| Research background:In recent years,due to the destruction of the natural environment,the ozone layer has been destroyed to varying degrees.In some places,there have been holes in the ozone layer,and the filtering effect of the destroyed ozone layer has been weakened.As a result,the amount of ultraviolet rays(UV)reaching the earth's surface has increased.Ultraviolet radiation can promote the body's absorption of vitamin D,but excessive UV radiation will affect human health.First,ultraviolet radiation will lead to photoaging of the skin,reduce collagen fibers in the cortex,reduce skin elasticity,and even severely damage the connective tissue of the skin,resulting in skin relaxation,sagging and deepening wrinkles and other adverse consequences;second,short-term exposure.In high doses of ultraviolet radiation,it will lead to apoptosis and necrosis of skin cells,which will damage the protective function of the natural barrier of epidermis,and then cause clinical characteristic changes,including deepened texture,speckle pigmentation,wrinkles increase,dry rough,skin relaxation,telangiectasia,elasticity decrease,benign and malignant tumors.Skin is the largest organ of human body.It can prevent ultraviolet radiation,regulate body temperature,prevent water loss,maintain appearance and resist microbial invasion.Skin is composed of non-homologous cells with strong self-healing ability.Skin,as the largest organ of human body,is directly in contact with the external environment,and is one of the main target organs of ultraviolet radiation.The biological effects caused by different bands of ultraviolet radiation are also different.UVB mainly causes skin erythema,immunosuppression and skin cancer,while UVA causes skin tanning,skin photosensitivity and skin photoaging.Among the three different wavelength ultraviolet rays,UVA has the strongest ability to produce free radicals and lipid peroxidation,which can affect collagen and elastic fibers in dermis tissue and result in skin photoaging.UVB mainly causes lesions in epidermis and superficial dermis,resulting in sunburn erythema,immunosuppression and skin cancer.The combination of UVA and UVB can cause a series of epidermal and dermal lesions,and the presence of UVA can also enhance the erythematous effect of UVB.In addition,UVB can induce the formation of Pyrimidine dimer in skin cell DNA photoadduct.If the latter is not repaired properly,it can lead to DNA mutation and skin cancer.Ultraviolet A causes secondary DNA damage mainly by oxidizing to produce peroxides,and then strengthens the carcinogenic effect of UVB.The self-repair and renewal of normal skin is the result of the interaction of different types of stem cells,which is the requirement of maintaining normal skin tissue structure and stabilizing intracellular environment.The skin is divided into dermis and epidermis.The epidermis is composed of basal layer,spinous cell layer,granular layer,transparent layer and cuticle.The basal layer contains a large number of stem cells,which are clusters of cells.They proliferate and develop into epidermal cells in vivo.Epidermal stem cells have a relatively slow division and proliferation cycle,which can cause a short expansion of autologous cells.After several divisions,epidermal stem cells can proliferate into epidermal cells.To complete the final differentiation.In vitro studies have shown that S14G-Humanin can significantly inhibit stem cell damage at low concentrations,and has significant inhibitory effects on the death of stem cells and primary cortical cells.S14G-Humanin can protect stem cells through anti-cell death pathway.S14G-Humanin can protect cells by binding with some receptors,including Bcl-2 family proteins Bim,Bid and Bax.Long-term skin exposure to UV radiation can cause DNA damage and mutation in cells.Once damaged,the body will initiate repair procedures,some genes in cells are inhibited,and some genes are activated.Failure to repair damaged DNA triggers apoptotic processes.Apoptosis is closely related to Bcl-2,an inhibitor of apoptosis,and Bax,an accelerator of apoptosis.Bcl-2 protein can inhibit cell apoptosis.After binding with Bax to form heterodimer,Bcl-2 protein can inhibit the activation of Caspase-3 downstream by inhibiting the release of cytochrome C,thus achieving the purpose of inhibiting cell apoptosis.In addition,Bax protein can also promote cell apoptosis by directly establishing transmembrane channels on mitochondrial membrane,promoting the release of cytochrome C,and promoting cell apoptosis by regulating cytochrome C.The ratio of Bcl-2 to Bax determines whether the cells are apoptotic or not,and a positive regulation of apoptosis is formed between them.JAK2 / STAT3 signaling pathway