## 3 Results
### 4 HR Increased OPG, RANKL, AP, and Runx2 Expression in Tissue  
The administration of 4 HR increased the expression of OPG, RANKL, Runx2, and AP in both sexes of the rats (Figure 4). Full length blots were shown in Figure S1. The relative expression of OPG, RANKL, Runx2, and AP in the male control group was \(1.00\pm 0.15\), \(1.11\pm 0.10\), \(1.12\pm 0.20\), and \(1.32\pm 0.42\), respectively. That in the male experimental group was \(1.90\pm 0.13\), \(1.78\pm 0.06\), \(1.83\pm 0.23\)  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Group** & **Male** & **Female** \\ \hline Control group & \(69.25\pm 1.51\%\) & \(68.68\pm 0.79\%\) \\ Experimental group & \(71.69\pm 0.88\%\) * & \(71.52\pm 1.85\%\) * \\ \hline \hline \end{tabular}  
* \(p<0.05\), comparison between the experimental group and control group. The values are presented as mean \(\pm\) SD.  
\end{table}
Table 1: Intergroup comparison of percentage root volume/total volume.  
Figure 3: The results of micro-computerized tomography (mCT) analysis. Compared to that in the experimental group, root resorption was more prominent in the control group. Particularly, central roots (arrow) showed extensive resorption in the control group. In a higher-magnification view of the mesial root, both groups showed external root resorptions (arrow heads). Interestingly, lacunas caused by root resorption were found not only on the compression side but also on the tension side.  
and \(2.34\pm 0.13\), respectively. The difference between the groups was significantly different for OPG, RANKL, Runx2, and AP (\(p\) = 0.014, 0.009, 0.015, and 0.016, respectively). The OPG-to-RANKL ratios for males were \(0.91\pm 0.17\) and \(1.07\pm 0.05\) for the control and experimental groups, respectively (\(p>0.05\)).  
The relative expression of OPG, RANKL, Runx2, and AP in the female control group was \(0.81\pm 0.04\), \(0.94\pm 0.13\), \(1.16\pm 0.75\), and \(1.52\pm 0.16\), respectively. That in the male experimental group was \(2.29\pm 0.38\), \(1.56\pm 0.13\), \(2.32\pm 0.60\), and \(2.01\pm 0.84\), respectively. The difference between the groups was significantly different for OPG and RANKL (\(p\) = 0.040 and 0.007, respectively). The OPG-to-RANKL ratios for females were \(0.87\pm 0.12\) and \(1.48\pm 0.34\) for the control and experimental groups, respectively (\(p=0.041\)).  
Upon histological analysis, the findings for active root resorption were similar in both groups. However, the area of active root resorption was more frequently observed in the control group (Figure 5a).  
Figure 4: The results of Western blot for tissue samples. (**a**) The representative blot images after 4-hexylesorcinol (4 HR) administration. Compared to that in the control group, the administration of 4 HR increased the expression of OPG, RANKL, alkaline phosphatase (AP), and Runx-2 (1: control; 2: experimental group). (**b**) Relative level of expression of \(\beta\)-actin for each protein was calculated. In addition, the OPG-to-RANKL ratio was calculated. When compared to the control group, the experimental group showed a significantly higher level of expression (\(\text{\textdagger}\,p<0.05\)).  
The thickness of the cementum was thinner in the middle area of the root surface. Discontinuous cementum was frequently found in the control group. The results of the immunohistochemical staining were in accordance with the results of the Western blotting (Figure 5b). Positive reactions to AP, OPG, RANKL, and Runx2 were mainly found in the periodontal ligament area.