Treatment of androgenetic alopecia by exosomes secreted from hair papilla cells and the intervention effect of LTF

Androgenetic alopecia (AGA) is the most common cause of chronic progressive hair loss in men, and AGA has a severe negative impact on the quality of life and physical and mental health of patients.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is the most common cause of chronic progressive hair loss in men, 1 with a global prevalence of approximately 20%, 2 and AGA has a severe negative impact on the quality of life and physical and mental health of patients. 3In the face of the need to continuously improve their quality of life, it is crucial to develop new therapies to inhibit hair loss and increase hair proliferation.
The hair follicle is an intact cutaneous microorganism that exhibits cyclic activity after birth.Hair follicle renewal occurs in three stable phases, namely proliferation (anagen phase), degeneration (regression), and rest (resting phase). 4Androgens cause miniaturization of the hair follicle and inhibit hair growth by binding to the androgen receptor (AR). 3 Excessive androgen receptor activation in the scalp accelerates hair loss by shortening the anagen phase of the hair growth cycle and thinning and trimming hair follicles.Hair loss is the result of apoptosis-induced premature termination of hair follicle growth and degeneration, 5,6 and dermal papilla cells (DPC) in scalp hair are androgen target cells. 7though two FDA-approved drugs (finasteride and minoxidil) have been widely used in the market for the treatment of AGA, the results of pharmacological testing show that at least 30%-40% of the population is not suitable for the treatment of hair loss with minoxidil.Finasteride is unsuitable for all patients with hair loss, and both have significant side effects after taking the drug. 8,9anwhile, another treatment modality, hair transplantation, has achieved some results in some patients, but it also has the disadvantage of insufficient hair source and a cumbersome procedure.Given their limitations, there is a need to develop a more effective material to prevent hair loss.
In the treatment of AGA or hair regrowth, stem cell therapy is used as a new approach to treating hair loss, using autologous stem cells to improve hair regrowth, including reversing the pathological mechanisms that cause hair loss, regenerating hair follicles or using tissue engineering methods to create hair.Essential cells are isolated from scalp tissue biopsies and matured in vitro for injection into the patient.Studies have shown that autologous suspension injections of human follicular mesenchymal stem cells (HFSCs) specifically injected into the frontal scalp of AGA patients increased hair density in the treated area by 29%. 10 Stromal vascular fraction (SVF) cells enhance the viability of adipose-derived stem cells and, when applied to grafts, produce better results in hair transplantation and provide a rich source of ADSCs. 11In addition, the preparation of platelet-rich plasma (PRP) for injection into the scalp of patients with alopecia areata improves skin ischemia and increases the vascular structure around the hair follicle and is also effective in the treatment of female androgenetic alopecia (F-AGA). 12Traditional Chinese medicine is an integral part of medicine in East Asia, and herbs are commonly used to treat patients with alopecia areata.In recent years, herbal medicine has received increasing attention as a source of drugs for treating AGA. 13 Based on relevant studies, 14 we selected commonly selected prescriptions for the treatment of androgenetic alopecia Exosomes are cell membrane-derived nano-vesicles with unique cell and tissue penetration capabilities and small particle sizes that can easily cross biological barriers such as capillaries and the bloodbrain barrier.In addition, exosomes have good biocompatibility, are easily stored and transported, can improve drug stability, reduce drug dose, and can be actively targeted through surface modification. 15,16The herbal compounds produced in the herbal formulations have beneficial effects as exosome modulators in a variety of diseases, inhibiting pathological processes such as tumors, cardiovascular dysfunction, kidney disease, and liver disease.It was found that exosomes from treated mesenchymal stem cells treated with Buyang Huanwu Decoction had the ability to upregulate VEGF and Ki-67 expression and increase vascular density in tissues, and protect mice from stroke. 17Zhen Wu Tang regulates exosome secretion and inhibits the NF-kB/NLRP3 signaling pathway to reduce the inflammatory response in IgA nephropathy, thereby alleviating renal dysfunction. 18Similarly, exosomes have shown promise in hair restoration, as they contain potent cytokines and growth factors that promote hair growth.As exosomes derived from mesenchymal stem cells (MSC-Exos) have been shown to stimulate skin fibroblast proliferation and migration and regulate scar formation, 19 exosomes released by keratin-forming cells affect melanocyte pigmentation. 20erefore, we investigated the mechanism of efficacy and therapeutic potential of exosomes extracted after herbal intervention on hair papilla cells in androgenetic alopecia by studying the common drugs used in herbal treatment of androgenetic alopecia, screening, and composing herbal complexes to culture hair papilla cells.(2) LTF tincture preparation: take dried He Shouwu, angelica, Chuanxiong, and Poria each 20 g cypress leaf herbs, crushed, through 20 mesh sieve, according to 1:5 material to liquid ratio to add 500 g concentration of 60% ethanol solution ethanol maceration 14d, daily shaking 3-4 times, every 2 min.14d after taking the maceration solution filtrate, supplemented with 60% ethanol solution, adjusted to the prescribed amount and stirred well.After 14 d, the macerated solution was filtered out, supplemented with 60% ethanol, adapted to the required amount, and mixed well.

