Osteoarthritis (OA) is a degenerative disease characterized by the wear, destruction, and loss of articular cartilage, accompanied by hyper osteogeny around the joint [1], which can cause the loss of joint function. The clinical symptoms of OA include joint pain, stiffness, limited mobility and deformity [2], which seriously affect the life quality of patients. However, the existing medical technology cannot cure OA, and the common oral drugs for treating OA, such as non-steroidal anti-inflammatory drugs [3], have many side effects on human body. Fortunately, many studies showed that oral administration of undenatured type II collagen can inhibit the occurrence of arthritis or alleviate arthritis symptoms without side effects [4,5]. Soluble undenatured type II collagen (SC II) extracted from terrestrial animals such as cattle, sheep, pigs and chickens or marine animals such as blue sharks and squid has been proven to have the effect of improving OA or RA [[6], [7], [8]]. Moreover, undenatured type II collagen acts on the associated lymphoid tissues of the small intestine after passing through the gastrointestinal digestive system and inhibits the occurrence and development of arthritis by inducing immune tolerance [9,10]. Importantly, Nagler-Anderson et al. [11] demonstrated that oral administration of SC II inhibited the occurrence of arthritis in mice, while the same dose of denatured type II collagen (i.e. SC II incubated at 56 °C for 45 min) had no significant effect on the incidence and severity of the arthritis.

Our previous research found that the gastric digestive stability of SC II under low pH conditions was poor, that is, the triple helix structure of SC II would be partially destroyed [12]. It might lead to the loss of active epitopes [13,14]. We subsequently found that anionic polysaccharides such as carrageenan and xanthan gum could form insoluble or soluble complexes with SC II through electrostatic interaction and significantly improve the gastric digestive stability of SC II under low pH conditions [15]. Therefore, it is speculated that the protective effect of these anionic polysaccharides on SC II during gastric digestion might have beneficial effects on the activity of SC II in the intestine. The above gastrointestinal digestive research of SC II was only explored in vitro, while the real digestive system in vivo is complex and variable, and there may be some differences between them [16]. In addition, to solve the problem of high salt content and time and energy consumption caused by salting-out and dialysis processes, we precipitated SC II from hydrolysate by the method of complex coacervation between SC II and anionic polysaccharides, and the complex of chondroitin sulfate-soluble undenatured type II collagen (CS-SC II) was finally obtained [17]. The collagen content of the complex was increased and the salt content was significantly reduced than the product directly freeze-dried after salting out. However, whether CS-SC II is superior to SC II in terms of gastrointestinal digestive stability and improvement of osteoarthritis is still unclear and urgently needs to be investigated. Viuda-Martos et al. [18] proved that anthocyanins, phenolic acids, and flavonoids were stabilized through the interaction with dietary fiber, thereby the bioavailability of phenolic and flavonoid compounds could be improved. Porporatto et al. [19] found that SC II and chitosan could form complexes through electrostatic interactions or hydrogen bonding, which can resist enzyme digestion. Moreover, co-administration of SC II with chitosan could alter the uptake and/or distribution of antigens, thus promoting an early anti-inflammatory environment in vivo. However, from their electrophoresis results, the protective effect of chitosan on SC II was not significant, and the effect of co-administration of SC II with chitosan was not explored in arthritis models.

In addition, the pathogenesis of OA is complex, mainly related to infection, metabolic disorder, trauma et al., in which immune response, inflammation, and signaling pathways may play important roles [20]. As to the effect of undenatured type II collagen on OA, most studies focused on behavior [21] and histopathological changes [4,22] in OA model animals, but its mechanism has not yet been deeply explored, for example, the role of undenatured type II collagen in regulating immune tolerance, inflammation, OA related signal pathways and key biomarkers.

Therefore, in this study, the gastrointestinal digestive characteristics of CS-SC II and SC II were investigated by in vitro and in vivo gastrointestinal digestion. SDS-PAGE and CD were used to determine the changes in α1 chain and triple helix structure of CS-SC II and SC II. The distribution of CS-SC II and SC II in vivo was analyzed through in vivo and in vitro imaging. Then, the OA model rats induced by sodium iodoacetate (MIA) were orally administrated with CS-SC II and SC II, and the improvement effect of CS-SC II and SC II on the symptoms of OA in rats was compared by the behavioral changes such as the width difference of the left and right joints, and the weight-bearing ratio of the right hindlimb. The effect of CS-SC II and SC II on the reduction of articular cartilage injury and the regulation of OA key biomarkers and signal pathway in rats were analyzed by histopathological observation, immunohistochemistry analysis, flow cytometry analysis and fluorescence quantitative polymerase chain reaction (qPCR). This study aimed to provide scientific guidance for the clinical application of CS-SC II and SC II.