Protective Effects of Korean Red Ginseng Against Oxidative Stress-Induced Damage in Human Hair

Abstract

Background

Oxidative stress causes fatal damage to follicular keratinocytes (FKCs) and is a common pathophysiology of many hair disorders.

Objective

This study investigated the protective effects of Red ginseng extract (RGE) and its main ginsenosides against oxidative hair damage using an in vitro organ model of human hair follicles.

Methods

We examined whether RGE and its constituent ginsenosides could prevent oxidative damage induced by H₂O₂ in FKCs by suppressing apoptosis and promoting hair growth.

Results

RGE and its main ginsenoside, G-Rb1, significantly inhibited reactive oxygen species production and apoptosis in FKCs. Furthermore, they effectively alleviated the inhibition of hair growth induced by oxidative damage and inhibited the transition of hair from the anagen to the telogen stage. The hair cycle and apoptosis were associated with the modulation of p53 and Bax/Bcl2 signaling.

Conclusion

RGE and G-Rb1 can effectively mitigate the oxidative damage caused by FKCs, thereby affecting hair growth and hair cycles.

INTRODUCTION

Hair growth is a significant contributor to scalp protection and aesthetics. Among the various factors for hair growth, the interaction between various components of hair follicles, such as the hair matrix composed of epithelial cells and the dermal papilla composed of mesenchymal cells, is critical¹,². The balance between the hair matrix and dermal papilla is related to hair formation and loss, and various types of alopecia occur when there is an imbalance³,⁴,⁵.

Oxidative stress is characterized by elevated intracellular levels of reactive oxygen species (ROS), which damage lipids, proteins, and DNA. It has been linked to a myriad of pathologies in various diseases⁶,⁷. Oxidative stress causes fatal damage to follicular keratinocytes (FKCs), which are rapidly proliferating cells⁸. Excessive ROS production is associated with alopecia. Cells from the balding scalp show increased sensitivity to oxidative stress, exhibiting decreased proliferation, increased senescence, and increased levels of hair growth inhibitory factors, such as TGF-β⁹. Patients with alopecia areata exhibit a substantial increase in the density, proliferation, and degranulation of perifollicular mast cells, which is expected to result in higher levels of ROS¹⁰. Furthermore, the excessive accumulation of ROS around hair follicles induces apoptosis in hair matrix keratinocytes during chemotherapy and radiotherapy, leading to hair loss¹¹,¹².

Red ginseng extract (RGE; the steamed root of Panax ginseng Meyer) is an herbal medicine actively studied for its antioxidant activity¹³. RGE protects against premature catagen development by restoring p53 and Bax/Bcl2 expression¹⁴. Furthermore, RGE has been reported as a potent regulator of hair growth¹⁴,¹⁵,¹⁶. A previous study in a model of antineoplastic chemotherapy-induced alopecia confirmed that RGE is mainly involved in the apoptosis of FKCs and exhibits an anti-apoptotic effect by modulating p53, Bax/Bcl2, and caspase-3 signaling¹⁴. RGE allows FKCs to form hair for a longer period, thereby exerting a protective effect on hair growth and the hair cycle¹⁴. Considering the role of ROS in the pathogenesis of various hair disorders, we aimed to identify the role of ROS in FKCs, the main targets of ROS in hair follicles. Specifically, this study investigated the effects of RGE and its main ginsenosides on hydrogen peroxide (H₂O₂)-induced oxidative hair damage in human FKCs and hair follicle organ culture models.

MATERIALS AND METHODS

Materials

The Korean Ginseng Corporation, located in Daejeon, South Korea, provided RGE extract prepared using a standardized and reproducible method. This extract was derived from the roots of 6-year-old red ginseng (Panax ginseng Meyer) cultivated in Korea. To create RGE, the red ginseng underwent steaming at temperatures of 90°C–100°C for 3 hours followed by drying at 50°C–80°C. The RGE extract was obtained from the red ginseng water extract through a triple extraction process using circulating hot water at temperatures of 85°C–90°C for 8 hours. The resulting concentrated extract retained 36% water content relative to its total weight. Analysis via high-performance liquid chromatography revealed the presence of major ginsenosides in the RGE, including G-Rb1 (7.44 mg/g), G-Rg1 (1.79 mg/g), G-Re (1.86 mg/g), G-Rc (3.04 mg/g), and G-Rb2 (2.59 mg/g) (Chengdu Biopurify Phytochemicals Ltd., Chengdu, China).

