## 4 Discussion  
This study demonstrated that rats exhibited locomotive dysfunction in the acute phase after DMM surgery and in the chronic phase due to the development of KOA. Oral administration of BO improved this disability and suppressed the phosphorylation of ERK1/2 in the dorsal horn of the spinal cord. Studies have shown that the DMM rat model exhibits osteoarthritis changes within 4 weeks after surgery [17; 29]. Surgical intervention and KOA development induce knee pain and decreased latency to fall off the rotarod apparatus. The rotarod test is an established method to measure the ability of coordinated movement, and it can also be used in measuring pain-related locomotive disability [25]. Interestingly, in this present study, the rotarorod test revealed that the latency to fall off the rotarod apparatus decreased not only in the chronic phase when KOA changes have occurred but also in the acute phase a few days after the DMM surgery. This result suggests that the postoperative acute pain caused by the DMM surgery hampered the locomotive function of rats. No difference was observed in the rotarod test between each group 7 days after DMM induction, which may be due to the healing of the surgical wound. It has been reported that ERK1/2 phosphorylation in the dorsal horn of the spinal cord is involved in the development of pain in the postoperative pain model [21], and it has also been related to neuropathic pain and aggravated pain-related behavior [28; 30]. Therefore, ERK1/2 phosphorylation in the dorsal horn of the spinal cord may lead to not only chronic pain, but also pain sensation and allodynia through various nociceptive stimuli. The stage in this present study is divided into acute phase 1 day after DMM induction and chronic phase 4 weeks after the surgery. In both phases, the phosphorylation of ERK1/2 in the  
Figure 6: Expression of pERK1/2 and the rotarod performance test after the intrathecal injection of the MEK inhibitor U0126. (**A**) Immunoblot images of pERK1/2 and total ERK1/2 in the right dorsal horn of the spinal cord 1 day after surgery. (**B**) The quantified data of the immunoblotlot for pERK1/2 normalized to total ERK1/2. Each \(n=5\), \({}^{*}p<0.05\). (**C**) Walking time in the rotarod performance test. Each \(n=5\), \({}^{*}p<0.05\). All data are presented as mean \(\pm\) standard deviation. pERK, phosphorylated extracellular signal-regulated kinase; DMM, destabilization of the medial meniscus. dorsal horn of the spinal cord was accelerated, indicating acute and chronic postoperative pain conditions. Intrathecal catheter injection of U0126, MEK inhibitor against ERK1/2 phosphorylation, was able to improve the rotarod test performance (Figure 6C). The phosphorylation of ERK1/2 induced by MEK may play a crucial role in pain-related behavior in the DMM model as well as in the postoperative pain model [21] and the chronic constriction injury model [28]. Therefore, this present study has demonstrated a novel therapeutic effect of BO, wherein the oral administration of BO reduced postsurgical or KOA-associated pain by inhibiting the phosphorylation of ERK1/2. The rotarod test performed on day 1 showed that BO administration recovered the rotarod test results to 26.7 \(\pm\) 6.7 s (Figure 3); however, the administration of MEK inhibitor recovered the results to only 23.3 \(\pm\) 2.0 s (Figure 6). Therefore, BO may possess other analgesic mechanisms in addition to the inhibition of ERK1/2 phosphorylation. [PERSON] et al. [15] reported that BO inhibited IL-1\(\beta\) secretion as well as joint fluid retention in the KOA rat model. Among the crude drugs present in BO, _Astragalus_ root [31], _Glycyrrhiza_[32], and ginger [33] have been reported to exert analgesic effects against neuropathic pain. [PERSON] et al. [34] demonstrated the inhibition of the calcium-activated chloride channel TMEM16A using liquitiigenin derived from _Glycyrrhiza_.