Traditional Chinese Medicine Treatment for Androgenetic Alopecia Based on Animal Experiments: A Systematic Review and Meta-Analysis

Background In the present study, we systematically evaluated the effects of Traditional Chinese Medicine (TCM) on androgenetic alopecia (AGA) in rodent models (rats and mice) to provide potential evidence for the treatment of AGA by TCM. Methods Previous research papers focusing on the treatment of AGA by TCM were retrieved from various electronic databases (PubMed, Embase, The Cochrane Library, CNKI, Vip, Wanfang data, and CBM) up to October 30, 2021. Screening of the literature was performed independently, and data were extracted and assessed. A meta-analysis was performed using RevMan 5.3 software. Results When compared with the model groups, a group of C57BL/6 mice treated with TCM showed an increase in the total number of hair follicles (mean difference [MD] = 11.99, 95% confidence interval [CI] [5.94,18.03], P=0.0001), as well as a decrease in serum testosterone (T) level (MD = −1.10, 95% CI [−1.43, −0.78], P < 0.00001), skin discoloration time (MD = −2.93, 95% CI [−4.03, −1.84], P < 0.00001), and skin hair growth time (MD = −3.16, 95% CI [−4.16, −2.16], P < 0.00001). Terminal hair/vellus hair also increased in TCM-treated AGA animals (MD = 3.02, 95% CI [2.05, 3.98], P < 0.00001). No significant difference was found in serum estradiol (E2) level, skin tissue E2 level, or skin tissue T level between the TCM-treated group and the model group. Conclusion TCM can increase the total number of hair follicles and terminal hair/vellus hair ratio, and reduce skin discoloration time and skin hair growth time in AGA animal models. These effects may be related to the reduction of the serum T level in AGA animals. These conclusions need to be verified by high-quality studies as the current analysis may be affected by the number and quality of the studies identified.


Introduction
Androgenic alopecia (AGA) is a common form of hair loss both in men and women and is likely due to an excessive response to androgens. AGA is characterized by the gradual miniaturization of hair follicles, shortening of the hair growth period, and reduced number of hairs [1]. Clinical manifestations of AGA include smooth skin in the alopecia area, increased skin sebum, dandruff, and pruritus. Hair in the frontotemporal region and on top of the head becomes increasingly sparse or even fully detached with time [2]. AGA is the most common type of hair loss in clinical practice and accounts for 84.8% of all hair loss [3]. e condition can impact the usual interpersonal communications and social relations of those affected.
AGA can be treated by medication, hair transplantation, low-energy laser therapy, mesoderm therapy, autologous platelet-rich plasma injection therapy, and TCM treatment. Drug therapy is an important treatment for AGA patients, with both minoxidil and finasteride approved by the Food and Drug Administration (FDA) for treatment of alopecia [4][5][6]. However, the clinical efficacy of these drugs is limited and they are also associated with certain side effects. us, it is essential to develop safer and more efficient drugs to manage this condition.
Seborrheic alopecia has been studied in TCM for thousands of years, and was first recorded in the "Yellow Emperor's Inner Classic" work. Based on the diagnosis and treatment characteristics of the condition as a whole, as well as its different syndromes, TCM has achieved satisfactory curative effects in the treatment of AGA. Recently, researchers working on clinical trials in TCM have shown that TCM offers advantages in treating the AGA; however, the mechanism of the effect is not yet clear. erefore, we performed a meta-analysis to evaluate treatment effects of TCM in AGA in order to investigate possible treatments. Evaluation of animal models is necessary to improve the quality of in vivo experiments as it connects basic research and clinical trials [7].

Materials and Methods
We followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines [8] (Supplementary Table 1) for this systematic review and meta-analysis.

Types of Study.
We collected previously published research reports from various electronic databases up to October 30, 2021, regardless of language. We also manually reviewed the bibliographies of narrative review articles which could not be retrieved by electronic searches. Conferences proceedings, dissertation abstracts, and other unpublished data were also considered in the analyses to ensure that no potential studies were overlooked.

Types of Subject.
Rodents (mice and rats) were selected as our research objects irrespective of their sex or strain. e rodents were developed as models for alopecia or seborrheic alopecia, with no limits placed on the methods by which the rodent models were developed.

