hepatocytes and certain subpopulations of leukocytes mediated by the membrane-bound b-receptor glycoprotein 130 (gp130) [51]. In a second pathway, which is called trans-signaling, IL-6 binds to soluble forms of the IL-6R (sIL-6R), and this agonistic IL-6/sIL-6R complex can, in principle, activate all cells due to the uniform expression of gp130 [52]. Most pro-inflammatory roles of IL-6 have been attributed to the trans-signaling pathway, whereas anti-inflammatory and regenerative signaling is mediated by IL-6 classic signaling.

This double face characteristic of IL-6 can explain its anti/pro-inflammatory activity in different conditions.

Systemic chronic inflammation involves progressive changes in inflammatory cells and the coexistence of tissue destruction and repair; it can become pathological because of the loss of tolerance or because of regulatory processes. Obesity and metabolic syndrome lead to an inflammatory state that differs from the classical response, with the inflammatory process being systemic and characterized by a chronic low-intensity reaction [53,54]. Chronic systemic inflammatory diseases like obesity, non-alcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) liver diseases, diabetes, cardiovascular diseases, pain, and other nervous system disorders are related to a mild chronic inflammation, which appears to start from an alteration in the metabolism of these patients and, for this reason, it is also named metaflammation, that occurs in several tissues, including adipose, pancreas, liver, muscle, brain, and heart tissues [55]. In these chronic inflammatory diseases, the use of bioactive compounds acting on CB2 cannabinoid receptors has shown therapeutic effects through the reduction in pro-inflammatory mediators such as TNF-α, IL-1β, and IL-6 [56,57] (Figure 2).

KOA is a chronic joint disease whose mechanisms of progression are not fully understood [4], but it is evidently the expression of a local disease rather than a chronic systemic one. As said above, this is also evidenced by the fact that, in the pre-surgical stages, treatment involves the on-site injection of substances such as corticosteroids [58], hyaluronic acid [59], platelet-rich plasma [60], or oxygen–ozone [61]. It has recently emerged that the role of muscle-derived IL-6 is unlike what was assumed in the past. Interestingly, it has been indicated that IL-6 plays opposing roles as an anti-inflammatory myokine and as a pro-inflammatory cytokine [25,62]. Skeletal muscles can release several bioactive substances, collectively named myokines, among which IL-6 is the most abundant. The endocannabinoid system (ECS) is involved in various processes, including brain plasticity, learning and memory, neuronal development, nociception, inflammation, appetite regulation, digestion, metabolism, energy balance, motility, and the regulation of stress and emotions [63]. The activation of CB2 cannabinoid receptors appears to induce the release of several mediators from muscle tissue which have been defined as exerkines, including IL-6 [64], that have anti-inflammatory activity. As a consequence, this change in IL-6 serum levels enforces the hypothesis that hemp seed oil and terpenes could represent a valid option in the complementary therapy of KOA. This could, therefore, become a new opportunity to personalize therapies, making the individual rehabilitation project more effective and multidimensional [64].

However, this study cannot be considered free of limitations. The main limitation is the short duration of the follow-up and the small patient sample. Therefore, additional studies are needed to monitor how serum cytokine levels change over time and how this finding affects pain and joint function. In addition, it should be considered that inflammatory cytokine serum levels depend on several factors such as smoking, diet, sedentary lifestyle, or frequent sport activity. However, it should be pointed out that in this study we were certain that these parameters remained constant in the sample analyzed between T0 and T1. In addition, we did not investigate the correlation between the blood chemistry data and the clinical data of these patients, but we believed it was essential in the first instance to verify the blood variations induced by terpenes; however, we intend to follow up on this research with further studies that also will include appropriate clinical evaluations. Finally, there was no placebo group, but this choice was due to the necessity of guaranteeing treatment for all patients since all of them suffered from significant knee pain. Nevertheless, future studies will overcome this limitation, in compliance with the necessary ethical rules.

On the contrary, we consider an important strength of this study is the fact that it highlighted the new and probably unexpected anti-inflammatory effects of terpenes, with particular reference to IL-6, thus opening further research scenarios on the therapeutic potential of these molecules.