Clinical Observation Of Therapeutic Effects Of The Combination Of A Microneedle Therapy And External Application Of 5% Minoxidil Tincture On Male Androgenetic Alopecia And Research On The Molecular Mechanism In Relevant Signaling Pathways

Introduction: Androgenetic Alopecia(AGA)is a clinically common alopecic disease that primarily occurs in 20-55-year-old men.With this disease,hair is lost from both sides of the forehead,where fine and thin hair gradually extends to the top of the head,and a high forehead is formed by an m-shaped receding hairline;or hair is lost from the top of the head.The skin of the area of hair loss is smooth with fine lanugos and its secretion of grease may increase.The condition of AGA is generally less severe than that of male AGA.In most female cases,hair is gradually lost from the top of the head and the frontotemporal area is generally not involved with a slow course of alopecia.According to histopathological examinations of AGA,it can be seen that in the area of hair loss,anagen hair follicles decrease,while telogen hair follicles increase;during the later period,volumes of hair follicles greatly reduce,the density of hair follicles decreases or even to zero,and there are fibrotic changes of connective tissue around hair follicles.Although AGA doesn’t affect people’s health,as a serious impediment to meeting patients’ aesthetic demands,it still attracts extensive attention because of its aesthetic demands.There are many therapies for AGA,including oral administration of finasteride,external application of minoxidil,the photoelectric therapy and hair transplantation etc.,but no single therapy is highly effective.Now many scholars are focusing on improvement of therapeutic effects of AGA.Though people look forward to the value of the microneedle therapy with minoxidil for hair loss treatment,its research is rare.AGA’s pathogenesis is that on the top of head,hair follicles gradually atrophy,volumes of hair follicles reduce,terminal hair follicles are gradually converted into lanugo follicles and hair follicles disappear in the end,caused by increase of androgen and 5α reductase type Ⅱ ihigh n blood circulexpression of ation and local androgen receptors.Wnt/β-Catenin pathway is the signaling pathway that is most closely related to the attack of AGA and plays an important role in inducing generation of hair follicles and promoting growth of hair follicles.JAK-STAT is a new-found signaling pathway that is closely connected with hair growth in recent years.By experiments,scholars found that local application of small-molecule inhibitors of JAK-STAT signaling pathway could make hair follicles enter into the anagen rapidly and promote hair growth.However,there are few studies relenting to it.The Longnon-coding RNA(Lnc RNA)is a non-coding RNA with a length of more than 200 nt.As it doesn’t encode protein,it was regarded as the “noise” in genetic transcription and not taken seriously.However,recent research shows that Lnc RNA contains rich information and has close relation to occurrence,development,prevention and treatment of human diseases.As research on Lnc RNA hasn’t been involved in experiments about AGA,it is significant to study relations between Lnc RNA and AGA.At present,literature reports of most studies on AGA treatment are evaluation of clinical effects with some qualitative explanation and mechanism research mainly focuses on animal experimental studies with a lack of deep research on quantitative and molecular mechanisms of human tissue.Therefore,we designed this experiment to observe the therapeutic effect of the combination of the microneedle therapy and 5% minoxidil on male AGA by external application.Its therapeutic effect and safety were observed by subjective and objective assessment and histopathological studies.Then genes that were differentially expressed in Lnc RNAs,m RNAs and related signaling pathways(Wnt/β-Catenin and JAK-STAT pathways),which were closely related to AGA,were identified by gene chips and PCRArray to find out possible pathogenesis.Afterwards,changes of m RNAs and protein levels of most differentially expressed molecules in relevant signaling pathways of scalp tissue before and after treatment were tested to provide a theoretical basis for clinical effectiveness.Materials and methods:1.Objects of studySixty 20-60-year-old(Mean 37.4±1.5 years old)male volunteers with Norwood-Hamilton III-VI Androgenetic Alopecia and a medical history of 1-30 years(Mean 8.1±0.5 years)were recruited.Exclusion criteria: patients who suffer from a clear