**1. Introduction**

Osteoarthritis (OA), also called degenerative joint disease, is a universal debilitating joint disease, and is the most prevalent type of arthritis characterized by synovial inflammation, the gradual loss of articular cartilage and degenerative changes in other surrounding tissues, including the synovium, menisci, ligaments, and subchondral bone, that are caused by multiple risk factors such as age, weight, excessive joint usage, and metabolic or genetic factors [1,2]. OA is the most common joint disorder in the elderly around the world, which can decrease quality of life due to pain, sti ffness, loss of function, and disability [3]. Chronic joint pain is the main symptom of OA and is the primary reason for patients to seek medical and pharmacological treatments. Although structural changes, cartilage degeneration, and subchondral bone remodeling are the main contributors to disease progression and joint pain in patients, inflammatory responses mediated by pro-inflammatory cytokines and inflammatory mediators following joint injury also contribute to these pathological events in OA [4–8].

There is no treatment to prevent the development of OA or cure it once it has set in; thus, the goal of OA treatments currently available in clinical practice is mainly aimed at reducing pain, minimizing cartilage damage, and improving or maintain functional status. Commonly recommended treatments include aerobic exercise, strengthening exercises, and medicinal agents, including non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or acetaminophen, which treat OA primarily by reducing pain and inflammation [9]. However, these medications cannot prevent progressive cartilage degradation or repair the damaged cartilage of OA patients, and long-term use of these drugs can lead to various adverse e ffects, including renal toxicity, gastrointestinal disturbances, diarrhea, nausea, vomiting, or increased cardiovascular risks [10–12]. In addition, a recent clinical study reported that chronic but not short-term use of NSAIDs was associated with no clinical improvement in pain and a minimal, non-statistically significant improvement in clinical outcomes in persons with sti ffness and functional and structural changes due to OA [13]. Despite their questionable e fficacy, prescription NSAIDs are widely used in OA treatment due to the lack of more e ffective medications.

Therefore, the chronic nature of the disease has encouraged researchers and patients to try various complementary and alternative approaches, including dietary supplements, functional health foods, and nutraceuticals, to relieve pain and improve joint function as well as to manage or slow disease progression [14–17]. The most widely used and studied dietary supplements or functional health foods for joint pain include those related to chondroprotection, such as glucosamine, chondroitin sulfate, methylsulfonylmethane (MSM), collagen hydrolysates, and hyaluronic acid [18]. However, their e ffectiveness remains controversial; moreover, they can cause multiple adverse e ffects, such as stomach upset, constipation, diarrhea, headache, and rash [19,20]. In addition, natural products and nutraceutical products, including curcumin and turmeric extract, green tea extract, resveratrol, citrus fruit extract, *Boswellia serrata*, omega-3 fatty acids, and many others, have been investigated as potential candidates for joint-health promoting dietary supplements [21]. Interestingly, several studies have hypothesized that gu<sup>t</sup> microbiota modulation through the supplementation of specific dietary ingredients including probiotics and prebiotics are able to modify the onset and the progression of OA [22]. Although previous studies have investigated the gut-joint axis in animal model, clinical studies in humans are lacking and no clear mechanism of action has been determined [23]. Therefore, future perspectives should focus on a more detailed understanding of the e ffects of microbiota modulation in OA patients. Nevertheless, dietary supplements with proven medical benefits, including managing and slowing disease progression and reducing the symptoms of OA, may o ffer safer alternatives to currently available pharmacological therapies [24,25].

FlexPro MD ® (FP-MD), a novel, patented multi-ingredient dietary supplement formulation consisting of krill oil, natural astaxanthin, and sodium hyaluronate, has been shown to markedly reduce pain in subjects su ffering from chronic mild-to-moderate knee joint pain [26]. Moreover, another study demonstrated that FP-MD could e ffectively inhibit lipopolysaccharide (LPS)-induced mRNA expression of pro-inflammatory cytokines and inflammatory markers by reducing nuclear factor-kappa B (NF-κB) activation, both in RAW264.7 cells and in an LPS-induced arthritis mouse

model. In addition, FP-MD e ffectively suppressed the expression levels of matrix metalloproteinases (MMPs) at the transcriptional level in inflamed knee joint tissues [27]. However, its e ffects on the structural joint damage and associated joint pain of OA have not been explored. In this study, we investigated the e ffects of FP-MD on pain, and the structural changes and inflammatory responses known to cause pain, in a rat model of OA induced by monosodium iodoacetate (MIA) injection.

