# Hair Regeneration Effects of _Lespedeza bicolor_ Extract In Vivo and In Vitro
## 4 Discussion  
Alopecia, which has various causes or interactions, including aging, endocrine imbalances, and genetic factors, is a common hair disease, especially in adult males [2]. It is not clear whether the proliferation rate or apoptotic change of DPCs increase could affect alopecia. For example, the front of patients with androgenic alopecia (bald) has a low proliferation rate, inducing HF miniaturization, which could be caused by an increase in DNA damage and imbalance in the cell's recovery capacity [24]. HF growth cycles include three consecutive phases, namely, growth, regression, and relative quiescence. HFs are mini organs with complex structures and uncertain functions that develop in dynamic and alternately changing environments of various molecular signals. Minoxidil, which is widely used as a treatment for alopecia, has been used for more than 30 years to promote hair growth, but it has side effects such as decreased sexual function. Additionally, minoxidil has a concentration-dependent effect on the differentiation and proliferation of normal human keratinocytes [25] and induces significant DPC proliferation [26]. Figure 6: qRT-PCR analysis of genes related to cell cycle arrest (ERK, p21, p53), differentiation (p38), mitosis (CCNB1), proliferation (p57), and apoptosis (Bax, Bcl-2) after 60 h of treatment of human dermal papilla cells with LBE, PCA, and minoxidil. Representative data from each group are expressed as means \(\pm\) SDs (\(n=3\)). * \(p<0.05\) compared with the control groups. This study clearly demonstrates that LBE can induce DPC proliferation and promote hair regrowth in C57 BL/6 mice. The results showed that LBE treatment enhanced the proliferation rate of DPCs at 0.8-20 \(\upmu\)g/mL, whereas its cytotoxic effects on DPCs appeared at higher concentrations (100-500 \(\upmu\)g/mL), indicating its high safety profile. Therefore, the results of this study suggest that 0.8-20 \(\upmu\)g/mL of LBE may stimulate DPC proliferation and may be safe for subcutaneous use, especially for the scalp, which has anti-inflammatory properties and for the increase in DPCs [27]. Cytotoxicity to human DPCs appeared at high concentrations (100-500 \(\upmu\)g/mL), confirming that the safety margin was high. Therefore, the results of this study demonstrated that LBE can stimulate DPC proliferation and may be safe for subcutaneous use, especially on the scalp. The function of DPCs is regulated by the altered shape of hair in the anagen and telogen phases. According to [PERSON] et al. [28], in mouse experiments, on day nine after hair removal, all HF entered the final stage of the growth phase of the hair cycle. After hair loss, spontaneous regression begins to appear around the 17 th day and enters the resting phase (telogen) around the 20 th day. HF circulation in young mice follows a rather precise time scale [29]. Therefore, the hair growth-promoting effect of LBE was observed using 6-week-old mice. A similar pattern to that of hair circulation due to hair loss was identified. Topical application of 10 mg/mL LBE can control the early anagen phase and extend the mature anagen phase better than minoxidil as a positive control. Our previous study showed that ginseng berry increased DPCs by regulating the expression of Bcl-2, p38, and ERK, which was strongly correlated with promoting hair regeneration in vivo [30]. Previous studies showed that topical administration of LBE could act as a potent hair cycle regulator by promoting DPC proliferation. In this study, the G1 phase increased, and the S and G2/M phases decreased after LBE treatment for 60 h, regardless of whether LBE treatment affected the cell cycle of human hair DPC. These results indicate the potential for cell cycle arrest regulation by LBE. Thereafter, cell cycle-related gene expression regulation was confirmed through LBE, and LBE regulated the expression of p21, p38, and ERK genes involved in cell cycle arrest.