| Animal model establishment and treatment
Twenty-five 6-to 8-week-old SPF-grade C57BL/6 female mice, body mass 16 to 18 g, were reared adaptively for 1 week, the mice used for hair loss experiments were treated with a shaving device and hair removal cream to remove hair, and the area of hair removal area was about 3 cm × 3 cm.In the model group, 25 mice were randomly divided into control group, model group, minoxidil treatment group, healing formula group, and healing formula-DPC-EXO treatment group, 5 mice in each group.Blank control group (Control), negative control group (Model), and positive control group (Minoxidil and LTF) were set up in the experiment.Except for the common control group, the animals in each group were injected subcutaneously with testosterone propionate (DHT) (at a daily dose of 5 mg/kg) for 28 consecutive d to establish a mouse hair loss model.At the end of the modeling period, 0.2 mL of 5% minoxidil (Mil) and tincture of LTF were evenly applied to the observation area of the hair loss experiment.In the control and model groups, 0.2 mL of saline was injected subcutaneously at the back of the neck and administered once a day for 15 d.In contrast, 0.2 mL of exosomes stained with DIR was injected subcutaneously for 7 d.

| CCK-8 assays for optimal drug intervention concentrations
Hairy papilla cells were cultured routinely and inoculated uniformly in 96-well plates, divided into a control group, an LTF group with different concentrations, and a blank group, and 5 replicate control wells were set up for each group.After the cells were plastered, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, and 1.5 mg of LTF Chinese medicine extract were added to each group, respectively.After 24 h incubation, 10 μL of CCK8 solution was added to each well and incubated for 3 h at 37°C incubator, and finally, the absorbance was measured at 450 nm wavelength by enzyme standardizer.

| Preparation of in vitro culture of LTF-DPC-EXO
Female C57BL/6 mouse primary DP cells were isolated and cultured in successive passages (≤4 generations) to obtain sufficient cell volume, followed by injection in T175 culture flasks for culture.When the cell confluence reached about 60%, Chinese medicine extract was added to the medium.The concentration of Chinese medicine stimulating DPC proliferation was determined to use CCK-8, and the final effective concentration was determined.After the final effective concentration was determined, the DPC was treated, and the cell growth status was observed every 12 h.When the cell confluence reached 90%, the DMEM base medium containing no exosome serum was replaced, the cell culture supernatant was collected for about 2 d, and the DPC-EXO was obtained by low-temperature differential centrifugation.
We cultured hair papilla cells with different drug concentrations and hair papilla cells had a high proliferation rate at a concentration of 1.3 mg/mL (Figure 1A).We cultured the hair papilla cells in the fourth generation with therapeutic formula, and the exosomes were extracted in the sixth-generation cells.The exosomes were enriched and prepared by ultrafiltration and centrifugation, and the DPC exosomes were observed by electron microscopy with typical exosomal features (stereotype or hemispherical with one side depressed).

| Hair growth observation in live and somatic animal models tracing
The hair growth on the back of each group of mice was photographed and recorded.The DPC-EXO was labeled with DIR, and then the DPC-EXO was injected subcutaneously into the dorsal skin of the AGA mice model.The IVIS in vivo imaging technique was used to observe the AGA mouse model continuously for one week, and in vivo tracer shots were taken at 0.5 h, 24 h, 48 h, 72 h, and 96 h after the last injection, mainly to detect the fluorescent dye signal distribution and intensity of exosomes and somatic animal models tracing.

