雄激素性秃发（Androgenetic alopecia，AGA）是一种非瘢痕性、进行性毛发减少的疾病，表现为雄激素依赖性脱发障碍。在AGA中，睾酮代谢物双氢睾酮（dihydrotestosterone，DHT）作用于易感毛囊（hair follicle, HF），使HF的生长期缩短，休止期延长，进而导致HF微型化。

皮肤表面有各种各样的皮肤微生物群，包括细菌、真菌和病毒。皮肤微环境和微生物群落之间复杂的相互作用对于维持皮肤稳态至关重要。同时，皮肤微生物组还可影响皮肤的生理病理过程。HF与皮脂腺共同组成毛囊皮脂腺单位，形成了特殊的高脂环境。许多研究已经证实，在HF开口和上部存在大量细菌和真菌，在深层的毛球部位也存在少量菌群。毛囊及毛囊周围正常菌群的定植对维护皮肤稳态具有重要作用。如果出现菌群紊乱，可能打破原有的免疫豁免状态，导致炎症浸润引发多种毛发疾病，例如细菌毛囊炎，慢性毛囊疾病如斑秃和扁平苔藓等。因此，探究头皮菌群在毛发疾病中的作用和机制非常重要。

目前尚无研究揭示头皮菌群异常在AGA发病中的作用，本课题拟探索皮肤菌群在健康者及AGA患者中的差异，探究头皮菌群失衡在AGA的发病中的作用和机制，以期为后续治疗提供方向。

第一部分 雄激素性秃发小鼠模型模型的构建

目的：构建雄激素性秃发小鼠模型，并进行不同造模方式的比较。

方法：通过对6周龄C57/BL6小鼠进行脱毛后，采用以下几种方式构建AGA小鼠模型，包括皮下注射DHT（1mg/天），腹腔注射DHT（1mg/天），皮下注射睾酮（0.5mg/天）及腹腔注射睾酮（0.5mg/天），通过大体形态学和组织病理学观察小鼠的毛发生长情况、毛囊数量、毛发直径等。

结果：与空白组相比，各处理组小鼠毛发生长均受到了不同程度的抑制，其中以腹腔注射DHT组的毛发生长程度抑制最为显著。在造模过程中，小鼠背部皮肤未出现明显的红斑、渗出、糜烂等不良反应。

结论：在小鼠AGA模型中，腹腔注射DHT（1mg/天）可能是效果最佳的造模方式。

第二部分 头皮菌群在雄激素性秃发中的变化及头状葡萄球菌促进生发的作用

目的：探究头皮菌群在AGA患者中的变化，及研究优势菌对毛发生长的调控作用。

方法：采集健康者及AGA患者的皮脂内容物、毛囊、头皮的微生物群落，进行宏基因组测序。利用小鼠AGA模型，通过比较小鼠的毛发生长情况、毛囊数量等方式，探究了头状葡萄球菌对AGA小鼠模型毛发生长的作用。

结果：

①分析皮脂内容物菌群：与健康对照相比，AGA患者头顶部皮脂内容物菌群的alpha多样性没有显著改变；枕部皮脂内容物菌群的香农指数（Shannon index）显著降低。在门水平，AGA患者枕部皮脂内容物中放线菌门（Actinobacteria）富集，对照组中变形菌门（Proteobacteria）富集；在头顶部皮脂内容物中厚壁菌门（Firmicutes）在对照组富集。在属水平，有3个差异物种在AGA患者枕部皮脂内容物中富集，其中痤疮丙酸杆菌属（Cutibacterium）的丰度最高。有6个差异物种在对照组中富集，其中鞘氨醇单胞菌属（Sphingomonas）的丰度最高；而头顶部皮脂内容物中检测到葡萄球菌属（Staphylococcus）在对照组中富集。在种水平，痤疮丙酸杆菌（Cutibacterium acnes）等在AGA患者枕部皮脂内容物中富集，少动鞘氨醇单胞菌（Sphingomonas paucimobilis）等在对照组中富集；在头顶部，头状葡萄球菌（Staphylococcus capitis）等在对照组皮脂内容物中富集，球形马拉色菌（Malassezia globosa）等在AGA患者中富集。

②分析毛囊菌群：与健康对照相比，AGA患者枕部和头顶的毛囊菌群的alpha多样性没有显著改变。在门水平，枕部和头顶部毛囊中都检测到软壁菌门（Tenericutes）显著富集在对照组。在属水平，支原体（Mycoplasma）等富集在对照组枕部毛囊中；AGA患者头顶部毛囊中检测到茧蜂病毒属（Bracovirus）富集，链球菌属（Streptococcus）富集在对照组。在种水平，猪鼻支原体（Mycoplasma hyorhinis）富集在对照组枕部毛囊中；口腔链球菌（Streptococcus oralis）等富集在对照组头顶部毛囊中，AGA患者出现群聚性囊状病毒（Cotesia congregata bracovirus）富集。

