*2.5. The IA Administration of 7 rh Decreases Chondrocyte Apoptosis*

The results of TUNEL staining exhibited that 7 rh treatment prevented cell apoptosis in the ACLT-OA cartilage (Figure 5A). The apoptotic rate of chondrocytes in the articular cartilage of the ACLT-OA mice (22.4 ± 3.3%) was significantly more increased than in the sham + saline group (4.5 ± 0.7%, *p* < 0.001) and in the sham + 7 rh 13.8 nM group (1.0 ± 0.5%, *p* < 0.001). After 7 rh treatment at 5 weeks, the apoptotic rate of chondrocytes in the 7 rh-treated articular cartilage was significantly reduced to 7.6 ± 1.4% in the 7 rh 6.9 nM group and 5.8 ± 0.8% in the 7 rh 13.8 nM group, respectively (Figure 5C).

**Figure 5.** Effect of 7 rh on chondrocyte apoptosis in articular cartilage. (**A**) TUNEL staining in the articular cartilages of the sham control groups (sham+ saline and sham+ 7 rh 13.8 nM), ACLT, and two 7 rh treating groups. The arrows mean positive stain of TUNEL. (**B**) The representative micrographs of immunolocalized activated caspase 3. (**C**) Quantitative analysis of the apoptotic rate. (**D**) Quantitative analysis of the immunohistochemical staining of activated caspase 3. Each bar represents the mean±SEM of 12 samples in each group. (\*\* *p* < 0.01 versus the sham control groups; \*\*\* *p* < 0.001 versus the sham control groups; # *p* < 0.05 versus the ACLT group; ### *p* < 0.001 versus the ACLT group).

Caspase-3 is considered as the primary executioner for apoptosis; therefore, we used IHC staining of activated caspase-3 for the analysis of apoptosis activity. The results revealed that activated caspase-3 protein expression in the ACLT group was significantly increased compared with the sham control groups, but was significantly decreased in the OA-induced mice treated with 7 rh (both 6.9 nM and 13.8 nM) at 5 weeks (Figure 5B,D). These results showed that 7 rh significantly reduced chondrocyte apoptosis in ACLT mice.