| Histopathological observation
At the end of the experiment on the 42nd day, the mice were put to death, and the skin tissue of their dorsal hair removal area was taken for HE staining to observe the hair growth.3 mice in each group were randomly selected to take the skin tissue of the experimental observation area, fixed with 4% paraformaldehyde solution, dehydrated step by step, transparent, and embedded.The sections were HE routinely stained to observe hair growth.4μm paraffin sections, routinely dewaxed with xylene, rinsed with tap water after all levels of ethanol to distilled water washing, routinely dehydrated, transparent, and sealed with neutral resin, observed under the microscope, and photographed.The relevant parts of the samples were collected and analyzed.

| ELISA assay
After 12 h fasting after the final administration, each mouse in each group was anesthetized intraperitoneally with pentobarbital sodium.Blood was taken from the eye and centrifuged at 3000 rpm for 10 min at 4°C to separate the supernatant.The levels of testosterone (T) and estradiol (E2) in the serum were measured using by ELISA kit method.

| Quantitative (q) RT-PCR to detect the expression of VEGF, AKT1, and CASP3 mRNA in each group
Total RNA was extracted from the back skin of mice, and a micro nucleic acid protein analyzer determined the sample RNA concentration and purity.The template cDNA was obtained by reverse transcription, and the target genes were amplified using SYBRGreen.A quantitative PCR instrument detected the fluorescence signal.Finally, the relative mRNA expression of the target genes was calculated using the 2-ΔΔCt method with GAPDH as the internal reference, and the primer sequences are shown in (Table 1).

| Western blot to detect the expression of VEGF, AKT1, and CASP3 protein in each group
The total proteins were extracted by lysis buffer, and BCA determined the protein concentration.Equal amounts of proteins were subjected to 5% SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane, and closed at room temperature for 3 h.Primary antibodies were added overnight at 4°C, and HRP-labeled secondary antibodies were incubated at room temperature for 2 h.ECL luminol was used for color development.Photographed protein bands with computer software and calculate grayscale values with ImageJ.The results were expressed as a multiple of protein expression relative to controls using β-Tublin as an internal reference.

| Statistical processing
Graph Pad Prism 8.0.2 was used for statistical processing, one-way ANOVA was used for comparing multiple data groups, and the twoway variance comparison was analyzed by the LSD method.The DunnettT3 test analyzed the variance disparity, which was considered statistically significant at p < 0.05 (Table 3).

| Results of CCK8 assay and the isolation and identification of therapeutic deconfusion-DPC-EXO
Using the western blot assay, we found that the DPC prepared by our group expressed the universal markers of exosomes CD63 and CD9, which were consistent with the exosome characteristics, and the concentration of exosomes after extraction was 1.494518256 (μg/ μL).The hairy papilla cells and exosomes are shown in (Figure 1B).96 h after the last injection of exosomes, the live tracing showed that there was still a strong fluorescence signal of exosomes in the dorsal skin of mice, and the sign of exosomes disappeared in the area where some of the mice had fully grown hair, and the effect of exosomes on hair growth (Figure 3).We measured the hair length and number of hair follicles in each group of mice and the results are shown in Table 2.

| Effects of serum hormone levels in mice with seborrheic alopecia model
After subcutaneous injection of testosterone propionate, the serum levels of hormone T in all experimental groups of mice were higher than those of the normal control group, and E2 was lower than that of the normal control group (Figure 4).The amount of testosterone in the serum appeared significantly higher, indicating a significant androgenic dysregulation in the androgenetic alopecia model mice.After pharmacological and exosomal interventions, all treated groups showed higher serum hormone T levels and slightly lower and lower E2 levels compared to the blank group.All treated groups showed a significant decrease in hormone T levels and a significant increase in E2 levels compared to the model group.All of them improved the hyperandrogenic status in blood to different degrees and restored the level of estradiol in mice, and the treatment effect was significant (p < 0.01).