is one of the most studied signaling pathways in recent years.STAT3 signaling pathway can directly transmit the signals sensed by cell membrane to the nucleus,activate the transcription of target genes,with few and simple links.Many cytokines(CNTF,IL2,IL6,etc.)and growth factors(PDGF,EGF,CSF,etc.)use this signal transduction pathway to induce cell differentiation,proliferation or apoptosis.They play a specific and multi-functional biological function in the regulation of immune function,the occurrence of tumor and the generation of hematopoietic cells.PI3 K belongs to phosphorylated phosphatidylinositol phosphokinase family located in D3 of inositol ring.The primary structure of PI3 K family is involved in extracellular signal transduction.Its reaction product is the second messenger,and its target is serine / threonine kinase.Akt is located at the downstream of PI3 K.It is activated by extracellular stimulation.Akt is transferred from cytoplasm to cell membrane.Two residues of Akt(threonine 308 and serine 473)are phosphorylated in the cell membrane,making Akt fully activated.Akt pathway can inhibit cell apoptosis and promote cell growth.PI3 K / Akt and JAK2 / STAT3 are important signaling pathways.They are involved in many pathophysiological processes such as cell activation,apoptosis,inflammatory response and chemotaxis.These two signal transduction pathways are involved in the mechanism of anti apoptosis of various interventions.Whether these two signaling pathways are involved in oxidative stress,free radical metabolism and DNA damage remains to be studied.1.Cytoprotective role of S14G-humanin(HNG)in ultraviolet-B induced epidermal stem cells injury.Objective:To investigate the protective effect of S14G-humanin(HNG)on ultraviolet-Binduced epidermal stem cell injury.Methods:The protective effect of HNG on epidermal stem cells was studied under UV-B treatment.The experimental cells were randomly divided into four groups: blank control group,ultraviolet group,100 ? m HNG pretreatment group and 200 ? m HNG pretreatment group,each group including 60 Petri dishes.Interventions: ESC was treated with 100 ? m and 200 ? m HNG for 12 hours,and stimulated with 50 MJ / cm2 ultraviolet for 12 hours.MTT cell survival and LDH cytotoxicity.MTT assay was used to test cell viability;real-time PCR was used to analyze the transcription levels of Wtn3 a,Myc and cyclin D1;protein separation and Western blotting were used to analyze the expression of Wtn3 a,Myc and cyclin D1;measurement of reactive oxygen species(ROS);TMRM staining;Determination of reduced glutathione(GSH).Results:HNG inhibited the production of ROS induced by UV-B and increased the expression of glutathione,an antioxidant.HNG pretreated cells showed very mild cytokine production after exposure to UV-B,including TNF-alpha,IL-1beta and IL-6.HNG pretreatment has protective effect on UV-B-mediated cytotoxicity and promotes ESC survival.In addition,HNG therapy attenuated the reduction of mitochondrial membrane potential(MMP)induced by UV-B,and maintained their identity and stem cell capacity.In mechanism,HNG treatment improved the reduction of Wnt/beta-catenin pathway proteins induced by UV-B,including Wtn3 a,Myc and cyclin D1.Conclusion:HNG plays an important role in promoting survival and antioxidant stress of embryonic stem cells,and may be used in ESC-mediated therapy.2.S14g-humanin(HNG)alleviates the molecular mechanism of UV-induced free radical metabolism and DNA damage in mouse skin cellsObjective:To explore the molecular mechanism of S14G-humanin(HNG)in alleviating the free radical metabolism and DNA damage induced by ultraviolet radiation in mouse skin cell.Methods:The experimental cells were divided into two groups: S14G-humanin plasmid transfection group and blank control group,each group consisted of 60 Petri dishes.Interventions: Cells in the S14G-humanin plasmid transfection group group were stimulated with ultraviolet radiation of 30 J/m2 for 12 hours,while those in the blank control group were not treated.Mouse skin cells were transfected,S14G-humanin target gene expression was analyzed by RT-PCR,colony formation test was carried out on mouse skin cells,apoptotic status of transfected cells was analyzed by flow cytometry,and apoptotic protein and cell pathway protein expression were measured by Western Blot.Results:Functional t S14G-humanin V143 A selectively enhanced S involved in cell growth arrest and DNA damage repair.The expression of 14G-humanin V143 A target was not apoptotic;the apoptosis or cell cycle distribution of t S14G-humanin group was not affected by ultraviolet radiation;ts S14G-humanin V143 A could protect rat skin cells from apoptosis induced no significantly by ultraviolet