systemic disease,infection or ulceration of scalp or other skin diseases;patients who have systematically applied glucocorticoid or immunosuppressors within four weeks;patients who have received local external application or oral administration of drugs or other systemic therapies within six months;non-compliant patients.Before participation,volunteers were informed of risks,benefits and possible postoperative complications and signed both an informed consent and an agreement of photo usage.2.Preparation before treatment and the therapeutic process(a)Preparation before treatmentEstablish personal files;take photos in a designated room;keep photos of subjects’ head;examine densities and diameters of hair of fixed areas of hair loss with the hair detection system.After one-hour anesthesia on treated regions,they were cleaned with normal saline and disinfected with 75% alcohol.(b)Therapeutic processVolunteers were randomly divided into three groups and each group consists of 20 people.The first group only received external application of 5% minoxidil;the second group got a single microneedle therapy;and the third group underwent the combination of the microneedle therapy and external application of 5% minoxidil.Volunteers received 8 times of microneedle therapy in 6 months with an interval of 3 weeks.The depth of the microneedle was 1-2mm and volunteers underwent 2-3 cycles per time.In the third group,volunteers received the microneedle therapy and 2ml Minoxidil Tincture at the same time.In the group with 5% Minoxidil Tincture,volunteers had external application of about 1ml 5% Minoxidil Tincture twice a day for six months.Volunteers who only received external application of minoxidil were paid a return visit once three weeks.During each visit,each volunteer’s adverse reactions were recorded in detail.In each group,5~6 subjects accepted pathologic sampling of scalp of hair loss before and after treatment voluntarily.The ethical approval number relenting to this research is: KLS[2017]No.136.3.Therapeutic evaluationSubjective evaluation from doctors and subjects;Objective evaluation of curative effects included photo taking with digital cameras before and after treatment and during return visits and evaluation of hair densities and diameters with the hair detector system;Evaluation and prognosis of adverse reactions;histological examinations,paraffin section making of scalp tissue before and after treatment,HE staining,observation with optical microscope for comparison of numbers and morphologic changes of hair follicle before and after treatment between groups.4.Preliminary screening and pathway analysis of long non-coding RNAs and their co-expression m RNAs in AGAComparison of expressions of Lnc RNA and m RNA in areas with and without hair loss of male volunteers with AGA and predictive analysis on pathways relenting to female AGA were carried out(where two signaling pathways were selected for follow-up experiments).Then,florescent real-time quantitative PCR was used to further confirm chip detection results.Volunteers included ten 30-60-year-old male patients with AGA of Norwood-Hamilton V-VI.Three pairs of samples of areas with and without hair loss were collected to conduct gene chip detection and the rest seven pairs of samples were applied to subsequent verification.5.Expression differences of scalp tissue of hair loss before AGA treatment,normal scalp tissue and scalp tissue of hair loss after AGA treatment in Wnt/β-Catenin and JAK-STAT signaling pathwaysIn the combination group,Comparisons of molecule expression in Wnt/β-Catenin and JAK-STAT signaling pathways between volunteers’ areas with and without hair loss before and after AGA treatment were carried out and florescent real-time quantitative PCR was used to further confirm experimental results of PCRArray.Volunteers for comparison consisted of six 30-60-year-old male patients with AGA of Norwood-Hamilton V-VI.After treatment,tissue in areas of hair loss on the top of the head was sampled.Three sets of samples of tissue of hair loss before treatment,normal tissue and tissue of hair loss after treatment were used for Gene pathway gene pathway examinations and the rest seven sets of samples were applied to subsequent verification.6.Changes of differentially expressed molecules in Wnt/β-Catenin and JAK-STAT signaling pathways of scalps before and after AGA treatment in each groupFlorescent real-time quantitative PCR,Westernblot and immunohistochemistry technique were used to further examine expression changes of differentially expressed