| Effect of HE staining on hair follicle tissue recovery in seborrheic alopecia model mice
The pathological sections were observed under an ordinary light microscope 40× field of view (Figure 5): the total number of hair follicles in the control group rats was more, the diameter of the hair trunk was more significant than the unilateral inner hair root sheath, and the pigment content was more.In the model group, the total number of hair follicles was significantly reduced, some of the hair follicles were atrophied and disappeared, and only their accessory sebaceous glands were visible.The hair follicles were located in the superficial dermis without a connective tissue sheath, the hair matrices were atrophied, the hair papillae were shrunken, the hair ends were pestle-shaped, and the hair bulb was in a regressive or resting phase.The hair follicles were more densely distributed in each treatment group, the inner hair root sheath and melanin granules were visible, the hair shaft was formed, and the hair bulb showed the morphology of the anagen phase.

| Effects of VEGF, AKT1, and CASP3 mRNA expression in seborrheic alopecia model mice
Compared with the control group, the mRNA expressions of VEGF and AKT1 in the model group were significantly reduced, and the expression of CASP3 was significantly increased.The mRNA expression of VEGF and AKT1 in all treatment groups was lower than that of the control group and higher than that of the model group; the expression of CASP3 was higher than that of the control and lower than that of the model group.Compared with the LTF and Mil groups, the LTF-EXO group promoted VEGF and AKT1 and decreased CASP3 expression more significantly.(p < 0.01), as shown in Figure 6.

| Effect of VEGF, AKT1, CASP3, and CASP3 protein expression in mice with seborrheic alopecia model
To investigate the potential role of DPC-EXO in promoting hair growth, we tested the levels of VEGF, AKT1, and CASP3 proteins in the skin tissue of the back of mice.Protein blot analysis showed that the expression levels of the apoptotic protein CASP3 were significantly reduced, and the expression levels of the anti-apoptotic factors VEGF and AKT1 were increased after LTF-DPC-EXO intervention compared to the model group; LTF and Mil reduced the expression to some extent and restored the expression of VEGF and AKT1, with insignificant differences between the two groups (Figure 7).This suggests that LTF-EXO may inhibit the CASP3mediated apoptotic pathway contributing to hair regeneration.The TCMSP (Traditional Chinese Medicine Systems Pharmacol) database was used to analyse the main drug components of the Liao Tuo FAang.The results are shown in Table 3.

| DISCUSS ION
Exosomes are tiny particles containing cytoplasmic components that are released by cells during cell growth and cell membrane fission 21 and are an important component of paracrine signaling.They regulate the biological activity of recipient cells and mediate intercellular communication, allowing cells to grow and communicate through proteins, nucleic acids, and lipids that carry. 22It was found that in vitro induction induced proliferation and migration of human dermal papilla cells and secretion of VEGF and IGF-1. 23Exosomes of hypoxia-pretreated adipose mesenchymal stem cells (ADSCs) improve neoangiogenesis and survival of transplanted tissue by regulating VEGF/VEGF-R, 24 and ADSC-Exos isolated from ADSCs promote follicle regeneration in vivo. 25Injection of dermal papilla cell-derived exosomes into mice accelerated and prolonged hair follicle hair growth, stimulated β-linked protein, and increased expression of Shh factor.An increase in mean hair density and thickness was clinically found in 20 patients after 12 weeks of treatment using exosomes. 26e PI3K/AKT signaling pathway is a bridge between extracel-   MSCs possess cytokines that provide antiviral therapy through immunomodulation, suppression of inflammatory responses, etc. 40,41 Using stem cell-derived exosomes with the same high secretory activity and immunomodulatory effects, stem cells and their derived exosomes are therefore expected to be the best carriers of antiviral drugs in the viral microenvironment and potential tools for potential cell therapy.
However, our study also has some limitations.Firstly, after collecting exosomes of cellular origin, it was found that they were not produced in satisfactory quantities.Secondly, the exosome isolation process is very costly and it still takes time to harvest large quantities of high-quality exosomes at low cost.Thirdly, current exosomebased storage techniques (4°C for short-term storage and − 80°C for long-term storage) cause inevitable damage to exosome concentration, content, and integrity due to repeated freeze-thawing and ice production.