Isolation of hair follicles and FKCs in the anagen stage

Skin samples were procured from the occipital scalp of adult male patients diagnosed with alopecia who had undergone hair transplantation and provided informed consent. The scalp tissue underwent horizontal division at the junction of the dermis and subcutaneous fat layer, after which the hair follicles were carefully extracted from the dermis, ensuring retention of the intact hair bulbs and associated dermal structures, including the dermal papilla and connective tissue sheath. Only actively growing hair follicles meeting these criteria were selected for further experiments involving cell separation.

Under the magnification of a surgical stereomicroscope, the dermal papilla was meticulously excised by delicately gripping the top of the hair bulb with microsurgical forceps and gradually applying pressure to create a slanted incision into the connective tissue sheath at the base of the hair bulb. Subsequently, following the removal of the connective tissue sheath from the follicle where the papilla was detached, FKCs underwent treatment with trypsinase at 37°C for 5 minutes. They were then isolated from the outer root sheath as individual cells using a pipette and subsequently cultured. Second-to-third-generation FKCs obtained through lineage culture were utilized for subsequent experimental analyses.

Culture media

Human hair follicles were maintained in Williams E medium (Gibco, Waltham, MA, USA), which was enriched with 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco), 10 µg/ml insulin (Sigma, St. Louis, MO, USA), 10 ng/ml hydrocortisone (Sigma), and 2 mM L-glutamine (Gibco). On the other hand, FKCs were cultured in Keratinocyte Basal Medium (Invitrogen, Waltham, MA, USA), supplemented with bovine pituitary extract, human epidermal growth factor, insulin, hydrocortisone, gentamicin, and amphotericin B.

FKC culture and human hair follicle culture

In this study, second-generation and third-generation FKCs were utilized. For human hair follicles in the anagen stage, each well of a 24-well plate (Corning, Steuben County, NY, USA) contained one hair follicle, to which 500 µl of complete Williams E medium was added. The cultures were then maintained in an incubator at 37°C under an atmosphere of 5% CO₂/95% air. The wells were categorized into 4 groups: control, H₂O₂ single-treatment, RGE treatment, and saponin treatment. The control group received only the complete culture medium, while the H₂O₂ single-treatment group was exposed to a medium containing 1 mM H₂O₂, as previously documented. To the RGE treatment group, 500 µg/ml of RGE was supplemented to a medium containing 1 mM of H₂O₂, following the established protocol¹⁴,¹⁷,¹⁸. For the saponin treatment group, either 20 µM or 200 µM of saponin (G-Rb1/G-Rb2/G-Rg1/G-Rc/G-Re), identified as appropriate concentrations through preliminary studies using serial doses up to 500 µM and utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which revealed that these concentrations did not exhibit cytotoxicity in FKCs and promoted their growth, was added to the cells, and the cultures were maintained accordingly.

Measurement of ROS levels

Free radicals within the FKCs were visualized using TCS SPE confocal microscope (Leica Microsystems, Bannockburn, IL, USA) with the aid of DCF and MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific, Waltham, MA, USA) introduced into the cells. Subsequently, the outcomes were assessed utilizing Metamorph Imaging Software (Molecular Devices, San Jose, CA, USA).

Western blot analysis

Total protein was extracted from FKCs or human hair follicles utilizing a Protein Prep Kit (Qiagen, Hilden, Germany), and the concentration of protein was determined using the Bradford assay. Subsequently, 30 µg of protein was subjected to separation on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skim milk, followed by overnight incubation with primary antibodies against caspase-3/β-actin/eIF2α/CHOP/XBP1/BiP (Cell Signaling Technology, Danvers, MA, USA) or p53/Bax/Bcl2/p21/p27/Cyclin D/Cyclin E. Afterward, the membrane was washed 3 times for 5 minutes each with Tris-buffered saline containing 0.1% Tween-20 detergent.

Immunohistochemical staining

On the fourth day of hair follicle organ culture, 10–15 hair follicles were collected from each group for paraffin block preparation. These follicular tissues were embedded in paraffin and longitudinally sectioned at a thickness of 4 µM, with the resulting sections mounted onto slides. Following deparaffinization and rehydration, the sections were treated with a peroxidase-blocking agent (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 minutes, followed by blocking in a solution containing 10% normal horse serum (Vector Laboratories, Newark, CA, USA) and bovine serum albumin (Sigma-Aldrich, Burlington, MA, USA) in phosphate-buffered saline (PBS) for 1 hour. After washing with PBS, the sections were incubated overnight at 4°C with mouse anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibodies (StressMarq, Victoria, BC, Canada), followed by incubation with the corresponding secondary antibodies. The sections were then counterstained with 0.05% DAB and analyzed using conventional immunohistochemical staining methods (Image analySIS 3.2 software; Olympus, Tokyo, Japan). Six hair follicles per group were subjected to analysis.