molecules(FZD3,CXCL9 and IL2RA)and molecules closely related to AGA(β-Catenin,LEF-1 及TGF-β1)in Wnt/β-Catenin and JAK-STAT signaling pathways before and after AGA treatment.7.Statistical analysisSPSS18.0 was applied to statistical analysis of data where P<0.05 indicated statistical difference.Results:1.Results of clinical observationThe combination group showed the best curative effect.According to doctors’ subjective evaluation,hair loss of 35% of volunteers were significantly improved and that of 60% of volunteers showed moderate improvement.According to volunteers’ subjective evaluation,hair loss of 85% of volunteers presented improvement of more than 50%.By comparison between conditions before and after treatment,after treatment,the terminal hair density increased by 55.7 ± 31.1 /cm2 and the average diameter of terminal hair increased by 21.1 ± 15.3μm.In respect of clinical manifestation,after treatment,hair loss on the top of volunteers’ head was obviously improved with significant increase of hair density.According to histopathological examinations,before treatment,there was little anagen hair in each group,while after treatment,hair follicles increased in each group with significant increase of anagen hair.During the return visit six months after treatment,volunteers’ hair was maintained well and only a small amount of hair was lost.Among three groups,adverse reactions showed no significant difference and volunteers’ adverse reactions could be relieved naturally without treatment.In the group of microneedle therapy,morbidities of scalp infection and lymphadenectasis were high.2.Results of preliminary screening and pathway analysis of long non-coding male-AGA-related RNAs and their co-expression m RNAs(a)Screening results of specific expression of expression profile of Lnc RNAs and their co-expression m RNAsBy gene chips,40173 Lnc RNAs and 20730 m RNAs were examined,in which 2143 Lnc RNAs and 2821 m RNAs showed differential expression.Compared with normal scalp tissue,tissue with hair loss had 770 Lnc RNAs of up-regulated expression,1373 Lnc RNAs of down-regulated expression,2141 m RNAs of up-regulated expression and 680 m RNAs of down-regulated expression(Times of differential expression >2.4).(b)Pathway analysis of co-expression m RNAs of Lnc RNAsKEGGpathway enrichment analysis was adopted to conduct pathway analysis of co-expression m RNAs of Lnc RNAs.Results showed that compared with normal scalp tissue,tissue with hair loss had 53 pathways that had relations with up-regulated co-expression m RNAs and 11 pathways connected with down-regulated co-expression m RNAs.JAK-STAT pathway from the up-regulated signaling pathways and Wnt/β-Catenin pathway from the down-regulated signaling pathways were selected for subsequent research.(c)Verification on specially expressed Lnc RNAs and their co-expression m RNA：Florescent real-time quantitative PCR was used to further confirm expression levels of differentially expressed Lnc RNAs and their co-expression m RNAs.We selected five types of Lnc RNAs(G001747,G012997,RP11-818024.3,RP11-76908.2 and G036413)and five types of co-expression m RNAs(PRAC2,KRTAP20-1,ARL6IP1,SPDYE4 and STARD9)from chip results.Results showed that compared with scalp tissue of hair loss,in normal tissue,among the five types of Lnc RNAs,expressions of G001747 and G012997 were up-regulated and those of RP11-818024.3,RP11-76908.2 and G036413 were down-regulated;among the five types of m RNAs,expressions of PRAC2,KRTAP20-1 and ARL6IP1 were up-regulated and those of SPDYE4 and STARD9 were down-regulated.Results of florescent real-time quantitative PCR were consistent with those of chips.3.Expression differences of scalp tissue of hair loss before AGA treatment,normal scalp tissue and scalp tissue of hair loss after AGA treatment in Wnt/β-Catenin and JAK-STAT signaling pathways(a)Expression results of related genes of scalp tissue of hair loss before AGA treatment,normal scalp tissue and scalp tissue of hair loss after AGA treatment in Wnt/β-Catenin and JAK-STAT signaling pathwaysPCRArray results showed that compared with areas of hair loss,there were 16 up-regulated genes and 2 down-regulated genes in Wnt/β-Catenin pathway and 10 down-regulated genes and no up-regulated gene in JAK-STAT pathway in normal areas.Compared with areas of hair loss,there were 8 up-regulated genes and 3 down-regulated genes in Wnt/β-Catenin pathway and 2 down-regulated genes and 17 up-regulated genes in JAK-STAT pathway in areas of hair loss after treatment.By comparison among normal tissue,tissue of hair loss before treatment and tissue of hair loss after treatment,they had no common down-regulated gene and 4 common up-regulated genes in Wnt/β-Catenin pathway,where FZD3 showed the largest expression difference;they had no common down-regulated gene or up-regulated genes in JAK-STAT pathway;6 genes that were significantly down-regulated in tissue of hair loss before treatment compared with the normal tissue showed significantly increased expression in tissue of hair loss after treatment,where CXCL9 and IL2 RA presented the biggest expression difference.