*2.6. The IA Administration of 7 rh Decreases mTOR Expression and Increases LC3 and Beclin 1 Expression*

To clarify if 7 rh treatment can rescue the autophagy effect in the ACLT-OA model, we examined the expression of autophagy markers, including mTOR, LC3 and beclin-1 in the articular cartilage (Figure 6). The results of IHC staining showed that the autophagy dysfunction in response to ACLT-OA induction was rescued by 7 rh. The expression of phosphorylated mTOR in the 7 rh treated group was significantly lower than that in the ACLT group (*p* < 0.05 in 7 rh 6.9 nM group and *p* < 0.01 in 7 rh 13.8 nM group). Meanwhile, the density of LC3 and beclin 1 in the cartilage of the 7 rh treated group was significantly higher than that in the ACLT group at 5 weeks (*p* < 0.05 in LC3 and *p* < 0.001 in beclin 1).

**Figure 6.** Effect of 7 rh mediated enhanced autophagy-related proteins in articular cartilage. (**A**) The representative micrographs of immunolocalized phosphorylation of mechanistic target of rapamycin (phospho-mTOR) in articular cartilage of the sham control groups (sham+ saline and sham+ 7 rh 13.8 nM), ACLT, and two 7 rh treated groups. (**B**) The representative micrographs of immunolocalized light chain 3 (LC3). (**C**) The representative micrographs of immunolocalized beclin-1. (**D**) Quantitative analysis of the immunohistochemical (IHC) staining of phospho-mTOR. (**E**) Quantitative analysis of the IHC staining of LC3. (**F**) Quantitative analysis of the IHC immunohistochemical staining of beclin-1. In quantitative analysis, each bar represents the mean±SE of 12 samples in each group. (\* *p* < 0.05 versus the sham control groups; \*\*\* *p* < 0.001 versus the sham control groups; # *p* < 0.05 versus the ACLT group; ## *p* < 0.005 versus the ACLT-OA group; ### *p* < 0.001 versus the ACLT group).

#### **3. Discussion**

The results of the current study are the first to show the central role of the Ddr1 inhibition in ameliorating OA progression in the ACLT induced OA model. By using a Ddr1 inhibitor (7 rh), we significantly slowed the articular cartilage degradation, improved the weight-bearing ability, and promoted the running endurance through treadmill tests at 5 weeks after ACLT. Histologically, the reduced expression of chondrocyte hypertrophic markers indicated the inhibition of chondrocyte terminal differentiation. Furthermore, 7 rh reduced the chondrocyte hypertrophic differentiation and apoptosis by enhancing autophagy.

Previous studies have indicated the similar biological behaviors of articular chondrocytes in OA progression with the chondrocytes of the growth plate in EO [2]. In the OA status, the articular chondrocytes become hypertrophic, with changes accompanied by the overexpression of the hypertrophic markers, including alkaline phosphatase, Col X, and MMP13 and subsequent chondrocyte mineralization and apoptosis, which is similar with the phenotype changes in EO [13]. It was already known that the chondrocyte differentiation and EO in the growth plate were regulated by the parathyroid hormone-related protein (PTHrP)-IHH feedback loop [14]. PTH 1–34, sharing the same receptors with PTHrP, was also reported to suppress the expression of Col X and IHH of hypertrophic chondrocyte in the fetal bovine growth plates [15]. In our previous study, we showed that IA injection of the PTH 1–34 at 3–5 weeks significantly attenuated the loss of proteoglycans and type II collagen, suppressed the expression of Col X, and reduced chondrocyte apoptosis in papain-induced OA in rats [3]. Similar results have also been reported in the systemic administration of PTH 1-34 [5] and in both surgical induced and spontaneous OA models [4]. These studies proved the concepts that treatments that can inhibit chondrocyte terminal differentiation in the growth plate and can also suppress chondrocyte apoptosis in articular cartilage and then inhibit the progression of OA.

Our recent research, investigating the role of Ddr1 in the regulation of EO, has indicated that the chondrocyte-specific Ddr1 knockout can delay the EO and accompany decreased chondrocyte proliferation, terminal differentiation, and apoptosis in growth plates of mice [8]. Accordingly, we hypothesized that the Ddr1 inhibitor (7 rh) may maintain the survival of articular chondrocytes and reduce the progression of OA. To test this hypothesis, we examined the efficacy of the IA injection of 7 rh on reducing the cartilage degeneration and inhibition of the chondrocyte apoptosis in the ACLT-induced OA model in mice. Our results indicated that the IA injection of 7 rh improved the function of the OA joint and reduced the OARSI score. We also found that 7 rh can decrease chondrocyte hypertrophic differentiation by inhibiting the expression of Col X, MMP13, and IHH. IHH signaling is not only essential for regulating normal chondrocyte proliferation and differentiation in the growth plate but also important in modulating chondrocyte terminal differentiation in OA cartilage. Lin A.C. et al. used genetically modified mice to exam the role of IHH signaling in OA chondrocytes and demonstrated that the expression of IHH was up-regulated by OA and was closely related to the severity of OA [13]. Moreover, the pharmacological inhibition of IHH signaling could decrease the hypertrophic differentiation of chondrocytes and ameliorate OA severity [13,14]. Accordingly, we reputed that the inhibition of the up-regulation of IHH signaling in OA plays a pivotal role in suppressing the degradation of cartilage.