TUNEL assay

FKCs were fixed in formaldehyde 24 hours post drug treatment on Chambered Cell Culture Slides (Falcon, Corning, NY, USA). After reaching the anagen stage on the 4th day of hair follicle culture, 10–15 hair follicles were collected, and paraffin blocks were prepared from these follicles. The hair follicles embedded in paraffin were longitudinally sectioned into 4-µM-thick tissues and mounted onto slides. Cell death assessment was conducted using the TUNEL assay with an ApopTag in situ apoptosis detection kit (Chemicon, Tokyo, Japan).

Analysis of the morphological changes in human hair follicles

In this research, the length of human hair follicles was determined as the distance from the base of the hair bulb to the tip of the hair shaft. Measurements of follicle length were conducted every 2 days utilizing a measuring scale affixed to the alternate lens of a stereomicroscope. Subsequently, the follicle cycle was assessed and categorized into stages: anagen VI (scored as 100), early catagen (scored as 200), mid-catagen (scored as 300), or late catagen (scored as 400).

Statistical analysis

Statistical tests for each variable were performed using SPSS software (version 20.0; SPSS Inc., Chicago, IL, USA). The parametric Student’s t-test was used to compare the differences between the control and experimental groups. Statistical differences were considered significant at p<0.05.

RESULTS

Effects of G-Rb1/G-Rb2/G-Rg1/G-Rc/G-Re on the oxidative stress-induced apoptosis of FKCs

Based on the concentration of H₂O₂ used in a preliminary study, we used 1 mM H₂O₂. Our results confirmed that H₂O₂ treatment increased the number of TUNEL-positive FKCs along with a significant increase in ROS levels (Fig. 1)¹⁶,¹⁹. Referring to the effective concentration of RGE in a previous study, pretreatment with 500 µg/ml of RGE significantly inhibited ROS production and apoptosis of FKCs induced by H₂O₂ (Fig. 1)¹⁴. Therefore, RGE may exert its protective effects on hair follicles by inhibiting ROS production and reducing oxidative damage.

Fig. 1. Protective effects of RGE on hair follicles by inhibiting H₂O₂-induced ROS production. (A) Results of the TUNEL assay revealed that pretreatment with 1 mM H₂O₂ significantly increased the apoptosis of FKCs, while pretreatment with 500 µg/ml of RGE significantly inhibited ROS production and H₂O₂-induced FKC apoptosis. (B) Pretreatment with 1 mM H₂O₂ increased p53/cleaved caspase-3 expression, while pretreatment with 500 µg/ml of RGE suppressed it.

RGE: Red ginseng extract, ROS: reactive oxygen species, FKC: follicular keratinocyte, PI: propidium iodide, H₂O₂: hydrogen peroxide.

^*p<0.05.

We also investigated the effects of G-Rb1, G-Rb2, G-Rg1, and G-Rc/G-Re, the main saponin components of RGE, on apoptosis of FKCs induced by oxidative stress with 1 mM H₂O₂. After pretreatment with 20 µM or 200 µM of ginsenosides (G-Rb1/G-Rb2/G-Rg1/G-Rc/G-Re), we found that G-Rb1 inhibited the apoptosis of FKCs most significantly (Fig. 2A). After treatment with H₂O₂ alone (1 mM), the expression of the apoptosis markers Bax and cleaved caspase-3 were significantly increased, while the expression of Bcl-2, an anti-apoptotic protein, was significantly decreased. Compared to that in the H₂O₂ alone treatment group, the expression of Bax/cleaved caspase-3 was significantly decreased, while the expression of Bcl-2 was increased in the RGE and G-Rb1 treatment groups (Fig. 2B).

