| Background:Photoaging is one of the main courses of skin aging,which is induced by long-term of ultraviolet(UV)radiation(UVR).The main manifestation are wrinkles,skin relaxation,telangiectasia and so on.In addition,photoaging can also induce skin tumors.Therefore,it is of great significance to study the treatment and mechanism of skin photoaging.The histological changes of skin photoaging include epidermal thickening,solar elastosis,and collagen reduction.Among them,collagen reduction is the characteristic histological manifestation of skin photoaging,and consistent to the seriousness of photoaging.Current treatments for skin photoaging include retinoids,laser,intense pulsed light,photodynamic therapy,injection therapy,and so on.More and more studies showed that Q-switched 1064 nm Nd:YAG laser is efficient in rejuvenating photoaged skin and promoting collagen synthesis,but the mechanism is unclear.MicroRNAs(miRNAs)are involved in the occurrence,development and repairment of photoaging.Various miRNAs play different roles in skin rejuvenation of lasers treatment.They affect skin barrier repairment and collagen synthesis by negatively regulating target proteins.Studies showed that miR-196b-5p regulates collagen synthesis in a variety of diseases,such as keloid and systemic sclerosis.Predicted by bioinformatics method,transforming growth factor-β receptor 2(TGFBR2)is one of the directly targets of miR-196b-5p.TGFBR2 is a receptor of transforming growth factor-β1(TGF-β1)on the surface of cell membrane,which is responsible for transmitting TGF-β1 signals,and the activation of TGF-β1/Smad signaling pathway promote collagen synthesis.Downregulation of TGFBR2 and upregulation of Smad7 lead to suppression of TGF-β1/Smad transduction and reduce collagen synthesis in photoaging skin.However,the effect of Q-switched 1064 nm Nd:YAG laser on miR-196b-5p,TGFBR2 and Smad7 in skin photoaging is unclear.In this study,skin photoaging model rats,human skin fibroblasts and HaCaT cells were selected as experimental subjects.The effects of Q-switched 1064 nm Nd:YAG laser on miR-196b-5p,TGFBR2 and Smad7 in photoaged skin was investigated.Objective:1.To establish and verify a skin photoaging model in Wistar rat,and to investigate the effect of Q-switched 1064 nm Nd:YAG laser on miR-196b-5p,TGFBR2 and Smad7 in photoaged rat skin.2.To verify the effect of Q-switched 1064 nm Nd:YAG laser on miR-196b-5p,TGFBR2 and Smad7 in human skin fibroblasts and HaCaT cells.3.To verify the targeting relationship between miR-196b-5p and TGFBR2.To explore the role and mechanism of miR-196b-5p-TGFBR2-Col3a1 axis in the irradiation of Q-switched 1064 nm Nd:YAG laser.Part Ⅰ Q-switched 1064 nm Nd:YAG laser rejuvenates photoaged skin of Wistar ratMethods:(1)The rats were randomized into two groups:a control group and an UVR group.The dorsal skin of the UVR group was radiated with UVA and UVB for 12 weeks.The total dose of UVA was 106.05 J/cm2,and that of UVB was 13.49 J/cm2.The photoaging model was verified by macroscopic observation,dermoscopy detection,hematoxylin-eosin(HE)staining,Sirius red staining,and hydroxyproline content detection.(2)The photoaged rats in part one was randomly divided into an untreated group and a laser group.Rat dorsal skin of the laser group was irradiated with Q-switched 1064 nm Nd:YAG laser.Laser irradiation was applied once every other day,three times,for a week,and the parameters were as follows:spot size,6 mm;fluence,2.5 J/cm2;pulse width,5 ns;frequency,10 Hz,five passes.HE staining and hydroxyproline content detection were used to detect the improvement of skin in photoaged rats.(3)The mRNA expression of TGF-β1,TGFBR1,TGFBR2,Smad2,Smad3,Smad4,Smad7,COL1A1,COL3A1 and miR-196b-5p was detected by the qRT-PCR method.(4)Western blot method was used to detect the protein expression of TGFBR2,Smad2/3,p-Smad2/3 and Smad7.Immunohistochemical(IHC)was used to detect the protein expression of TGFBR2 and Smad7.Results:(1)Skin photoaging model was successfully established by UVR.Winkles was observed on the dorsal skin of rat.Observed by a dermoscope,rat skin of the UVR group showed typical skin photoaging manifestations:rough skin texture,scattered distribution of linear or branched blood vessels.HE stained skin sections of the UVR group showed that the epidermis was thickened(P<0.01),the dermal fibers were tiny and curly.Sirius red stained skin sections of the UVR group showed that the dermal fibers were decreased,the proportion of type I collagen was decreased(P<0.0001),and the proportion of type III collagen was increased(P<0.01).Compared with the control group,the hydroxyproline content of the UVR group was decreased(P<0.001).