Studies On The Mechanisms Of Basic Fibroblast Growth Factor In The Treatment Of Chemotherapy-induced Alopecia

Background and objective: Hair loss can be mainly divided into anagen effluvium and telogen effluvium.Anagen effluvium is mainly caused by chemotherapy and radiotherapy,etc.In the study,chemotherapy induced alopecia(CIA)was studied to understand the mechanism of anagen effluvium and to find the key cytokines or pathway.The incidence of tumor is increasing year by year,and the main treatment is surgical resection,radiotherapy and chemotherapy at present.CIA is a common chemotherapy side effect.CIA has a high incidence and great influence in tumor patients undergoing chemotherapy.However,there is a lack of radical treatment for CIA at present,so how to treat CIA is an urgent and difficult problem to be solved.Sonic hedgehog pathway plays a very important role in in development of hair and cycle transformation..Sonic hedgehog(Shh)is a key gene in its pathway.Shh is up-regulated in the early anagen and is one of factors regulate the development of hair and cycle transformation.Although no direct link has been found between chemotherapeutic-induced hair follicle cell apoptosis and SHH,the destruction of SHH signal has been identified as a key event in the CIA mechanism.Basic fibroblast growth factor(b FGF),also known as fibroblast growth factor-2(FGF-2).At present,some literatures have reported its effects on hair follicle proliferation and cycle transformation in hair follicle organ culture models,animal models and human experiments.But there has been no literature to report how it can take effect on CIA.We has tested a variety of growth factors in the process of hair growth and hair follicle cycle adjustment on CIA preliminarily,including KGF-2,b FGF,EGF,adipose stem cells culture medium and so on.We found some growth factors alone or together to promote hair growth,especially b FGF has a potential role for the treatment of CIA.Studies have shown that FGF can induce β-keratin and SHH pathways in mice,accelerating the transition of hair follicles from telogen to anagen.However,the specific regulatory effect and mechanism of b FGF in the process of CIA remain to be further studied.We intend to investigate the role of b FGF in hair follicle growth and cycle transformation in mice models of CIA by incutaneous injection of b FGF,and explore the mechanism of action of sonic hedgehog signaling pathway in it.Material and Methods: 1.Subjects: C57BL/6 mice.2.Evaluate the process of CIA in the mice model: A mouse model of CIA was constructed.C57BL6 mice of 6-8 weeks were divided into two groups.Group A was the blank control group.Group B was the CIA group.Rosin: paraffin = 1:1 was prepared for back depilation of mice.150mg/kg cyclophosphamide diluted by phosphate buffer saline(PBS)was intraperitoneally injected into group B on the 9th day after depilation,and the same amount of PBS was injected into group A.(1)digital camera was used to observe the hair follicle cycle on the back of mice(2)hematoxylin-eosin(HE)staining was used to observe the morphological changes of hair follicles at different time points(days 1,4,7,11,13 and 16)(3)ki67 was used to detect the proliferation of hair follicle stromal cells at different time points(days 1,4,7,11,13 and 16)by immunohistochemical technique.(4)Western blot detection of P53 protein levels 3.Integrated transcriptomic,proteomic and metabolomic bioanalysis to investigate the mechanism of CIA:(1)RNA-sequencing data of CIA on mice was downloaded from the GEO database for differential analysis,The results of the two groups of RNA-sequencing datasets were intersected by Venndiagram.(2)Establish a mouse model of CIA.C57BL6 mice of 6-8 weeks were divided into 2 groups.Group A was the blank control group;Group B was CIA group.Rosin: paraffin = 1:1 was prepared for back depilation of mice.On the 9th day after depilation,150mg/kg cyclophosphamide diluted by PBS was injected intraperitoneally into group B,and the same amount of PBS was injected into group A.Total proteins were extracted from the back skin on day 4 after administration,and i TRAQ technique was used to detect the expression of differential proteins.KEGG and GO enrichment were used to analyze the effects of CIA in various aspects such as biological function.Protein interaction analysis was performed on the differential proteins by String and Cytoscape website to find the key proteins that may be related to hair cycle.The intersection of RNA sequencing results and i TRAQ differential proteins was obtained by Venndiagram.The differential protein expression was verified by Western blot.(3)Based on RNA sequencing and proteomics results,non-targeted metabolomics sequencing and differential metabolite analysis were further performed on the dorsal skin of mice on day 4 after administration.4.Study on the mechanism of b FGF in treating CIA: Construct a mouse model of CIA.C57BL6 mice were divided into 3 groups at 6-8 weeks.Group A was CIA group.Group B was b FGF treatment group.Group C was treated with b FGF and GDC-0449 at the same time.