radiation(P>0.05).Western blot results showed that the expression level of p-c-jun in s14g-humaninv143 a group was significantly lower than that in the control group(P<0.05).The recombination of wild-like S14G-humanin activity in cells inhibits the JNK activity stimulated by ultraviolet radiation and the transmission of JNK signal.Conclusion:S14G-humanin(HNG)can alleviate the free radical metabolism and DNA damage induced by ultraviolet radiation in mouse skin cell skin cells by inhibiting JNK signaling pathway.3.Study on the mechanism of S14G-humanin(HNG)in alleviating the damage of epidermal stem cells induced by oxygen-glucose deprivation and reoxygenationObjective:To study the mechanism of S14G-humanin(HNG)in relieving oxygen-glucose deprivation and reoxygenation on epidermal stem cells.Methods:Epidermal stem cell ESC were isolated from the back skin of mice for cell culture and randomly divided into 3 groups: control group(16 hours of cells cultured in normal cell culture plates);induction group(deprived glucose and serum in sealed chamber)The culture was performed under hypoxic conditions at 37 °C,followed by an additional 9 hours to maintain the oxygen glucose deprivation/reoxygenation model in the normal medium)and the HNG + induction group: 1 ?g / l of HNG was added to the induction group to continue the culture.5 hours;HNG+ induction group + inhibitor group(specific inhibitors such as FLLL32,LY294002 and MK-2206 were added to study the activation of PI3 K / AKT pathway and Jak2 / Stat3);mitochondrial transmembrane potential was measured by TMRM staining Level;determination of reactive oxygen species by DCFH-DA;detection of apoptosis and expression of related factor proteins by q RT-RCR and Western blotting;cell viability assay using cell counting kit,Annexin V-FITC apoptosis detection reagent.The cell was tested for apoptosis rate.Results:Under the conditions of oxygen and glucose deprivation/reoxygenation,HNG increased ESC activity,reduced apoptosis rate,reduced intracellular ROS level,promoted the generation of GSH,and prevented the decrease of mitochondrial transmemmene potential.HNG increased the expression levels of Jak2,Stat3,p-jak2,and p-stat3 proteins,decreased the expression of TNF-?,IL-1?,and IL-6 m RNA,and the expression of apoptotic proteins Bax and caspase.In terms of mechanism,after Jak2/Stat3 pathway was inhibited,the expression levels of apoptotic proteins Bax and caspase and the apoptosis rate were increased.After inhibition of the PI3K/AKT pathway,the expression of Jak2 and Stat3 proteins was significantly reduced..Conclusion:S14G-humanin(HNG)passed PI3 K / AKT Pathway activates the expression of Jak2/Stat3 to alleviate oxygen-glucose deprivation Dry skin cell damage.4.S14g-humanin protects epidermal cells from oxidative stress by enhancing the antioxidant defense system and activating pparaObjective:To investigate the mechanism by which s14g-humanin protects epidermal cells from oxidative stress by enhancing antioxidant defense system and activating ppar alpha.Methods:cell culture was conducted by subculture,cryopreservation and resuscitation,and the oxidative stress injury model of epidermal cells was established by using H2O2.The experiment was divided into normal group,model group and s14g-humanin experimental group.During the study,cell proliferation activity,cell morphology and other growth state evaluation were detected,and then the activity of SOD,CAT,GSH,GPx and GST in the cells were measured with the kit.PPAR-m RNA expression was detected by gel imaging using agarose electrophoresis.PPAR-m RNA expression was detected by cell proteins.PPAR-protein expression was detected by Western blot.Results:Under the stimulation of H2O2,the cell morphology of the S14G-humanin group was improved,the proliferation activity was increased,and the activity of antioxidant enzymes(SOD,CAT,GSH,GPx and GST)was enhanced.The ppar-m RNA expression in human epidermal cells was significantly higher,and the expression level of ppara protein was increased.Conclusion:H2O2 can significantly stimulate human epidermal cells and inhibit their growth,and s14g-humanin can improve oxidative stress injury of human epidermal cells to some extent.S14g-humanin can enhance the enzymatic activity of antioxidant activity systems such as SOD activity,CAT activity and GSH activity,which are the main components of the antioxidant defense system in the organism.The increased activity of s14g-humanin can help prevent the organism from being hurt by stress.At the same time,s14g-humanin can increase ppar-m RNA level and promote the expression of ppar-protein,thus protecting epidermal cells from oxidative stress. |
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