③分析头皮菌群：与健康对照相比，患者枕部和头顶的头皮的alpha多样性没有显著改变。在门水平，AGA患者与健康对照相比，头顶部与枕部无差异菌群。在属水平，木糖氧化无色杆菌噬菌体（Phikmvvirus） 等富集在对照组枕部头皮；孪生球菌属（Gemella） 和马赛菌属（Massilia）富集在对照组头顶部。在种水平，假单胞菌病毒（Pseudomonas_virus_LKA1）富集在对照组枕部头皮；头状葡萄球菌（Staphylococcus capitis）和蒂莫内马赛菌（Massilia timonae）富集在对照组头顶部。

通过对优势菌的比较发现，相较于AGA患者，头状葡萄球菌（Staphylococcus capitis）在健康者的皮脂中和头皮表面的富集程度明显增高。健康者来源的头状葡萄球菌外用于小鼠AGA模型后，可明显促进小鼠的毛发生长，毛囊数量明显增多。

结论：头状葡萄球菌在AGA患者头皮微环境中的富集程度明显降低；头状葡萄球菌外用可促进AGA小鼠模型的毛发生长。

第三部分 头状葡萄球菌代谢产物富马酸相关衍生物促进毛发生长的作用与机制研究

目的：探究头状葡萄球菌代谢物对毛发生长的作用和机制。

方法：利用HUMAnN的MetaCyc差异功能分析及靶向中心碳代谢组学技术检测来源于AGA患者和健康人头皮微环境的头状葡萄球菌代谢产物如富马酸相关衍生物的变化。基于AGA小鼠模型，探究富马酸相关衍生物fumaric acid促进毛发生长的作用和机制。

结果：通过MetaCyc分析发现，头状葡萄球菌主要介导还原性三羧酸循环生物学过程。根据KO分析及EGGNOG功能注释推测，头状葡萄球菌与富马酸相关代谢途径密切相关。通过靶向中心碳代谢组学分析发现，AGA的头皮微环境可影响头状葡萄球菌富马酸相关衍生物fumaric acid的产生，富马酸相关衍生物fumarate acid能够促进AGA小鼠模型的毛发生长，这一过程与富马酸相关衍生物fumarate acid影响皮脂腺细胞分泌NGF及PGF密切相关。

结论：头状葡萄球菌代谢产物富马酸相关衍生物fumaric acid能够通过影响皮脂腺细胞分泌NGF及PGF，进而影响生发生长。

Androgenetic alopecia (AGA) is a non-scarring type of hair loss, with progressive loss of hair. It is an androgen-dependent hair loss disorder. In AGA, the anagen phase of hair follicles gradually shortens, while the telogen phase lengthens due to the dihydrotestosterone (DHT). DHT, as a metabolite of testosterone, could influence susceptible hair follicles and leads to follicular miniaturization.

The skin surface harbors microbiota, including bacteria, fungi, and viruses. The complex interactions between the skin microenvironment and microbiota are crucial for maintaining skin homeostasis. Simultaneously, the skin microbiome could affect the physiological and pathological processes of the skin. Hair follicle, forms the pilosebaceous unit with sebaceous gland, creating a high-fat microenvironment. Numerous studies have confirmed the presence of bacteria and fungi at the hair follicular opening and upper regions, and a smaller number of microbiome at the deeper hair bulb region. The colonization of microbiota in hair follicles and around hair follicles plays an important role in maintaining skin homeostasis. If the ecosystem was disturbed, it may contribute to disruption of immune privilege and inflammatory infiltration, which could lead to various hair disorders, such as bacterial folliculitis, chronic follicular diseases like alopecia areata, and lichen planopilaris. Therefore, it is important to explore how the scalp microbiota participates in hair cycle regulation and immune cell interactions, as well as the regulation in pathogenesis of hair disorders.

However, there is no literature revealing the role of the scalp microbiota in the pathogenesis of AGA. Therefore, our study aims to explore the differences in skin microbiota between healthy individuals and AGA patients, elucidate the mechanisms and clarify the role of microbiota in AGA pathogenesis, and therefore to provide the direction in AGA treatment.

Part I: Comparison of modeling methods for androgenetic alopecia in mice

Objective: To establish mouse model of AGA and explore the effects of different modeling methods for AGA in mice.

Methods: 6-week-old C57/BL6 mice were depilated, common induction methods for AGA mouse models were established, including subcutaneous injection of DHT (1 mg/day), intraperitoneal injection of DHT (1mg/day), subcutaneous injection of testosterone (0.5mg/day), and intraperitoneal injection of testosterone (0.5mg/day). The hair growth, follicle count, and hair diameter of the mice were observed through histopathological methods.