Fig. 2. Protective effects of G-Rb1 on hair follicles by inhibiting H₂O₂-induced ROS production. (A) Among the main saponin components of RGE, G-Rb1 most effectively prevented the H₂O₂-induced apoptosis of FKCs. (B) Pretreatment with 1 mM of H₂O₂ increased cleaved caspase-3 and pro-apoptotic Bax expression and decreased anti-apoptotic Bcl2 expression, while pretreatment with 500 µg/ml of RGE or 200 µM of G-Rb1 restored the H₂O₂-induced effects on caspase-3 and Bax/Bcl2 expression.

H₂O₂: hydrogen peroxide, ROS: reactive oxygen species, RGE: Red ginseng extract, FKC: follicular keratinocyte.

^*p<0.05.

Effect of G-Rb1 on free radical production

We then analyzed the effects of pretreating FKCs with 200 µM of G-Rb1 or 500 µg/ml of RGE on the ROS production induced by 1 mM H₂O₂. We found that pretreatment with G-Rb1 or RGE significantly inhibited ROS overproduction (p<0.05). Furthermore, the expression of CHOP/xbp1/eIF2α/BIP, which are markers of endoplasmic reticulum stress, was significantly increased in the H₂O₂ alone treatment group; the expressions of these markers tended to decrease after pretreatment with G-Rb1 or RGE (Fig. 3A).

Fig. 3. Protective effects RGE and G-Rb1 on the production of free radicals and the expression of cell cycle-related markers in hair follicles. (A) DCF-DA staining and western blot were used to determine the expression of CHOP/xbp1/eIF2α/BIP, markers of endoplasmic reticulum stress. The expression of these markers significantly increased after pretreatment with 1 mM of H₂O₂, while their expression tended to decrease again after pretreatment with 500 µg/ml of RGE or 200 µM of G-Rb1. (B) The effects of oxidative stress on the cell cycle of FKCs induced by 1 mM of H₂O₂ were determined after pretreatment with 200 µM of G-Rb1 or 500 µg/ml of RGE. The expression of the cell cycle markers p21/p27 was significantly increased, while that of Cyclin D/Cyclin E was significantly decreased upon treatment with 1 mM of H₂O₂. Pretreatment with 200 µM of G-Rb1 or 500 µg/ml of RGE restored their expression.

RGE: Red ginseng extract, DCF-DA: 2’,7’-dichlorofluorescein diacetate, H₂O₂: hydrogen peroxide, FKC: follicular keratinocyte, DCF: 2’,7’-dichlorofluorescein.

^*p<0.05; ^**p<0.01.

Effect of G-Rb1 on the expression of cell cycle-related markers

The effects of oxidative stress (treatment with 1 mM H₂O₂) on the cell cycle of FKCs were analyzed after pretreatment of 200 µM of G-Rb1 or 500 µg/ml of RGE. The results showed that the expression of the cell cycle markers p21/p27 was significantly increased, while the expression of Cyclin D/Cyclin E was significantly decreased upon H₂O₂ treatment alone; pretreatment with G-Rb1 or RGE reversed these trends (Fig. 3B).

Effect of G-Rb1 on the length and cycle of human hair follicles

The effects of G-Rb1 and RGE on human hair follicle growth were investigated in a human hair follicle injury model induced with 1 mM H₂O₂ ¹⁹. On the second day of culture, the lengths of the hair follicles in the control, H₂O₂ alone, G-Rb1, and RGE treatments were 710.52±53.05 µm, 528.07±57.91 µm, 603.35±36.12 µm, and 626.24±49.72 µm, respectively; a significant difference in length was found between the control and H₂O₂ alone treatments (p<0.05). There was no significant difference in length growth among the H₂O₂ alone, G-Rb1, and RGE treatments (p>0.05). On the 4th day of culture, the lengths of the hair follicles in the control, H₂O₂ alone, G-Rb1, and RGE treatments were 1,136.59±89.32 µm, 669.64±57.80 µm, 809.15±77.47 µm, and 989.03±83.42 µm, respectively; there was a significant difference in length between the control and H₂O₂ alone treatments (p<0.01). Furthermore, there was a significant difference in growth length among the control, G-Rb1, and RGE treatments (p<0.05). On the 6th day of culture, the lengths of the hair follicles in the control, H₂O₂ alone, G-Rb1, and RGE treatments were 1,469.06±88.33 µm, 705.73±113.39 µm, 1,055.78±116.93 µm, and 1,132.66±105.89 µm, respectively. There was a significant difference in length growth between the H₂O₂ alone and G-Rb1 and RGE treatment groups (both p<0.01) (Fig. 4A).

