The Mechanism Of Hypoxic Induced AQP1 Expression In Schwann Cells

?Background?BELL's palsy is the most common facial nerve injury.The initial cause is not yet clear,but according to the results of laboratory anatomy and clinical facial nerve canal decompression surgery,researchers found that ischemia and hypoxia of facial nerve caused by the change of those nourishing blood vessels.The vicious cycle of "edema-ischemia and hypoxia-edema" formed by the stenosis of the facial nerve canal is the direct cause of neurodegeneration and necrosis.The degree of swelling of the facial nerve is Negatively correlation.Thus,termination of the vicious cycle of "edema-ischemia and hypoxia-edema" appears to be more important in the treatment of facial nerve injury.In recent years,it has been found that aquaporin is a kind of membrane channel protein family with fast transport function for water molecules,which is closely related to the formation and elimination of edema of various tissues and organs(AQP0-12),and has been found in various tissues and organs.Our previous work have confirmed that AQP1 is present in Schwann cells of facial nerve and is associated with the edema of facial nerve and Schwann cells.Mitogen-activated protein kinase(MAPK)signaling pathway which is widely present in all kinds of cells,and is the intersection of cell signaling,can transmit extracellular information to the nucleus,can directly or indirectly regulate the transcription factor activity and gene expression.MAPK is the main signal pathway in cell stress and injury feedback.Studies have shown that AQP1 expression can be induced by hypoxia,osmotic stress,inflammatory mediators and other regulation.Studies have shown that under different stress conditions,AQP1 gene expression and MAPK signal cascade have a certain correlation,but it is unclear whether MAPK signaling pathway is involved in the expression of hypoxia-induced Schwann cell AQP1 expression.Therefore,the hypoxia model of Schwann cells has been constructed by simulating under the hypoxic environment after facial nerve injury.The morphological changes of AQP1 and MAPK and the expression of AQP1 in Schwann cells induced by hypoxic environment have been studied by using SB203580,U0126 and SP600125,the inhibitors of p38,ERK and JNK signaling pathways,respectively.The effects of MAPK signal,Whether is related to the expression of AQP1 induced by hypoxia and reveal the molecular mechanism of AQP1 expression induced by hypoxia injury in Schwann cells,and provide a theoretical basis for the "edema-ischemia-hypoxia-edema" vicious cycle after facial nerve injury,have been observed.?Objective?1.In vitro construction of Schwann cell hypoxia model,simulated facial nerve damage after the formation of hypoxic environment for the follow-up experiment to provide a reliable and stable cell model;2.To study the changes of AQP1 expression in protein level and gene level of Schwann cells under hypoxia condition;3.To study the expression of P-p38,P-ERK and P-JNK in Schwann cells under hypoxic conditions;4.To explore the relationship between hypoxia-induced AQP1 expression and MAPK signaling pathway,and to reveal the possible molecular mechanism of hypoxia-induced changes in AQP1 expression in Schwann cells.?Methods?1.Schwann cell hypoxia model(1)Cell culture;(2)Immunofluorescence staining was used to identify the expression of AQP1 in RSC96 cells;(3)CCK8 cell proliferation-toxicity test;2.Hypoxia on the AQP1 and MAPK signaling pathways in Schwann cells(1)Western Blot was used to detect the expression of AQP1 in hypoxic condition;(2)Real time-PCR was used to detect the expression of AQP1 mRNA in hypoxic condition;(3)Western Blot was used to detect the expression of P-p38,p38,P-ERK,ERK,P-JNK and JNK in hypoxic condition.3.Hypoxia induced schwann cell AQP1 expression mechanism(1)Study on the expression of AQP1 in Schwann cells induced by hypoxia by P38 signaling pathwayWestern Blot was used to detect the expression of P-p38 in SBOO3580 pretreated with hypoxia;Western Blot was used to detect the expression of AQP1 in Schwann cells pretreated with SBOO3580 under hypoxia.(2)Study on the expression of AQP1 in Schwann cells induced by hypoxia by ERK signaling pathwayWestern Blot was used to detect the expression of P-ERK in Schwann cells pretreated with U0126 under hypoxia;Western Blot was used to detect the expression of AQP1 in Schwann cells pretreated with U0126 under hypoxia.(3)Study on the expression of AQP1 in Schwann cells induced by hypoxia by JNK signaling pathwayWestern Blot was used to detect the expression of P-JNK in Schwann cells treated with SP600125 under hypoxia;Western Blot was used to detect the expression of AQP1 in SP600125 pretreated with hypoxia.?Results?1.Immunofluorescence showed RSC96 expression of AQP1;2.The proliferation of CCK8 cells showed no significant difference between the two groups(P> 0.05),indicating that the hypoxia time was less than 9 hours and the cell proliferation was not significantly different from that of the control group,indicating that RSC96 cells in the hypoxic environment for more than 12 hours of its proliferation will be inhibited;in the hypoxia for 12 hours and 24 hours compared with the control group,the difference was significant(P <0.05);3.The expression of AQP1 in RSC96 cells was significantly increased after 1 hour of hypoxia(P> 0.05).AQP1 expression was gradually increased at 3 hours and 6 hours,AQP1 content was the highest at 6 hours of hypoxia,The difference was statistically significant(P <0.05).The expression of AQP1 began to decrease after hypoxia 6 hours;4.The mRNA content of AQP1 mRNA in RSC96 cells increased gradually,and the AQP1 mRNA level increased gradually at hypoxia 1h,3h and 6h,and the AQP1 mRNA content was the highest at 6 hours of hypoxia,the difference was statistically significant(P <0.05),And then gradually reduced;5.The P-p38,P-ERK and P-JNK were activated after hypoxia in RSC96 cells,and gradually increased from hypoxia for 15 min to 1 hour,and reached the maximum at 1 hour,The difference was statistically significant(P <0.05),and then gradually decreased;6.The phosphorylation of p38,ERK and JNK in the hypoxia + inhibitor group was inhibited by the combination of p38 MAPK inhibitor SB203580,ERK inhibitor U0126 and JNK inhibitor SP600125(P < 0.05);7.The expression of AQP1 was inhibited by hypoxia + SB203580,ERK inhibitor U0126 respectivel,the difference was statistically significant(P <0.05).There was no significant difference in AQP1 expression between hypoxia + SP600125 group and hypoxia group(P> 0.05).?Conclusions?1.AQP1 is expressed in RSC96 cells;2.Hypoxia could induce the expression of AQP1 in Schwann cells at the m RNA and protein level;3.Hypoxia could induce the phosphorylation of p38,ERK and JNK in Schwann cells;4.Hypoxia may regulate the expression of AQP1 through p38 and ERK signaling pathway.