The Effect Of Oxidative Damage On Golgi And Rab30 In HT22 Cells

Objective:To elucidate the effect of oxidative damage on Golgi and Rab30 in HT22 cells, we use the thapsigargin(TG) intervened oxidative stress model. Our study will focus on the relationship of Rab30 and Golgi fragmentation and the possible mechanisms of Golgi fragmentation.Experimental Methods:1.The cell culture: HT22 cells derived from mouse neural cells,with DMEM medium containing 10% FBS and 100 U penicillin- streptomycin solution performed culture.When cells were grown to 80%-90%confluence,with 0.25% trypsin to digestion cells for the experiment.2.Experimental groups: The HT22 cells of logarithmic growth phase in 96-well plates were randomly divided into three groups as follows:control group,0.1μg/ml TG intervention 3h group and 0.1μg/ml TG intervention 5h group.3.Real-time PCR was used to detect the expression of total Rab30 mRNA changes and Western blotting to detect the expression of Intracellular Rab30,GM130, and JNK3.4.Immuno-fluorographs of Rab30 and GM130 were taken to determine the sub-cellular location and fragment of Golgi in oxidative stress.Results:1.PCR to detect the expression of cell Rab30 mRNA: after exposure upon 0.1μg/ml TG,the expression of Rab30-mRNA increased in a time manner.After exposure upon TG 5 hours, the expression of Rab30-mRNA decreased remarkably compared with TG 3hours group(p<0.05).2.Western blot show: the expression levels of Rab30,GAAP and GM130 present the same trend: the normal control group> thapsigargin3 h group> thapsigargin 5h group, while JNK3 level is:thapsigargin 5h group> thapsigargin 3h group> the normal control group, among all group, the four protein show significant difference(P <0.05).3.Immuno-fluorescence staining: In control group, GM130 labeled Golgi apparatus is compact, crescent-shaped, locating at the perinuclear region, and the expression region of Rab30 coincide with GM130.4.In TG injury group,expressioon of GM130 and Rab30 was significantly decreased,and Golgi apparatus exhibited scattered structure and irregular morphological changes.Conclusion:1.Nerve cells express Rab30 protein, and mainly localized in the Golgi apparatus.2. The level of expression of Rab30 is reduce in the process of oxidative stress along with the fragment of Golgi.3. The Rab30 may mediate Golgi fragmentation, probably through the pathway of JNK3 and GM130.