Fig. 4. Effects of RGE and G-Rb1 on the growth length and cycle of human hair follicles. (A) RGE and G-Rb1 reduced the hair growth inhibition induced by pretreatment with 1 mM of H₂O₂. (B) RGE and G-Rb1 protected against premature catagen development after pretreatment with 1 mM of H₂O₂. All the values are the mean ± standard error of the mean.

RGE: Red ginseng extract, H₂O₂: hydrogen peroxide.

^*p<0.05; ^**p<0.01.

The effects of G-Rb1 and RGE on the human hair follicle cycle were investigated using a model of human hair follicle damage induced by 1 mM H₂O₂. In this study, hair follicles were categorized into anagen VI, early catagen, mid-catagen, or late catagen hair follicles, as described previously¹⁹. The hair cycle scores of the control, H₂O₂ alone, G-Rb1, and RGE treatments were 190.89±20.90, 330.69±25.89, 245.68±22.74, and 235.32±21.68, respectively. There was a significant score difference between the control and H₂O₂ alone groups (p<0.05), as well as between the H₂O₂ alone and G-Rb1 and RGE groups (p<0.05) (Fig. 4B).

Effect of G-Rb1 on cell death in human hair follicles

FKCs are among the most rapidly proliferating cells in the hair and are, therefore, highly affected by oxidative stress. Excessive ROS accumulation in the body during chemotherapy and radiation therapy induces FKC death and hair loss. TUNEL staining was performed to investigate the effect of G-Rb1 treatment on cell death in human hair follicles. Compared to H₂O₂ treatment alone, the number of TUNEL-positive apoptotic cells was significantly reduced in the RGE and G-Rb1 treatment groups (Fig. 5).

Fig. 5. Suppressive effects of RGE and G-Rb1 on the apoptosis of FKCs in human hair follicles. (A) Hair follicles were labeled with TUNEL reagent (green) and counterstained with PI (red). (B) Pretreatment with 500 µg/ml of RGE or 200 µM of G-Rb1 suppressed the apoptosis of FKCs induced by pretreatment with 1 mM of H₂O₂.

RGE: Red ginseng extract, FKC: follicular keratinocyte, PI: propidium iodide, H₂O₂: hydrogen peroxide.

^*p<0.05.

Effect of G-Rb1 on the expression of 8-OHdG

The molecule 8-OHdG is well recognized as an indicator of oxidative DNA damage. Compared with the control group, immunohistochemical staining showed that the expression of 8-OHdG was significantly increased throughout the epithelial cell layers, including the outer and inner hair bulbs and hair matrix in the H₂O₂ alone treatment group. In contrast, the expression of 8-OHdG was significantly decreased in the RGE and G-Rb1 treatment groups compared to that in the H₂O₂ alone treatment group. There was no difference in 8-OHdG expression between the RGE and G-Rb1 treatment groups (Fig. 6A). These results indicate that G-Rb1 and RGE significantly inhibited H₂O₂-induced oxidative DNA damage in hair follicles.

Fig. 6. Suppressive effects of RGE and G-Rb1 on oxidative DNA damage and the cycle of human hair follicles. (A) Representative photographs of 8-OHdG immunostaining to determine the degree of oxidative damage to the DNA. Pretreatment with 500 µg/ml of RGE or 200 µM of G-Rb1 suppressed the activation of 8-OHdG induced by 1 mM of H₂O₂. (B) Western blot results showed that pretreatment with 1 mM of H₂O₂ increased cleaved caspase-3 and Bax expression and decreased Bcl2 expression. Pretreatment with 500 µg/ml of RGE or 200 µM of G-Rb1 restored the changes in H₂O₂-induced caspase-3 and Bax/Bcl2 expression.

RGE: Red ginseng extract, 8-OHdG: 8-hydroxy-2'-deoxyguanosine; H₂O₂: hydrogen peroxide.

^*p<0.05.

Effect of G-Rb1 on the expression of apoptosis markers

The transition of hair follicles from the anagen to the senescent phase is a well-known apoptosis-driven process. We collected hair follicles after 4 days of human hair follicle organ culture and examined the changes in the expression of several apoptosis markers, the mechanisms of which are well-known during the transition from anagen to senescence. H₂O₂ treatment alone significantly increased the expression of caspase-3, p53, and Bax, whereas the expression of Bcl-2 was significantly decreased. In the RGE and GRb1 treatment groups, the expression of caspase-3, p53, and Bax significantly decreased, whereas the expression of Bcl-2 increased (Fig. 6B).

