Effects Of HMGB1 On The Migration Of Rat Astrocytes Induced By Magnetic Stimulation In Vitro

Background and ObjectHigh-mobility group box 1(HMGB1)is a non-histone DNA-binding protein that regulates gene expresson and nucleosome stabilization.HMGB1 has involved in the startment and development of stroke,it was considered as an inflammatory factor that aggravated the damage of stroke in the early stage,while it also can promote the regeneration of Neurovascular that conducive to the recovery of neural function.HMGB1 was involved in neurite outgrowth and cell migration in embryonic brain.The activated astrocytes can secrete HMGB1,but the effects of HMGB1 on the migration of astrocytes is still unclear.Previous studies have shown that Magnetic stimulation can activate and regulate the migration of astrocytes.In this study,we further investigate whether HMGB1 affect the migration of astrocytes induced by magnetic stimulation in vitro experiments and explore on its mechanism.Materials and Methods1.The extraction,culture and identification of astrocytes: Brain cortex tissue was separated from Sprague-Dawley(2-3 day)rat,the astrocytes were isolated and cultured.Observed the growth and morphology of astrocytes.Using Immunofluorescence to observed the expression of GFAP and identified the purity of astrocytes.2.Experimental groups:there were three groups according to different frequency of Magnetic stimulation.(1)control group;(2)Low-frequency MS group(1HZ);(3)High-frequency MS group(10HZ).Migration assay was performed to observed the cell migration,and Western Blot was applied to research the phosphorylation of ERK1/2 and the expression of HMGB1.3.Experimental groups after knockdown of HMGB1 expression:(1)control group(MS);(2)Si-RNA group(MS+HMGB1 Si-RNA).Migration assay was performed to observed the cell migration,and the interference effect was measured by Western Blot4.Experimental groups after adding U0126 :(1)control group(MS);(2)experimental group(MS+U0126).Migration assay was performed to observed the cell migration,and Western Blot was applied to observe the phosphorylation of ERK1/2 and the expression of HMGB1.Result1.The isolated astrocytes were passaged twice,and determined by GFAP immunofluorescence staining.The purity of astrocytes was >95%.2.High-frequency MS significantly promoted astrocyte migration compared to control and low-frequency MS group(p<0.01),and enhanced the phosphorylation of ERK1/2 and the expression of HMGB1,and has significant difference compared with the control and low-frequency MS group(p<0.01).3.The expression of HMGB1 and migration of astrocytes was significantly dereased compared with the control group after knockdown of HMGB1 exoression(p<0.01).4.The expression of HMGB1 and migration of astrocytes was significantly dereased compared with the control group after adding U0126(p<0.01).Conclusion1.High-frequency(10HZ)MS promotes the migration of astrocytes,and enchances the phosphorylation of ERK1/2.2.High-frequency(10HZ)MS promotes the expression of HMGB1,and it mediated migration of astrocytes induced by MS through an autocrine mechanism.3.The phosphorylation of ERK1/2 is beneficial to the expression of HMGB1,and HMGB1 is the downstream signal of ERK1/2.