**1. Introduction**

Osteoarthritis (OA) is a multifactorial joint disease and a common disabling condition affecting the global population [1,2]. The increasing joint pain and stiffness gradually leads to reduced physical function, quality of life, and frequent physician visits [3,4]. Up to 54.4 million adults experienced doctor-diagnosed arthritis during 2013–2015 in United States, and 23.7 million had arthritis-attributable activity limitations. In addition, adults with heart disease, diabetes, and obesity have a higher prevalence of OA (49.3%, 47.1%, and 30.6%, respectively) and consequently arthritis-attributable activity limitations [5]. Therefore, the managemen<sup>t</sup> of these associated factors are recommended to potentially reduce symptomatic knee and hip OA incidences [6,7].

OA pain is the predominant limiting factor for a patient's activity and life quality, and it leads those individuals affected to seek medical care [8]. The pain tends to be localized to the affected joint and aggravated by joint use while relieved by rest. The ultimate goal of nonsurgical treatment modalities is to reduce the pain and restore function while delaying total knee replacement (TKR), a substantial direct health-care cost in OA patients with end-stage disease. Researchers have urged into the preventive managemen<sup>t</sup> of OA and development of disease-modifying OA drugs [9].

HA (hyaluronic acid) is an intrinsic component within the knee joint and provides viscoelastic properties to synovial fluid. Increasing HA levels through intra-articular (IA) injection restores the viscoelasticity of the synovial fluid, which aids shock absorption, lubrication, and protection of the joint, along with a good safety profile [10–12]. Moreover, several retrospective studies have shown the potential of IAHA (intra-articular hyaluronic acid) to delay the time for TKR in patients with OA [13–16].

Shea nut oil (SNO), extracted from the African shea tree (Vitellaria paradoxa), contains a high nutritional value with high triterpene and oleic/stearic fatty acid concentration, vitamins, and minerals. Importantly, their high triterpene alcohol and tocopherol content are considered to have anti-inflammatory and anti-oxidant properties [17]. Clinical evidence suggests the bioactive triterpene concentrate has anti-inflammatory effects under daily oral supplementation in OA patients [18]. Additionally, a clinical report demonstrated a decrease in pain and stiffness in patients after daily oral SNO supplement for 16 weeks [19]. We previously reported that preventive oral administration of SNO dose-dependently reduces cartilage degeneration in a rat model of anterior cruciate ligament transection plus medial meniscectomy (ACLT + MMx)-induced OA [20]. It also reduces pain and provides differential cartilage protection in both acute and chronic OA rats [21].

Regarding the complexity of this natural plant oil, the overall protective effect may be derived from a combined mechanism of actions of the different triterpene concentrates (primary α, β-amyrin, lupeol, and butyrospermol), monounsaturated oleic acid, or tocopherol found in this shea nut oil product. For instance, in mouse inflammation models induced by complete Freund's adjuvant and by partial sciatic nerve ligation, daily oral intake of α and β-amyrin showed long-lasting antinociceptive and anti-inflammatory effects via direct activation of cannabinoid receptors and a concomitant inhibition of inflammatory NF-κB, cyclic adenosine monophosphate response element binding (CREB) pathway [22]. Otuki et al.'s report suggested that the antinociceptive properties of mixed amyrins may be involved in the inhibition of protein kinase A and protein kinase C pathways [23]. Others researchers found that lupeol acetate ameliorates collagen-induced arthritis through suppression of inflammatory cytokines and inhibition of bone erosion [24]. Indeed, the potential antioxidant and free radical scavenging effects of amyrin and lupeol have been demonstrated both in vitro and in vivo [25–28]. A recent report showed increased antioxidant activity and suppressed proinflammatory cytokine expressions in obese OA rats fed with SNO, further consolidating our previous findings [29]. Either of the triterpenoids and their potential target mechanisms may play a key regulatory role in our OA model and contribute to the antiarthritic action of SNO.

Although the protective potential of oral SNO supplementation differs from that of IAHA in many aspects (route, dose frequency, and mechanism of action). But the combination of both therapeutic models, like IA corticosteroid/anti-inflammatory drugs combined with IAHA, can lead to significant improvement of the clinical outcome of either agen<sup>t</sup> alone [30]; this however only provides short duration, and is not feasible for persisting injection. Therefore, we sought to determine the role of oral SNO as an adjuvant in combination with IAHA injections and compare the effectiveness in the OA rat model. In this study, we compared the differences in protective potential between SNO and IAHA in OA rats, and the hypothetical synergetic effect of SNO with IAHA on the prevention of OA progression was also examined.

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

#### *2.1. ACLT* + *MMx-Induced OA Animal Model*

A rat model of surgically-induced OA was proceeded as described previously [20,21], and all animal care and experimental protocols complied with institutional and international standards (Principles of Laboratory Animal Care, National Institutes of Health) and were approved (Institutional Animal Care and Use Committee [IACUC] no. 107-030) by the IACUC of Cathay General Hospital (Taipei, Taiwan). Adult male Wistar rats were purchased from BioLASCO Taiwan Co., Ltd. (Yilan, Taiwan) and housed

in Cathy Medical Research center with free access to the standard diet and water with a 12-h light/dark cycle at a temperature of 22 ± 2 ◦C and 55% humidity.

All ACLT + MMx surgeries were performed on the right knee of the rats by a single research specialist. Briefly, male Wistar rats (330–350 g) were anesthetized in an induction chamber using 5% isoflurane and then maintained with 2% isoflurane via a custom-made facemask. The right knee joint skin was shaved, and sterilized with povidone-iodine solution. An incision was made in the medial aspect of the joint capsule; the anterior cruciate ligament was transected using a scalpel, and the medial meniscus was removed completely using a tenotomy scissor. Following surgery, the joint was irrigated with normal saline; the joint capsule was sutured with 4–0 Vicryl, and 4–0 monofilament nylon was used for skin closure. Next, the wound area was sterilized, and cefazolin (100 mg/kg/day) was administered intramuscularly for 3 days to prevent infection. For the sham-operated rats, the same procedure was repeated, but neither ACLT nor removal of the medial meniscus was performed.