DISCUSSION

Hair loss negatively affects patients’ quality of life and is further aggravated by the lack of clinical improvement with currently known treatments²⁰. However, no effective treatment or prevention strategies for hair loss have been established yet. Free radicals are the major contributors to aging and several other diseases²¹. In dermatology, an increase in ROS levels causes gray hair, hair loss, and skin aging²². FKCs, which form hair, are one of the most rapidly proliferating cell types and are, therefore, highly affected by oxidative stress²³. Excessive ROS accumulation induces FKC cell death, leading to hair loss. Furthermore, increased neurogenic inflammation around hair follicles due to stress, chemotherapy, radiation therapy, or prolonged ultraviolet exposure is also known to cause excessive ROS accumulation²⁴,²⁵,²⁶,²⁷. ROS also induces cellular aging in normal hair follicles in androgenic alopecia, promoting hair loss progression. Hence, oxidative stress is actively being studied for its mechanisms of action and treatment of hair loss²⁸,²⁹,³⁰,³¹.

RGE is widely recognized as a food product for preventing hair loss in Oriental medicine, and its effect on promoting hair growth has recently been reported¹⁶. A previous study confirmed that RGE is mainly involved in the apoptosis of FKCs and exhibits an anti-apoptotic effect by modulating p53, Bax/Bcl2, and caspase-3 signaling, enabling FKCs to form hair for a longer period and showing a protective effect on hair growth and the hair cycle¹⁴. In a preliminary study, pretreatment of FKCs with 4-hydroperoxycyclophosphamide, a natural metabolite of the anticancer drug cyclophosphamide, significantly increased the number of TUNEL-positive FKCs, with a significant increase in ROS levels. The expression of p53/cleaved caspase-3 was also significantly increased (data not shown). To determine the effect of inhibiting ROS on the apoptosis of FKCs, we pretreated them with 500 µg/ml of RGE and 10 mM N-acetylcysteine, an inhibitor of ROS production. We found that the production of ROS and the number of TUNEL-positive FKCs were significantly reduced (data not shown). These results suggest that the accumulation of excessive ROS due to chemotherapy induces the death of FKCs, causing hair loss. Moreover, considering the role of ROS in the pathogenesis of various hair loss disorders, such as androgenic alopecia and stress-induced hair loss, we aimed to identify the role of ROS in FKCs, which are the main targets of ROS in hair follicles.

In this study, RGE and G-Rb1 significantly inhibited the death of FKCs, suppressed apoptosis markers, and increased the expression of anti-apoptotic proteins. Several studies involving human and animal models have shown that G-Rb1 is a major saponin that promotes hair growth³²,³³,³⁴. In addition, RGE and G-Rb1 significantly promoted hair growth and inhibited the transition from the anagen to the telogen phase. During this process, p53 in FKCs transforms into an active form that induces cell growth arrest and apoptosis. It then becomes associated with cytoplasmic or mitochondrial proteins, increasing the expression of Bax, a pro-apoptotic protein, and decreasing the expression of Bcl2, an anti-apoptotic protein.

As FKC death is a common pathophysiology in many hair disorders, the development of treatment and prevention strategies to prevent FKC death is of great clinical and economic value³⁵,³⁶,³⁷. In recent years, there has been an increasing interest in the development of novel, safe, and potent antioxidants from natural sources. Because FKCs are sensitive to oxidative stress, strategies to identify the cytopathological mechanisms of oxidative stress in FKCs are also of great value. Based on the results of this study, we expect to contribute to the development of drugs that can alleviate or prevent hair loss through the antioxidant effects of RGE.

In light of our results, future studies on the involvement of other ginsenosides besides G-Rb1 in the protection of RGE against oxidative stress-induced damage to human hair follicles, the existence of synergistic effects of multiple ginsenosides, and the antioxidant mechanisms of ginsenosides are warranted, which we plan to address.

In conclusion, RGE and its main ginsenoside, G-Rb1, effectively alleviated the inhibition of hair growth induced by H₂O₂ and inhibited the transition of hair from the anagen to the telogen stage. In addition, the hair growth cycle regresses through the modulation of p53 and Bax/Bcl2 signaling during apoptosis. Therefore, RGE and G-Rb1 can effectively mitigate the oxidative damage in FKCs caused by H₂O₂, thereby affecting hair growth and hair follicle cycles by allowing hair follicles to form hair for extended periods.