Experimental Study On The Expression Mechanism Of AQP1 In Rat Schwann Cells And Facial Nerve Edema

[Background]According to anatomical study we found that walking in the temporal bone of the facial nerve through the rigid stenosis of the facial nerve tube, edema and ischemic after injury can not be timely relieved, eventually lead to degeneration and necrosis, formation of varying degrees of facial nerve paralysis . Because of the lack of ideal treatment, so the study of facial nerve edema mechanism, try to find the formation of edema damage and continuous method to avoid further degeneration and necrosis of facial nerve is one of the direction of treatment.Aquaporins (AQPs) as a kind of specific channel protein which highly involved in transmembrane water transport, play an important role in the regulation of water metabolism, participate in the formation and elimination edema of various tissues and organs. Previously, study with animal facial nerve injury model ,we found the expression of AQP1 in facial nerve tissue was similar with the degree of nerve edema. The expression of AQP1 was mainly located in Schwann cells. The expression of AQP1 could regulate the swelling of cells. According to the study reported that acetazolamide can effectively inhibit the expression of AQP1, we also confirmed that acetazolamide inhibited Schwann cells expressed a negative regulatory role. However, there is no report on the mechanism of AQP1 expression in Schwann cells and facial nerve edema.Due to the lack of study on overall level of aquaporins function and its expression regulation mechanism, therefore, combined with the previous research basis, this study through Schwann cell edema model and facial nerve injury animal model, according to cell biology,molecular biology and other methods in vitro and in vivo edema models states ,to study the expression and regulation mechanism of AQP1.Acetazolamide intervention mechanism on the regulation of AQP1 expression, and aims to explore their relationship between each other for the early treatment of facial nerve edema.[Objective]1. To study the relationship between expression of AQP1 and Schwann cell edema,further to study its expression regulation mechanism.2. To study the effect of acetazolamide on the relationship between AQP1 expression and the Schwann cells edema.3. According to the mechanism of AQP1 expression in facial nerve edema model, which provided a new idea for clinical prevention and treatment of early facial nerve edema with aquaporins as a target.[Methods]A. Experimental study on the effect of osmotic pressure on expression of AQP1 in Schwann cells and its regulation.1. Effects of hypotonic solution on the biological Characteristics and AQP1 expression of Schwann Cells.1) The cells were adherently cultured and the morphology was observed under microscope.2) Immunofluorescence staining was used to identify the expression of S-100 and AQP1.3) Western-blot was used to verify AQP1 expression in RSC96.2. Construction of a hypotonic induced cell edema model.1) Two groups of hypotonic medium :240 mmol/L,150 mmol/L.2) CCK-8 test and flow cytometry apoptosis evaluation of standard time points.3) morphological changes with hypotonic-induced cell edema and confocal dynamic monitoring of cells volume.3. Effect of osmotic pressure on MAPK signal pathway in Schwann cells1) To replicate the cell edema models.2) Cell edema model group: 2 groups of hypotonic medium 240mmol / L, 150mmol / L,the control group to conventional serum-free DMEM culture.3) Western- blot was used to detect the phosphorylation of MAPK.4. Experimental study on the regulation of AQP1 expression with MAPK in Schwann cells1) Replicated the cell edema model.2) The cells were divided into normal control group (ERK/p38), hypotonic group,hypotonic intervention group, intervention group. The cells to be treated were previously added with MAPK-specific inhibitor for 1 h, then hypotonic medium was added.3) Western -blot was used to detect the expression of AQP1.B. Study the Regulation of Acetazolamide on AQP1 Expression in Rat Schwann Cells1. Experimental study on the effect of lentivirus-mediated AQP1 overexpression with the biological characteristics in Schwann cells.1) Construction and identification of rat AQP1 gene lentivirus overexpression vector: The rat AQP1 gene was cloned into the lentivirus, and the correct clones were obtained by PCR and sequencing. The recombinant plasmid was co-transfected with 293T cells to prepare the AQP1 gene overexpression Schwann cell models.2) Cell cycle detecting was used to illustrate the difference between overexpression cells and negative control cells.3) Immunofluorescence semi-quantitative method and Western-blot were used to detect the expression of