(1)rosin: paraffin = 1:1 was prepared for the back depilation of mice.On the 9th day after hair removal,each group was intraperitoneally injected with 150mg/kg CYP configutated by PBS.PBS / b FGF / GDC-0449+b FGF were injected into the back of mice every day from the 4th day after CYP administration for 14 consecutive days.(2)Take pictures by digital camera every day to record the cycle changes of hair follicles of the back skin.(3)Collect the back skin of each group at the different time point after the treatment and make paraffin sections to observe the number,shape,distribution and cycle of hair follicles in each group by HE staining.(4)Collect the back skin of each group at the different time point after the treatment and make paraffin sections.The expression level of Ki67 was detected by immunohistochemistry to detect the proliferation ability of hair follicle stromal cells.(5)The back skin of mice on the 10 th day after treatment was collected,and the expression levels of key proteins in the hedgehog pathway were detected by Western blot.Results: 1.Hair follicle cells may be affected more than other time points on the 4th day after administration in the CIA model.(1)Gross observation showed that the hair removal in CYP group almost started from the 4th day,and the neck was the most significant,which could be used as the starting point for observation of hair removal.(2)HE staining showed that the number of hair follicles on the fourth day of the CYP group was significantly reduced,the morphology was significantly changed and the arrangement was disordered.(3)Immunohistochemistry showed that the degree of Ki67(+)hair follicle cell on the 4th and 7th day is more than on other time points which showed that proliferation on the 4th and 7th day of the CYP group were affected more than on other time points.(4)Western blot showed that the expression of P53 protein in the CYP group was higher on the 1st day and the 4th day than on the 7th day which showed that the degree of apoptosis of hair follicle cells on the1 st day and the 4th day may more serious than other time points.2.Pathways related to cell metabolism may mediate the occurrence of CIA.CTPS may be used as one of the biomarkers to judge the severity of CIA.The SHH pathway may be the key pathway in CIA.(1)In the two RNA sequencing dataset,compared with the control group,the changes at the RNA level in the CYP group on the second day of administration were more significant than those on the first day.There were 24 genes simultaneously up-regulated and 59 genes simultaneously down-regulated in the two dataset,and the down-regulated genes accounted for a larger proportion.(2)At the 4th day of administration,there were 220 differential proteins in protein level,including 76 up-regulated proteins and 144 down-regulated proteins,with a larger proportion of down-regulated proteins.(3)On the 4th day of administration,there were 680 differential metabolites in metabolic levels,of which 458 were down-regulated and 222 were up-regulated,and down-regulated metabolites accounted for a larger proportion.3.b FGF accelerated the proliferation and transformation of hair follicles in mice model of CIA: Gross observation and HE staining showed that on the third day after treatment,hair follicles of mice’s back in both groups were in the catagen of malnutrition with no significant differences.On the 10 th day after treatment,most of the back hair follicles of the control group were still in the telogen of malnutrition,while some of the b FGF group had entered anagen.On the 14 th day after treatment,back hair follicles in the control group entered the early and middle anagen,while those in the b FGF group entered the late anagen.The immunohistochemistry results of Ki67 showed that the expression level of b FGF group was higher than in the control group at the 10 th and 14 th days after treatment.4.b FGF may play a role in CIA through the Shh pathway: The changes in the expression levels of key factors in the Shh pathway were detected,and it was found that the expression levels of SHH,PTCH1,GLI1 and GLI2 were increased in the b FGF group compared with those in the CYP group at the 10 th day after treatment.GDC-0449(Vismodegib)+b FGF group showed that the expression of PTCH1 is less than b FGF group.But compared with CYP group,the differences of GLI1 and GLI2 were not sttistically significant.Conclusion: 1.Hair follicle cells may be affected more than other time points on the 4th day after administration in the CIA model.2.Pathways related to cell metabolism may mediate the occurrence of CIA.CTPS may be used as one of the biomarkers to judge the severity of CIA.Shh pathway may be a key pathway in the CIA process.3.b FGF accelerated the proliferation and transformation of hair follicles in mice model of CIA.4.b FGF took effect in CIA through the Shh pathway.