Types of Intervention.
e models developed for alopecia or seborrheic alopecia were all treated with TCM. Drug dosage and modes of drug administration were excluded from the analysis. Untreated model groups were used as controls, and experiments were not limited to a specific time period.

Types of Outcome.
Outcomes analyzed were total number of hair follicles, serum estradiol (E 2 ) level, serum testosterone (T) level, skin tissue E 2 level, skin tissue T level, skin discoloration time, skin hair growth time, and terminal hair/vellus hair.

Exclusion Criteria.
e following exclusion parameters were set (i) reprinted literature; (ii) original and unpublished data that could not be obtained and extracted after contacting the authors; (iii) in vitro studies; (iv) intervention measures including other nontraditional Chinese medicine treatments such as acupuncture.

Search Strategy.
We retrieved previous research papers from different electronic databases including the CNKI database, Wanfang database, Vip database, CBM, PubMed, Embase, and the Cochrane Library up to October 30, 2021 in both English and Chinese, irrespective of publication status. e collected research reports focused on the use of TCM in rodent models for the treatment of seborrheic alopecia. Chinese search terms included zhongyi, zhongyiyao, xiongjisuxingtuofa, zhiyixingtuofa, and fazhutuofa and English search terms included Traditional Chinese Medicine, rodents, androgenic alopecia, and seborrheic alopecia.

Literature Screening and Data
Extraction. Two independent investigators screened, extracted, and crosschecked the data across the entire study period. In case of disagreement, a third party was consulted to arbitrate. e following data were extracted using a pre-established data extraction table i) basic study information (first author, title, publication year, animal species, body weight, sample size, modeling method, and composition of TCM); (ii) specific details of the intervention measures, including the dosage and course of treatment; (iii) relevant information on the assessment of risk of bias risk; and (iv) outcome indicators and outcome measurement data.

Methodology Quality
Evaluation. Ten items recommended by the SYRCLE and animal experiments bias risk assessment tools were used for evaluation, including generation and/or application of distributed sequences; whether each baseline was identical; allocation concealment; whether animals were randomly allocated during the experiment; whether researchers were blinded; whether outcome evaluation was randomly selected; and whether a method of blinding was adopted for the results evaluator. It was also noted whether incomplete data were reported and whether study reporting was irrelevant to selective outcome reporting, as well as any other biases. e evaluation results were denoted as "yes", "no," and "uncertain", representing low risk of bias, high risk of bias, and uncertain risk of bias, respectively.

Statistical
Analysis. RevMan 5.3 software was used to perform the meta-analysis. e relative risk was calculated as an effective indicator to count the data, and the mean difference (MD) was used as an effective indicator for data measurement. Point estimates and 95% confidence intervals (CIs) of each effect quantity were given. Heterogeneity among included studies was analyzed using the χ 2 test, and the magnitude of heterogeneity was judged quantitatively using I 2 . If no obvious heterogeneity was found among the results of each study (I 2 ≤ 50%), a fixed-effect model was used for the meta-analysis. If there was significant heterogeneity obtained among the studies (I 2 > 50%), the source of heterogeneity was further analyzed. After excluding obvious 2 Evidence-Based Complementary and Alternative Medicine methodological heterogeneity, a random-effects model was used for meta-analysis, and a descriptive analysis was performed.

Literature Screening Process and Results.
e results of the literature screening are shown in Figure 1. Twenty-one literature searches were included in the qualitative research, and 15 searches were included in the quantitative analysis.

Evaluation of Methodological
Quality. Only three out of 15 studies used a random number table, and it was not possible to determine whether animals were randomly assigned during the rest of the experimental period. e selected studies did not mention allocation concealment or blinded methods. All collected research data reports were complete and unrelated to the results of selective reports (Table 2). Other sources of bias related to this study could not be determined.
① Whether the generation or application of allocation sequence was adequate; ② Whether each baseline was identical; ③ Whether the allocation concealment was Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine   sufficient; ④ Whether the animals were randomly allocated during the experiment; ⑤ Whether the researchers were blinded; ⑥ Whether the outcome evaluation was randomly selected; ⑦ Whether the result assessors were blinded; ⑧ Whether incomplete data were reported; ⑨ Whether the research report was irrelevant to the selective resulting report; ⑩ Whether there was no other bias.