( 1 )
Drugs required for LTF: 15 g of He Shouwu, 15 g of Radix Angelicae Sinensis, 15 g of Rhizoma Chuanxiong, 15 g of Poria Cocos, 20 g of Phellodendron Bidentatae, taken as daily dosage for adults.Multistage membrane separation and concentration combined with the technology for extraction.Hair removal cream, testosterone propionate 2 g; 5% minoxidil 90 mL, injectable grade soybean oil 500 mL.
TTC AAG CCGTC GCTCA GCA GTA GTA ACG AAGGA AKT1 TGCAC AAA CGA GGG GAA TATAT CGTTC CTT GTA GCC AAT AAAGG CASP3 GAAAC TCT TCA TCA TTC AGGCC GCGAG TGA GAA TGT GCA TAAAT GAPDH CCTGG CAC CCA GCA CAAT GGGCC GGA CTC GTC ATAC TA B L E 1 PCR primer sequence information.F I G U R E 1 (A) Effect of LTF on hair papilla cell proliferation detected by CCK-8; (B) Electron microscopy of hair papilla cells and exosomes.

3. 2 |
Hair growth and in vivo tracing in mice with seborrheic alopecia modelAfter two weeks of treatment, mice in the therapeutic formula group, the minoxidil group, and the therapeutic formula-exosome group all showed significant hair regrowth, with the skin of the depilated dorsal area covered with black hairs.In contrast, the model group showed hair growth only locally on the skin, and the skin was almost hairless in most depilated areas.(Figure2) The live tracing technique showed the dynamic changes in the fluorescence signal of subcutaneously injected exosomes in the dorsal region.