Results: Compared to the blank group, hair growth inhibition in all treatment groups were observed. As we found, the intraperitoneal injection of DHT group showing the most significant inhibition. During the modeling process, no allergic reactions such as erythema, exudation, or erosion were observed on the dorsal skin of the mice.

Conclusion: In the AGA mouse model, intraperitoneal injection of DHT (1 mg/day) may be the most effective modeling method.

Part II:  Metagenomics reveals the distribution of scalp microbiota in AGA and the hair-promoting effects of Staphylococcus capitis

Objective: To investigate the impact of scalp microbiota on AGA pathogenesis and the regulatory effects of bacteria on hair growth.

Methods: Metagenomic sequencing were conducted to explore the microbiome of sebum content, hair follicles, and scalp surface lipids in healthy individuals and AGA patients. Subsequently, we used the AGA mouse model and histopathological methods by comparing hair growth and follicle count to explore the regulatory effects of Staphylococcus capitis on hair growth in AGA mice.

Results: Analysis of microbiome of sebum content revealed that there were no significant changes in alpha diversity in the sebum content of the vertex region of AGA patients compared to healthy controls; however, the Shannon index of the occipital microbiota was significantly reduced. At the phylum level, Actinobacteria were enriched in the occipital region of AGA patients, while Proteobacteria were enriched in the control group. Firmicutes were enriched in the vertex region of the control group. At the genus level, three species were enriched in the occipital sebum content of AGA patients, with Cutibacterium exhibiting the highest abundance. Six species were enriched in the control group, with Sphingomonas being the most abundant. In the vertex region, Staphylococcus was enriched in the control group. At the species level, Cutibacterium acnes was enriched in the occipital region of AGA patients, while Sphingomonas paucimobilis was enriched in the control group. In the vertex region, Staphylococcus capitis was enriched in the control group, while Malassezia globosa was enriched in AGA patients.

In hair follicles, there were no significant changes in alpha diversity in the occipital and vertex regions of patients compared to healthy controls. At the phylum level, Tenericutes were significantly enriched in the control group in both the occipital and vertex regions. At the genus level, Mycoplasma was enriched in the occipital hair follicles of the control group, while Bracovirus was enriched in the vertex hair follicles of AGA patients. Streptococcus was enriched in the control group. At the species level, Mycoplasma hyorhinis was enriched in the occipital hair follicles of the control group, while Streptococcus oralis was enriched in the vertex hair follicles of the control group, while Cotesia congregata bracovirus enriched in AGA patients.

In the scalp surface lipids, there were no significant changes in alpha diversity in the occipital and vertex regions of AGA patients compared to healthy controls. At the phylum level, no differential microbiota was found between the vertex and occipital regions of AGA patients and healthy controls. At the genus level, Phikmvvirus was enriched in the occipital scalp of the control group, while Gemella and Massilia were enriched in the vertex scalp of the control group. At the species level, Pseudomonas_virus_LKA1 was enriched in the occipital scalp of the control group, while Staphylococcus capitis and Massilia timonae were enriched in the vertex scalp of the control group.

Therefore, by comparing dominant bacteria, we found that Staphylococcus capitis was significantly enriched in the sebum and scalp surface of healthy individuals compared to AGA patients. We collected Staphylococcus capitis from healthy individuals and applied to the AGA mouse model, we found it could significantly promoted hair growth and increased follicle counts.

Conclusion: The enrichment of Staphylococcus capitis in the scalp microenvironment of AGA patients is significantly reduced. Additionally, Staphylococcus capitis can promote hair growth in the AGA mouse model.

Part III: The role and mechanism of fumarate-related derivatives from Staphylococcus capitis in promoting hair growth and the review of clinical applications

Objective: To explore the metabolites involved in the regulation of hair growth by Staphylococcus capitis.

Methods: HUMAnN-based MetaCyc functional analysis and targeted central carbon metabolomics were used to investigate the production of metabolites such as fumarate-related derivatives by Staphylococcus capitis. We applied fumarate-related derivatives, such as fumaric acid, to the AGA mouse model, to clarify hair-promoting effects. Results: MetaCyc analysis of metagenomic sequencing revealed that Staphylococcus capitis primarily mediates the reductive tricarboxylic acid cycle (TCA). Based on KO analysis and EGGNOG functional annotation, fumarate-related metabolic pathways were enriched in Staphylococcus capitis. Targeted central carbon metabolomics confirmed that the scalp microenvironment in AGA may influence the production of fumarate-related derivatives, such as fumaric acid. Fumaric acid can promote hair growth in the AGA mouse model, by influencing sebaceous gland cell secretion of NGF and PGF, thereby regulating hair growth.

Conclusion: The metabolites produced by Staphylococcus capitis during the reductive TCA cycle, such as fumarate-related derivatives, can influence hair growth by affecting sebaceous gland cell secretion of NGF and PGF.