Total Number of Follicles.
Five studies reported the total number of hair follicles, with subgroup analyses based on animal species [9,12,17,19,23]. e results of a randomeffect model meta-analysis showed that the total number of hair follicles increased in the TCM group in C57BL/6 mice (MD  (Figure 2).

Serum T Levels.
Five studies reported serum T levels with subgroup analyses performed according to animal species [11,13,14,18,20]. e results of the random-effects model meta-analysis showed that TCM reduced serum T levels in C57BL/6 mice (MD � −

Skin Tissue E 2 Levels.
Four studies reported skin tissue E 2 levels with subgroup analyses based on animal species [10,[12][13][14]. e results of the random-effects model metaanalysis showed that TCM had no statistically significant effect on skin tissue E 2 levels in C57BL/6 mice (MD � −0.

Skin Tissue T Levels.
ree studies reported skin tissue T levels with subgroup analyses based on animal species [12][13][14].
e results of fixed-effect model meta-analysis showed that TCM reduced skin tissue T levels in

Skin Hair Growth Time.
A total of three studies reported skin hair growth time with subgroup analyses based on animal species [10,12,13]. e results of the randomeffect model meta-analysis showed that TCM reduced skin hair growth time in C57BL/6 mice (MD � −3.16, 95% CI    (Figure 9).

Publication Bias.
A funnel plot was drawn based on the outcome index of the total number of hair follicles in the TCM group versus the model group ( Figure 10). e results demonstrated that the distribution of each study on both sides of the funnel plot was asymmetric, thus indicating the existence of publication bias.

Discussion
To the best of our knowledge, this article is the first to use a meta-analysis to conduct a systematic review of the effects of TCM in animal models of AGA. Previous meta-analyses by You [24] and Wang [25] 30 19.6% Heterogeneity:  2 = 0.00;  2 = 1.31, df = 2 (P = 0.52); I 2 = 0% Test for overall effect: Z = 3.20 (P = 0.001)  Evidence-Based Complementary and Alternative Medicine density, thereby improving hair loss [5,7]. However, owing to the publication bias in the outcome index, additional largescale and high-quality trials are required for further verification and to obtain more reliable evidence.