#### **2. Materials and Methods**

#### *2.1. Preparation of FP-MD*

FlexPro MD ® (FP-MD) is a commercially available dietary supplement containing a proprietary combination of Superba ® Antarctic krill (*Euphausia superba*) oil (Aker BioMarine Antarctic US LLC; Metuchen, NJ, USA), Zanthin ® natural astaxanthin derived from *Haematococcus pluvialis*, Flexonic ® sodium hyaluronate (the sodium salt of hyaluronic acid) produced from fermentation by *Streptococcus zooepidemicus* (Valensa International, Inc.; Eustis, FL, USA), and food vehicles (Table 1). A sample of FP-MD was produced as reported previously [28,29], then stored at room temperature until use. The FP-MD sample consisted of 70% krill oil, 7% *Haematococcus pluvialis* extract, and 7% sodium hyaluronate, along with 16% various excipients (beeswax, olive oil, etc.), as determined by high-performance liquid chromatography (HPLC) and gas chromatography (GC), using the methods recommended by the United States Pharmacopeia (USP) and Korean Pharmacopoeia (KP), respectively.


**Table 1.** Composition of FlexPro MD ®.

1 adjust the amount depending on the amount of *Haematococcus pluvialis* extract.

#### *2.2. Animals and Ethics Statement*

Adult male Sprague-Dawley (SD) rats (aged 7 weeks with body weights ranging 200–214.0 g) were obtained from Orientbio Inc. (Sungnam, Gyeonggi, Korea) and housed in the animal facility at Biotoxtech Co. Ltd. (Cheongju, Chungbuk, Korea), a non-clinical Good Laboratory Practice (GLP)-certified Contract Research Organization (CRO). The rats were acclimated for 7 days and maintained under conventional housing conditions at 23 ± 2 ◦C with a controlled 12 h light/dark cycle, and were provided filtered tap water and a rodent chow diet (Envigo RMS Inc., Indianapolis, IN, USA) *ad libitum* throughout the experiment. All animal experimental procedures complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Korean National Animal Welfare Law. The experimental animal facility and all protocols involving animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Biotoxtech (IACUC approval number 180644).

#### *2.3. Experimental Design and Administration*

The experimental design of the study is shown in Supplementary Figure S1. Rats were randomly distributed in six groups of eight rats each, consisting of a normal sham control (Sham) group; an MIA-induced OA control (MIA) group; a positive control (PC) group; and three groups treated with increasing doses of FP-MD (25 mg/kg, 50 mg/kg, or 100 mg/kg). All of the groups received treatments via oral gavage once daily for 7 days before OA induction and then for 21 days thereafter until the end of the experiment. The PC group received Celecoxib administered orally at a dose of 3 mg/kg. The Sham and MIA groups received equivalent volumes of vehicle (corn oil). The administration volumes were 5 mL/kg body weight for all experimental groups. The animals were closely monitored for individual clinical signs and other complications by a veterinarian. Body weight was measured weekly, and the weight-bearing levels of the two hind limbs were measured at 0, 3, 7, 14, and 21 days post-OA induction.

#### *2.4. MIA-Induced OA Animal Model*

Following the 7-day pretreatment period, the animals were anesthetized with 2% isoflurane delivered via a nose cone and then their right hind knees were injected, either with 3 mg MIA (Sigma-Aldrich Inc., St. Louis, MI, USA) in 50 μL saline or an equivalent volume of sterile saline vehicle, through the infrapatellar ligament as previously described [30–32] while contralateral knees remained intact. The choice of MIA dose was supported by previous work demonstrating that 3 mg of MIA not only induced joint degeneration but also produced significant axonal injury to dorsal root ganglion cells, reproducing the neuropathic pain component typically observed at the later stages of OA development [33,34]. Following injection, the animals were allowed to fully recover from the anesthesia and were monitored appropriately before returning them to the cages, and then continued to receive the same oral doses of FP-MD, Celecoxib, or vehicle, respectively, for 3 weeks.

#### *2.5. Weight-Bearing Test (Pain Assessment)*

The weight-bearing distributions between the postoperative and normal hind limbs were determined at 0, 3, 7, 14, and 21 days post-OA induction by using an incapacitance meter (Bioseb Co.; Pinellas Park, FL, USA) as previously described [35,36], which independently measures the weight, in grams, that the animal distributes to each hind paw. Generally, normal animals distribute their weight equally on both hind paws, while animals with MIA-induced joint pain tend to favor the normal limb. The rats were placed in a container with their hind paws comfortably resting on two separate sensor plates. When the rats stand, they make natural adjustments to the weight distribution on both rear paws based on the level of joint pain they are experiencing. The equipment was set to average the weight measured by each sensor over a 5-second period, and a total of three measurements were taken and averaged for each rat. The result was expressed as the mean of weight ratio between injured and non-injured limbs.