F I G U R E 3
lular signals and intracellular responses and is involved in critical signaling pathways that regulate cell growth, proliferation, and differentiation.AKT1 is an important serine/threonine protein kinase that has a regulatory role in cell proliferation and apoptosis and acts as a major downstream molecule of the PI3K signaling pathway.When activated by extracellular signals, AKT1 can participate in positive or negative regulation of the PI3K/Akt signaling pathway through PI3K, thereby regulating AR protein expression. 27VEGF is widely present in different tissues of the body.In human skin, VEGF is made by hair papilla cells and is responsible for maintaining hair follicle growth and increasing the size of the follicle and the diameter of the hair shaft.In contrast, in hair follicle diseases such as AGA, the expression of VEGF is significantly reduced and even disappears from the scalp of patients with alopecia areata.VEGF can induce the proliferation, migration, or induce the formation of perifollicular vessels in DPCs, regulate the cyclic cycle of the hair follicle, and promote hair growth 28 ; and high expression of VEGF can significantly reduce DHT-induced apoptosis. 29During the degenerative phase of the hair follicle, degenerative apoptosis produced by the cells is activated by factors such as caspase drive Cell death-associated proteins inhibit the proliferation of dermal papilla cells, leading to premature maturation of the hair follicle and further hair loss. 30CASP3 plays an important role in apoptosis and is activated in apoptotic cells via apoptotic ligands and mitochondrial pathways, and is overexpressed in areas of alopecia in AGA patients early in the disease, indicating the presence of apoptosis and inflammation early in the disease.5a-DHT-induced apoptotic processes can block CASP3 activation by being inhibited by VEGF.Conversely, inhibition of the PI3K/AKT signaling pathway by activated CASP3 prevents the transition of hair follicles from the resting phase to the anagen phase, inhibits cell proliferation, induces apoptosis, and ultimately Hair follicles degeneration.Therefore, inhibition of CASP3 activation would help to reverse hair follicles entering the degenerative and abnormal anagen phases and induce hair regrowth in response to treatment.We first screened the remedies with good clinical feedback for the treatment of AGA and formed a Chinese herbal combination formula for the treatment of AGA, Liao Tuo Fang, based on the frequency of use of Chinese herbal medicines in conjunction with Chinese medical theory.By stimulating the hair papilla cells, which themselves have the ability to induce the hair growth cycle through paracrine action, the LTF enhances the activity of the hair papilla cells, thereby producing potentially specific exosomes.In this study, following topical application of testosterone propionate solution, serum levels of androgens were elevated in C57BL/6 mice and the growth cycle of dorsal hair was disrupted and inhibited in the early stages.In C57BL/6 mice injected with LTF-acquired DPC-EXO, the dynamics of the fluorescent signal reflected the process of restoration of hair growth in the dorsal skin of the mice, with melanin deposition at the site of loss of exosomal fluorescent signal, a change in skin color from flesh-pink to gray and black, and successful hair growth at a later stage, and a decrease in androgen levels in vivo.These results indicate that AKT1 Dynamic distribution of DIR-labeled exosomes revealed by in vivo tracer technique.TA B L E 2 Comparison of hair length and number of hair follicles in each group of mice.
expression in mouse skin tissues was significantly upregulated by the therapeutic degludec extract after drug and exosome interventions, and that activated VEGF and AKT1 had significant antiapoptotic effects in various cell types and were able to effectively inhibit the apoptotic process.This suggests that the activation of VEGF and AKT1 by DPC-EXO obtained through LTF may be mediated through the PI3K/Akt pathway to counteract CASP3-induced apoptosis.Previous studies have shown that platelet-rich plasma (PRP) and micrografts containing human hair follicle mesenchymal stem cells (HF-MSC) and adipose-derived mesenchymal stem cells (AD-MSC) contain a variety of cytokines such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF), which are involved in hair repair in AGA.31,32 Follicular augmentation cells and the number of hair follicles in PRP-, HF-MSC-and AD-MSC-treated scalp tissues were improved, and the mean number of hairs and hair density of patients were increased.33The results of animal experiments showed that MSC-Exos modulation of the TGFβ signaling pathway and PI3K/Akt pathway led to a nearnormal return of hair coverage on the back of mice.34These findings are generally consistent with our experimental results and F I G U R E 4 Serum hormone T and estradiol levels in mice of androgenetic alopecia model after testosterone propionate injection, compared with the model group *p < 0.01.F I G U R E 5 HE staining results of mouse skin histopathological sections.those of other treatments for AGA and show similar biomolecular pathways of action.It has been found that exosomes can be easily disseminated to different areas of the body via blood, cerebrospinal fluid, and saliva, and it can be used as a promising drug delivery system to study various physiological and pathological processes.35Loading drugs into cells, these hydrophobic or small molecule drugs can be incubated directly with the cells or exosomes, with hydrophilic compounds forming pores in the exosomes, allowing the hydrophilic drug to enter and then the cell to secrete the exosomes with the drug.36Paclitaxel (PTX) was isolated from the Chinese herb Taxus brevifolia and has anticancer activity.37PascucciL et al. demonstrated that mesenchymal stromal cells (MSC) can package and deliver PTX through their exosomes.38Wang et al. isolated the exosome M1-Exos from M1 phenotype macrophages that could activate the NF-κB pathway and enhance the anti-tumor activity of PTX.Wang et al. isolated the exosome M1-Exos from M1 phenotype macrophages that could activate the NF-κB pathway and enhance the anti-tumor activity of PTX.During the experiment, we observed that hair growth in the LTF-DPCs-EXO group was initially slower but faster in the later stages, which we speculate is due to the time required for activation of the exosomes and the drugs they are loaded with to enter the animals after being stored under frozen conditions (−80°C) and injected into the mice.Because exosomes are stable over time and temperature as drug delivery systems, 39 they are able to encapsulate drugs in lipid bilayer structures to prolong drug half-life and increase drug release stability.Thus, when the exosomes containing the components and biological information of the herbal compounds enter the bloodstream and become active again, they are able to show high stability and strong biocompatibility in the body, accelerating hair growth by inhibiting local inflammation, regulating signaling through expression, modulating immune responses and antioxidant effects.Stem cell therapy also has great therapeutic potential for other diseases.Clinical results have demonstrated the safety and efficacy of this therapeutic approach in a wide range of diseases.Studies have reported improvement in respiratory activity and worsening of symptoms following intravenous administration of MSCs to patients affected by coronavirus disease 2019 (COVID-19), suggesting that

F I G U R E 6 F I G U R E 7
Relative gene expression of VEGF, AKT1, and CASP3 in mouse skin tissue compared with the model.Analysis of protein expression of VEGF, AKT1, CASP3 in mouse skin using protein blotting.TA B L E 3 Composition of LTF Chinese yao and its main components.