(b)Differential expressions of FZD3,CXCL9 and IL2 RA in tissue with and without hair lossFlorescent real-time quantitative PCR was further used to examine expressions of FZD3,CXCL9 and IL2 RA in tissue with and without hair loss.Results showed that the expression of FZD3 in normal tissue was higher than that in tissue of hair loss;expressions of CXCL9 and IL2 RA in tissue of hair loss were higher than those in normal tissue with significant statistical differences of expression.4.Changes of differentially expressed molecules in Wnt/β-Catenin and JAK-STAT signaling pathways before and after AGA treatment(a)Changes of m RNA levels of FZD3,CXCL9,IL2 RA,β-Catenin,LEF-1 and TGF-β1 before and after AGA treatmentIn the group of single application of minoxidil,FZD3 and β-Catenin increased after treatment with statistical significance;in the group of single microneedle therapy,expressions of CXCL9 and IL2 RA increased after treatment with statistical significance and expressions of other genes showed no difference;in the combination group,expressions of FZD3,β-Catenin and LEF-1 significantly increased,while that of TGF-β1 significantly reduced after treatment.Expression differences were more significant in groups of single application of minoxidil and single microneedle therapy.Expressions of CXCL9 and IL2 RA showed no difference before and after treatment,but reduced after treatment.(b)Changes of protein levels of FZD3,CXCL9,IL2 RA,β-Catenin and LEF-1 before and after AGA treatmentImmunohistochemistry results indicated that in the group of single application of minoxidil,expressions of FZD3,LEF-1,CXCL9 and IL2 RA decreased after treatment without significance of expression changes before and after treatment;in the group of single microneedle therapy,expressions of FZD3,LEF-1,CXCL9 and IL2 RA increased but without significance of expression changes before and after treatment;in the combination group,expressions of FZD3 and LEF-1 increased and those of CXCL9 and IL2 RA decreased after treatment where expression changes of all indexes excluding IL2 RA had no significance.Westernblot results indicated that compared with the condition before treatment in single microneedle threatment group and combination threatment group,protein expressions of FZD3,β-Catenin and LEF-1 increased.In the group of single minoxidil application,all the indexes decreased,the expression of proteinβ-Catenin and LEF-1 were significant different between groups.In the combination threatment group,the expression of FZD3 was significant different before and after treatment.Conclusion:1.The combination of a microneedle therapy and external application of 5% Minoxidil Tincture could treat Androgenetic Alopecia effectively and the combination showed better curative effects than a single microneedle therapy or single external application of minoxidil.2.Compared with normal tissue,expressions of Lnc RNAs and their co-expression m RNAs showed significant differences in AGA tissue of hair loss.The relation between differential genes and AGA still needs a further study and may play a great role in diagnosis of AGA and evaluation of curative effects on AGA as a new biomarker.3.Up-regulated expression in Wnt/β-Catenin pathway and down-regulated expression in JAK-STAT pathway could inhibit hair loss.High expression of FZD3 could promote hair growth and its possible mechanism was that binding of Wnt5 a protein and FZD3 protein enhanced expression of LEF-1 to activate transcription of related target genes.Morbidity of AGA was related to CXCL9 and IL2 RA,indicating that occurrence of AGA is closely associated with autoimmune response and imbalance of immune regulation.4.The combination of a microneedle therapy and external application of 5% Minoxidil Tincture could enhance m RNAs and proteins of FZD3 and reduce m RNAs and proteins of IL2 RA and CXCL9 in scalp tissue of AGA,suggesting that the combination could treat Androgenetic Alopecia by activating Wnt signaling pathway and inhibiting JAK-STAT pathway.