| Background and objective:Excessive exposure to ultraviolet B(UVB)can damage various biological macromolecules,induce inflammatory responses,inhibit proliferation,and promote cell aging and death.UVB is a major factor in carcinogenesis of ultraviolet rays in sunlight.The skin is the largest organ of the human body and the most vulnerable to UVB damage.UVB has weak penetrating power and can only reach the epidermal layer.Therefore,related research mainly uses keratinocytes,which account for the main body of epidermal cells.The various damages of keratinocytes induced by UVB are closely related to the oxidative stress caused by the accumulation of reactive oxygen species(ROS).90%of ROS in cells come from mitochondria,and mitochondria are also the main sites for ROS clearance.A variety of enzymes in mitochondria are involved in the elimination of ROS,including superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase.Under normal physiological conditions,ROS levels are maintained at extremely low beneficial levels.UVB irradiation can cause a significant increase in ROS levels in keratinocytes,which is closely related to the decrease in antioxidant enzyme activity in mitochondria.Acetylation modification is the main way to affect the activity of antioxidant enzymes in mitochondria,and the deacetylase SIRT3 located in mitochondria has not been studied in keratinocytes.In this study,HaCaT,a mature in vitro model related to the effects of UVB irradiation on epidermal keratinocytes,was selected.The effects of UVB on the transcription and translation of SIRT3 were examined by qRT-PCR and Western blot.Since the distribution of SIRT3 in cells is not limited to mitochondria,we first tested the expression level of SIRT3 in nuclear plasma protein and mitochondrial protein and the effect of UVB on it by Western blot.In order to investigate the role of SIRT3 in UVB-induced oxidative damage,the effects of SIRT3 overexpression on UVB-induced ROS generation and SOD2 expression were examined.In addition,the effect of SIRT3 overexpression on HaCaT migration capacity and related Twist expression was also tested.Result1.qRT-RCR and Western blot results showed that low-dose UVB(30m J/cm~2)did not change,while high-dose UVB(120m J/cm~2)significantly down-regulated the m RNA and protein levels of SIRT3.2.Western blot was used to detect the expression of SIRT3 in nuclear plasma protein and mitochondrial protein.The results showed that in normal culture and UVB irradiation of HaCaT cells,SIRT3 was located in mitochondria.After high-dose(120m J/cm~2)UVB irradiation,SIRT3 protein levels in plasma and mitochondrial proteins decreased.3.Using DHE(dihydroethyl ingot)-reactive oxygen species detection and Western blot detection,it was found that the levels of ROS in HaCaT cells increased and the levels of SOD2 in total protein decreased after UVB irradiation at 30m J/cm~2and 120m J/cm~2.4.The scratch test results showed that the cell migration did not significantly slow down after 30m J/cm~2UVB irradiation,and 120m J/cm~2UVB irradiation significantly inhibited HaCaT cell migration.Western blot results showed that the expression of Twist protein decreased after 120m J/cm~2UVB irradiation,and the expression of Twist did not change after 30m J/cm~2UVB irradiation.5.SIRT3 overexpression down-regulates 120m J/cm~2UVB irradiation,which increases ROS levels and decreases SOD2 protein and reverses down-regulation of UVB on cell proliferation and migration.Conclusion1.Physiological dose of UVB did not affect SIRT3 transcription and protein levels in HaCaT cells,while high dose of UVB significantly down-regulated SIRT3 expression.2.SIRT3 is mainly located in mitochondria in HaCaT cells.The increase of ROS level after UVB irradiation may be related to the decrease of SIRT3 and SOD2 expression.3.SIRT3 may protect HaCaT cells from UVB-induced oxidative stress and its ability to inhibit cell proliferation and migration by up-regulating SOD2 expression.4.The inhibition of UVB on HaCaT migration is related to the decrease of Twistlevel. |
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