A Study On The Effects Of Melatonin On The Proliferation And Apoptosis Of Dermal Papilla Cells In Primary Hair Follicle Of Cashmere Goats

Cashmere goat is a kind of economic animal with diversified uses and the cashmere produced by cashmere has high economic value.the cashmere is produced by hair follicles,an accessory organ of the skin.Hair follicles are composed of more than 20 kinds of cells,which are mainly divided into epidermis and dermis.Among them,hair follicle stem cells belonging to the epidermis play an important role in the maintenance of hair follicle cycle and tissue formation.Dermal papilla cells exist in the dermis and are called "signal centers" of hair follicles.they control the proliferation and differentiation of epidermal cells of hair follicles by secreting many signal molecules such as cytokines and growth factors.at the same time,it regulates the development of hair follicles and further affects the growth of goat villi.Melatonin is an ancient and conservative indole with a highly lipophilic structure.it is easy to pass through the cell membrane and organelles to regulate metabolism inside and outside the cell and affect a variety of physiological processes of the body.including immune regulation,anti-inflammation,anti-apoptosis,inhibition of cancer cell proliferation and so on.Some studies have shown that subcutaneous implantation of melatonin in cashmere goats can increase the number of hair follicles and promote the growth of hair follicles and stimulate the elongation of hair follicle fibers.At present,the studies of melatonin in cashmere goats are mostly focused on villus growth and hair follicle elongation,but the role and molecular mechanism of melatonin in regulating the development of cashmere goat hair follicle cells are not clear.In this study,primary hair follicle dermal papilla cells of cashmere goats were treated with melatonin,and a variety of methods were used to explore the effects of melatonin on the proliferation and apoptosis of hair papilla cells,and to explore the molecular mechanism of melatonin regulating the growth of cashmere hair papilla cells.The test results are as follows:1.The primary dermal papilla cells of cashmere goats were obtained by microdissection and identified by microscopic morphological observation and cellular immunofluorescence staining.The results showed that the cells had agglutinative growth characteristics,flattened and polygonal,and α-SMA,VIM and other molecular markers of dermal papilla cells were positive by immunofluorescence.2.After dermal papilla cells were treated with different concentrations of melatonin,molecular biological techniques were used to detect the regulatory effects of melatonin on the viability,cycle,proliferation and apoptosis of dermal papilla cells.QRT-PCR technique was used to detect the effect of melatonin on the mRNA expression of Ki67 and PCNA,which are important proliferation markers of dermal papilla cells.The results showed that the proliferation ability of dermal papilla cells treated with melatonin was significantly enhanced,the proportion of cells in S phase was significantly increased,and late apoptosis was inhibited(P < 0.01),and the effect of 500pg/mL melatonin treatment was the most significant.Compared with the control group(0pg/mL),melatonin group(500pg/mL)could significantly up-regulate the mRNA expression of Ki67 and PCNA,which are important proliferation markers of dermal papilla cells.The above results showed that melatonin could significantly enhance the proliferation and inhibit the late apoptosis of dermal papilla cells.3.The mRNA libraries of 8 samples of dermal papilla cells in control group(concentration 0 pgshock ml,repeat number 4)and melatonin group(concentration 500 pg shock ml,repeat number 4)were successfully constructed and analyzed by RNA-seq sequencing.There were 421 differentially expressed genes between the control group and melatonin group,including 273 up-regulated genes and 148 down-regulated genes,GO analysis and KEGGPathway enrichment analysis showed that the differential genes were mainly concentrated in PI3K-Akt,MAPK,Cellcycle,FoxO,mTOR and other signal pathways,related to intracellular glucose metabolism,apoptosis,cell proliferation,transcription,cell migration and differentiation,stress,inflammation and so on.4.QRT-PCR assay was used to detect the effect of melatonin on the proliferation of dermal papilla cells and inhibition of mRNA expression of apoptotic genes.Western Blot method was used to detect the effect of melatonin on the activity of PI3K-Akt-mTOR pathway in dermal papilla cells.The results showed that after melatonin treatment,the mRNA expression levels of ZBED6 CL,TKTL1 and mTOR genes related to promoting cell proliferation and resisting inflammation were significantly higher than those in the control group,while melatonin treatment significantly decreased the phosphorylation level of PI3Kp85 regulatory subunit(p-P85)and Akt protein phosphorylation level(p-Akt),mTOR protein phosphorylation level(p-mTOR))in dermal papilla cells.PI3K-Akt-mTOR signal pathway is related to the proliferation and growth of dermal papilla cells.To sum up,this study demonstrated that melatonin promotes the growth of dermal papilla cells by mediating the expression of proliferation gene and anti-apoptosis gene mRNA through PI3K-Akt-mTOR signal pathway in dermal papilla cells,which provides a theoretical basis for further study on the effect of melatonin on hair follicle growth and development of Shanbei white cashmere goat.