The hypertrophy-like changes in chondrocytes secreted a number of proteins that are involved in tissue remodeling, and calcification has been associated with the early and late stage of OA [13]. The inhibition of chondrocyte hypertrophic differentiation was considered as a therapeutic target for OA progression. The Ddr2, a receptor tyrosine kinase, that can be activated by degrade type II collagen has been reported to play a role in chondrocyte differentiation in OA. The increased expression of Ddr2 was reported to be associated with OA progression and elevated MMP13 expression in the surgically induced OA in mice [14]. Moreover, Ddr2 modulated Runx2 activity and stimulated the expression of Col X in hypertrophic chondrocytes (ADTC5 cells) [15]. The role of Ddr1 in chondrocyte hypertrophic differentiation is still not clear. Col X and MMP13 were the mostly widely used markers for hypertrophic chondrocytes [13]. Apoptosis is also an important marker for detecting hypertrophic chondrocytes in

late staged OA cartilage. The TUNEL staining of late-staged human OA cartilage showed that the chondrocytes in the upper zone of cartilage undergo terminal differentiation and eventually die by apoptosis [16]. In the current study, 7 rh treatment significantly decreased the expression of Col X and MMP13 and also reduced chondrocyte apoptosis by decreasing the expression of activated caspase 3 in OA cartilage. These findings suggested that the IA injection of 7 rh reduced chondrocyte hypertrophic differentiation and apoptosis in the OA cartilage, thereby reducing OA progression.

Autophagy is an essential process in maintaining cellular metabolism and homeostasis. Emerging evidence suggests that the deregulation of autophagy is a crucial factor in the pathogenesis of OA [17,18]. Caramés, B. et al. studied the expression of autophagy regulator and chondrocyte apoptosis in the articular cartilage of human OA and the experimental OA, and the authors demonstrated that the autophagy regulators were reduced in the human OA and the aging-related or surgically induced OA in mice accompanied by an increase in chondrocyte apoptosis [17]. Recently, the same researchers used green fluorescent protein–light chain transgenic mice to detect autophagy activation in normal and aging cartilage and indicated that the autophagy regulators were reduced before the onset of cartilage degradation and the decrease in chondrocyte cellularity [18]. The mTOR signaling pathway plays a central role in regulating the initiation, processing, and termination of autophagy. The cartilage-specific deletion of mTOR upregulated autophagy and protected mice from OA [10]. In addition, autophagy activated by rapamycin (an inhibitor of mTOR) reduced the severity of experimental OA [19]. Similar to previous studies, our results showed significantly increased mTOR expression after experimental OA induction, indicating that the reduction in the autophagy process leads to articular cartilage degeneration [11,19]. Furthermore, we also found that beclin1 and LC3, the major indicators for autophagosome formation, were expressed in normal cartilage, indicating a functional autophagy process. However, the expression of autophagy markers (beclin1 and LC3) was significantly suppressed after ACLT, implying insufficient autophagy in OA cartilage. These findings indicated that articular cartilage degradation after ACLT was related to autophagy dysfunction. In this study, our results showed that the IA of 7 rh significantly restored autophagy function and further reduced the progression of OA, as shown by the reduced immunostaining of activated mTOR, the increased immunostaining of beclin1 and LC3, and histologically lower GAG loss and a lower OARSI score. Based on these findings, we suggested that the reduction in chondrocyte apoptosis, mediated by the activation of autophagy, may be a part of the mechanism of action of 7 rh on preventing OA progression.

In conclusion, our results showed that the IA injection of 7 rh reduced cartilage degradation in ACLT-induced OA animals. The inhibition of Ddr1 can reduce chondrocyte hypertrophic differentiation and chondrocyte apoptosis in the OA cartilage, as well as recover the autophagy function that was impaired by OA. We demonstrated that the inhibition of Ddr1 in articular cartilage could modulate OA progression and prevent chondrocyte apoptosis by promoting autophagy in OA cartilage. These findings suggested that 7 rh may be a potential disease-modifying drug to prevention OA progression.