(2)Detected by the qRT-PCR method,the mRNA expression of TGFBR2(P=0.01),COL1A1(P=0.04)and COL3A1(P=0.04)were downregulated by UVR,while the mRNA expression of Smad4 was upregulated(P=0.02).IHC showed that the protein expression of TGFBR2 was downregulated in the photoaged rat skin,while Smad7 was upregulated(P<0.01).(3)The laser irradiation rejuvenated the photoaged rat skin.HE staining and Sirius red staining showed that the epidermal thickness of the photoaged rat skin were gradually became thinner by laser irradiation(P<0.05).Compared with the untreated group,the dermal thickness of the laser group was increased(P<0.01).Compared with the untreated group,the hydroxyproline content in skin of the laser group was increased(P=0.01).(4)Compared with the untreated group,the mRNA expression of miR-196b-5p(P=0.04)and Smad7(P=0.01)was downregulated by laser irradiation,and the mRNA expression of TGFBR1(P=0.04),TGFBR2(P=0.04),Smad4(P=0.02)and COL3A1(P=0.03)was upregulated.Western blot analysis showed that the protein expression of p-Smad2/3(P=0.04)was upregulated by the laser irradiation.Western blot and IHC analysis showed that the protein expression of TGFBR2 was upregulated by the laser irradiation(P<0.05),while Smad7 was downregulated(P<0.05).Conclusions:The Q-switched 1064 nm Nd:YAG laser may rejuvenates photoaged rat skin by regulating the expression of miR-196b-5p,TGFBR2 and Smad7.Part Ⅱ The effect and mechanism of miR-196b-5p-TGFBR2-COL3A1 axis in the irradiation of Nd:YAG LaserMethods:(1)Primary human skin fibroblast was cultured and passaged,and was exposed to UVA and Q-switched 1064 nm Nd:YAG laser.CCK-8 experiment was detected after 24 hours of irradiation.RNA was collected after 48 hours of irradiation.The mRNA expression of miR-196b-5p,TGFBR2,Smad7,MMP-1 and COL3A1 was detected by qRT-PCR method.(2)Human immortalized keratinocyte HaCaT cell line was cultured and passaged.Cells were transfected with miR-196b-5p mimics NC,miR-196b-5p mimics,miR-196b-5p inhibitor NC,or miR-196b-5p inhibitor.Q-switched 1064 nm Nd:YAG laser was irradiated after 2 hours of the transfection of miR-196b-5p mimics.(3)After 48 hours of cell transfection,qRT-PCR was used to verify the transfection efficiency and the expression of miR-196b-5p and mRNAs.Western blot method was used to detect the expression of TGFBR2,p-Smad2/3,Smad2/3,Smad7 and COL3A1.Results:(1)The effect of UVA radiation on human skin fibroblast cells.After UVA radiation,human skin fibroblast cells became short and slender,and the cell viability was decreased(P<0.05).The results of qRT-PCR showed that the mRNA expression of TGFBR2(P<0.05),COL3A1(P=0.03)and Nrf2(P<0.05)was downregulated by UVA radiation,while the expression of miR-196b-5p(P=0.03),Smad7(P<0.01)and MMP-1(P<0.01)was upregulated.(2)The effect of laser irradiation on human skin fibroblast cells.The laser irradiation did not change the morphology and cell viability(P>0.05)of human skin fibroblast cells.The results of qRT-PCR showed that the laser irradiation upregulated the expression of TGFBR2(P=0.03),COL3A(P<0.01)and Nrf2(P=0.02),while the expression of miR-196b-5p(P=0.02),Smad7(P=0.03)and MMP-1(P=0.03)was downregulated.(3)MiR-196b-5p directly targets and regulates TGFBR2 in HaCaT cells.Through bioinformatics searches and literature review,it is known that miR-196b-5p directly targets TGFBR2.The results of qRT-PCR and western blot showed that the overexpression of miR-196b-5p downregulates the expression of TGFBR2 in HaCaT cells(P<0.05),while the inhibition of miR-196b-5p upregulates the expression of TGFBR2 in HaCaT cells(P<0.05).(4)The overexpression of miR-196b-5p decreased the expression of p-Smad2/3 protein(P=0.01)in HaCaT cells.It suggested that the overexpression of miR-196b-5p inhibit the activation of TGF-β1/Smad signaling pathway.(5)The Q-switched 1064 nm Nd:YAG laser activates the TGF-β1/Smad signaling pathway by downregulating miR-196b-5p.The results of qRT-PCR showed that the laser irradiation downregulated the expression of miR-196b-5p(P=0.01),and upregulated the expression of TGFBR2(P=0.02)in the miR-196b-5p overexpressed HaCaT cells.The results of western blot showed that the expression of COL3A1(P=0.04),Smad2/3(P<0.05),p-Smad2/3(P=0.02),TGFBR2(P=0.01)was upregulated,and the expression of Smad7 was downregulated(P=0.04).Conclusions:MiR-196b-5p regulates the TGF-β1/Smad signaling pathway and the expression of COL3A1 by targeting TGFBR2.Q-switched 1064 nm Nd:YAG laser improves photoaging by regulating the expression of miR-196b-5p-TGFBR2-COL3A1 axis and Smad7. |
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