Molecular Mechanism Of Collagen Type Ⅰ Synthesis Regulated By Melatonin Receptor 1a In Human Dermal Fibroblasts

Objective:The relationship between the circadian rhythm genes PER2 and CRY1 in Human Dermal Firbroblast（Normal,Human,Adult,HDFa）and skin aging were studied in this study.Meanwhile,the cell signaling pathways of Melatonin Receptor 1a（MT1）and circadian rhythm genes CRY1 and PER2 on skin aging were tested.Based on above pathways,natural plant extracts were screened,validated,and evaluated.Methods:1.In this study,by using small interfering RNA（si RNA）to interfere with the expression of rhythm gene CRY1,RT-PCR was used to detect the expression of rhythm genes CRY1,PER2 and aging-related indicators-matrix metalloproteinase1（MMP1）and Collagen Type 1（Collagen I）genes.2.After selecting the endogenous ligand of MT1 receptor,Melatonin（Melatonin）to act on HDFa,the expression of CRY1 and PER2 rhythm genes within 24 hours was detected,and the gene and protein expression levels of MMP1 and Collagen I were calculated.3.Living cell MT1 overexpression system:MT1 receptor is overexpressed on293T cells,and the level of live cell c AMP is detected by Glo-sensor experiment after melatonin.4.The gene expression levels of CRY1,PER2,MMP1 and Collagen I were detected after the activation of c AMP/PKA cell signal transduction pathway activator Forskolin and inhibitor H-89 respectively.5.GPCRome experiment was used to screen Epiphyllum extract,Glo-sensor experiment was used to detect the relationship between ephemeral extract and MT1receptor,and RT-PCR,immunofluorescence,Elisa were used to detect the expression of CRY1,PER2,Collagen I after Epiphyllum extract treated HDFa cells.Results:1.The relationship between rhythm gene and collagen type I synthesis:In the small interfering RNA knockdown experiment,when si CRY1 was transfected for24 h,the expression level of CRY1（p<0.0001）gene was significantly reduced,which proved that the knockdown model was established.When CRY1 was knocked down,the m RNA level of PER2（p=0.003）increased significantly,while that of MMP1（p=0.0019）and Collagen I（p=0.0111）decreased and increased respectively.2.Effect of melatonin on collagen type I synthesis:melatonin acted on HDFa and m RNA levels were detected by RT-PCR.The results showed that CRY1 was decreased（p<0.0001）,PER2 was increased（p<0.0001）,MMP1 was decreased（p=0.0022）and Collagen I was increased（p<0.0001）.3.Establishment of MT1 overexpression system:MT1 receptor was overexpressed in 293T cells,and c AMP of 293T cells was increased by Glo-sensor detection after melatonin treatment.4.Signal transduction pathway and molecular mechanism of CRY1,PER2 and MT1 receptor cells:HDFa cells treated with 5μM Forskolin can up-regulate PER2（p<0.0001）,down-regulate MMP1（p<0.0001）,and up-regulate Collagen I（p<0.0001）;10μM H-89 can up-regulate CRY1（p=0.0245）,MMP1（p<0.0001）,and Collagen I（p=0.0001）after acting on HDFa cells.5.Effect of Epiphyllum on rhythmic genes CRY1 and PER2:Epiphyllum extract organic phase（EPI-DMSO phase）and Epiphyllum extract aqueous phase（EPI-H₂O phase）at different concentrations:0.125%（v/v）,0.5%（v/v）,after 24 hours of exposure to HDFa cells,RT-PCR detection found that the transcription level of PER2in Epiphyllum extract aqueous phase（EPI-H₂O phase）0.125%（v/v）group and Epiphyllum extract aqueous phase（EPI-DMSO phase）0.5%（v/v）group increased（p=0.0116,0.0412,respectively）;The transcription level of CRY1 was decreased in the Epiphyllum extract aqueous phase（EPI-H₂O phase）0.5%（v/v）and Epiphyllum extract organic phase（EPI-DMSO phase）0.125%（v/v）groups（p=0.0088,0.0137,respectively）.Conclusions:1.After using si RNA to knock down CRY1 gene in HDFa cells,the expression of CRY1 gene decreased and the expression of PER2 gene increased at the transcriptional level,suggesting that CRY1 may form negative feedback regulation on PER2 gene.2.Melatonin,the endogenous ligand of MT1 receptor,can bind MT1 receptor in293T MT1 receptor overexpression system,recruit downstream G_S protein,and activate downstream c AMP/PKA pathway.The experiment verified that CRY1/PER2 gene is located downstream of the signal transduction pathway of c AMP/PKA cells.After melatonin acts on HDFa cells,the expression of CRY1 and PER2 genes was detected.It was found that melatonin can reduce the expression of CRY1 and increase the expression of PER2.It can be concluded that melatonin can activate the c AMP/PKA pathway by binding to MT1 receptor,thus promoting the expression of PER2 and reducing the expression of CRY1.3.The above experiments show that the increased expression of PER2 gene is an important factor to promote collagen type I synthesis,and the loss of PER2 gene expression may lead to decrease collagen type I synthesis.Melatonin can inhibit the expression of MMP1,promote the expression of Collagen I and play an anti-aging role in HDFa cells after 24 hours of treatment,which is related to the decrease of CRY1expression and the increase of PER2 expression.4.Epiphyllum extract may have the function of promoting collagen type I synthesis.Its mechanism is that Epiphyllum extract can activate MT1 receptor,promote the production of c AMP in cells,thus activate c AMP/PKA pathway,affect the expression of CRY1 and PER2,and regulate the synthesis of Collagen I.