TCM Understanding of Hair Loss.
Chinese classic herbal formulas documented in the ancient Chinese medical literature have been widely used in AGA for centuries [24].
Examples include the "Yellow Emperor's Inner Classic" during the Warring States Period [26], "Treatise on Febrile Diseases" during the three Kingdoms period, and "Compendium of Materia Medica" during the Ming Dynasty [27]. For example, the "Yellow Emperor's Inner Classic" states that "the hair is outside the kidney essence, and when the essence and blood are sufficient, the hair is thick and shiny." is clearly suggests that the growth of hair is related to the rise and fall of the essence and blood in the kidney and the filling of qi and blood.  14 Evidence-Based Complementary and Alternative Medicine Modern TCM has summarized and developed previous knowledge of seborrheic alopecia, and believes that this disease is closely related to the spleen, lung, kidney, liver, phlegm stagnation, and dampness-heat [28][29][30][31][32][33]. Most practitioners divide seborrheic alopecia into four syndrome types as follows (i) damp and heat fumigation; (ii) blood heat and wind dryness; (iii) blood deficiency and wind dryness; and (iv) liver and kidney deficiency. e corresponding treatment methods can be summarized as invigorating the spleen, clearing dampness, and clearing heat; cooling blood and moistening dryness; nourishing blood and removing wind and moistening dryness; and nourishing the liver and kidney. A number of researchers have looked at the causes and treatments of AGA within the context of TCM. For example, Liu suggests that the etiology of AGA is responsible for pathogenic dampness, while basic pathogenesis is pathogenic dampness obstructing sweat pores [34]. Jia believes that AGA is caused by deficiency of the spleen and stomach, up-flooding of damp-turbidity, lung-wei insecurity, and disharmony of blood based on the theory of "spleen and stomach deficiency with lung disease" [35]. Xuan formed the academic process of "regulating yin and yang" in the diagnosis and treatment of skin diseases through clinical practice and scientific research exploration [36]. Yang showed that seborrheic alopecia is caused by heat from the stomach and intestine dampness fumigating upward to the head and face, thereby invading hair roots and causing the hair to fall out [37]. Treatment is based on heat clearing methods and removal of dampness, strengthening the spleen and expelling wind, supplemented by cooling blood and promoting blood circulation. According to the investigations of Wang, seborrheic alopecia is caused by problems in the spleen and kidney, suggesting that the spleen should first be strengthened and warmed to eliminate dampness, resolving phlegm, eliminating lipids, and increasing blood flow [38]. Finally, the studies of Cheng presume that dampness-heat constitution is the main cause for alopecia seborrheic, while stasisturbidity causing decayed hair is the main pathogenesis [33].    [39], caused by the action of androgens on the epithelial cells of genetically susceptible hair follicles in androgendependent areas [40]. e overall goal of AGA treatment is to arrest miniaturization and improve hair density. Systemic and local conversion of testosterone to dihydrotestosterone (DHT) by 5-α-reductase (5AR) enzyme is the main factor responsible for the underlying pathological mechanism in AGA [41,42]. erapeutic targets reduce DHT production, cause vasodilatory effects, trigger anagen, prolong anagen, and subdue inflammation [43]. Other factors that affect seborrheic alopecia include genetics, endocrine, mental stress, overuse of the brain, staying up too late, and improper diet, for example. However, the effects of androgen and 5α-reductase on hair follicles have always been considered the most important factors in AGA [11,44].
Regarding the signaling pathway of TCM for hair loss, a study by Dou [59] demonstrated that monomeric components in Chinese medicine can act through various pathways such as Wnt, MAPKs, PI3K/Akt, androgen receptors, Fas/FasL to initiate or terminate a program of related factors. ese factors can competitively inhibit or synergistically activate related receptors and link proliferation or apoptosis of the hair follicle cells or dermal papilla cells.
erefore, a number of hairs remain in the anagen phase and achieve the effect of hair loss treatment and hair loss prevention.

Limitations.
Our present study has a number of limitations. (i) All studies included in this analysis were sourced from the Chinese literature. As no manual search was  conducted, some of the literature may have been overlooked, and the research conclusions may therefore be subject to publication bias. Indeed, a funnel plot of the total number of the hair follicles showed publication bias. (ii) e included literature did not always mention allocation concealment and blinding methods, potentially presenting selection and implementation bias. (iii) ere was no consistency in the types of model animals included in this study, as well as inconsistency in the treatment cycles. Also, the interventions measured in the model groups were not similar, potentially leading to high heterogeneity and low research quality. (iv) Lack of reports of morbidity and mortality rates and adverse reactions of experimental animals were another significant drawback to this meta-analysis. Taking all the limitations into account, the conclusions of the present study require further corroboration.

Implications for Future Trials.
More scientific, rigorous, and reasonable high-quality animal experiments should be carried out in the future. e studies should include a standardized and scientific experimental scheme, describing in detail the methods of generating allocation sequences, as well as methods of concealing the random allocation and blinding. Further reports of adverse reactions and morbidity and mortality rates are also required. Selection of standardized markers with important effects on AGA should be undertaken so that the mechanism of action of TCM on AGA may be fully understood, allowing TCM to be applied in clinical practice.

Conclusions
Androgenic alopecia is a genetically predetermined disorder that is caused by an excessive response to androgens. e condition has attracted considerable attention in society as it has a substantial effect on the social confidence of individuals. e clinical efficacy of drugs approved by the FDA is limited, and these drugs lead to side effects. Although TCM has been used to address these issues for centuries, the mechanism of action of TCM remains unclear. Our present study is the first of its kind and demonstrates that TCM can be potentially used to treat the AGA, although further studies are essential to validate the results.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest regarding the publication of this manuscript.

Authors' Contributions
Yanbo Liang and Jing Yuan contributed equally to this work, and both acted as first authors. ey were responsible for the literature research, data extraction, and data analysis.
Nururshopa Eskander Shazada refined the first draft in English and provided language help. Jun Jiang participated in the revision of the first draft. Prof. Jianlin Wu provided guidance and financial support for this study. All authors reviewed the content and approved the final version.