AQP1 protein in schwann cells after infection.2. Inhibitory effect of acetazolamide on the expression of AQP1 and MAPK signal cascade.1) The cytotoxicity of acetazolamide on Schwann cells was determined by CCK-8, and the appropriate drug concentration was selected.2) AQP1 overexpression was used to detect the changes of 12h AQP1 protein by immunoblotting.3) After processing the AQP 1 overexpressing strain with acetazolamide, the phosphorylation level of 0-12h MAPK protein was observed by immunoblotting.4) The cells were divided into normal control group, acetazolamide inhibition group,acetazolamide inhibition intervention group. After treatment with MAPK-specific inhibitor for 1 h, the treated cells were added with acetazolamide to observe the changes of AQP 1 protein content.3. Study on the mechanism of acetazolamide on the expression of AQP1 in Schwann cells1) Immunoprecipitation (IP) was carried out by using the constructed AQP1 overexpression strain to analyze the cytoskeletal proteins that could interact with AQP1. The immunofluorescence staining was used to verify that AQP1, MYH14 and Ub were co-located in Schwann cells. Repeating the IP test to verify the interaction of AQP 1 with MYH14 under the intervention of acetazolamide.2) Repeat the AZA intervention tests 12h. The cell membrane and cytoplasmic protein were extracted by membrane protein extraction kit separately. The expression of protein translocation was observed by immunoblotting.3) Construction of MYH14-shRNA: The rat objective gene was cloned into the lentivirus,and the correct clones were obtained by PCR and sequencing. The recombinant plasmid was co-transfected into Schwann cells. Immunofluorescence was used to observe the transfection efficiency. The level of MYH14 knockout was verified by immunoblotting.4) CCK-8 test was performed to test the cell viability and MG 132 concentration under MYH14-shRNA influence.5) The cells were divided into normal control group, acetazolamide inhibition group,acetazolamide inhibition intervention group. The untreated cells were knocked out MYH14, then acetazolamide was added to observe the translocation of AQP1 protein.6) To investigate the effect of acetazolamide on the level of ubiquitination in the cells.The cells were divided into normal control group, acetazolamide inhibitor group and acetazolamide inhibition group. The intervention group was treated with the pre-ubiquitination inhibitor for 1 h. Then addition of acetazolamide was used to observe the changes of AQP1 protein.C. Experimental study on expression and regulation of AQP1 in facial nerve edema in rats.1. Construction and evaluation of facial nerve injury model in rats.1) Wistar rats were treated with facial nerve axotomy of the left side and the right side was the control side. The rats were treated with the posterior incision approach. After 14 days, the rats were perfused and fixed. Continuous frozen sections were performed of rat brain stem part. Toluidine blue staining was used to count the neuron number of the facial nucleus.2) The distribution and expression of AQP1 were observed by immunohistochemistry to determine the phase change of AQP1. The expression of AQP1 was detected by immunohistochemistry.3) The expression of AQP1 protein in the facial nerve tissue of the temporal part was detected by immunoblotting to verify the trend of AQP1 protein level.2. Study on the MAPK signal cascade changes in facial nerve injury model Through repeated construction of animal model, timely MAPK protein phosphorylation changes of injured nerve were detected by immunoblotting.3. Regulation mechanism of MAPK on AQP1 expression in facial nerve edema.Animal model was divided into normal control group, model group, model intervention group and inhibitor blank control group. Intervention group was administrated with proper drug concentration and then received surgery. Immunoblotting was used to observe AQP1 protein changes.D. Statistical analysis of the results which will supply new ideas on follow-up studies.[Results]1. AQP1 expressed in RSC96. AQP1 reached the peak at 6h. At the same time point, the expression level of AQP1 was up-regulated by 150mM (P < 0.001), and the expression of AQP1 by 240mM (P < 0.01).2. p-ERK and p-p38 protein phosphorylation levels were modulated by different concentration of hypotonic solution. 150mM :The expression of p-ERK was the highest at 30 min (P < 0.001), p-p38 was highly expressed at 1 h (P < 0.001). 240mM :The expression of p-ERK was the highest at 15 min (P < 0.001),The p-p38 reached the strongest at 5 min (P <0.001). In U0126, an ERK inhibitor processed group, AQP1 expression of 2 treated groups showed significance decreased(150mmol/L: P<0.05;240mmol/L: P<0.001) , but showed no difference compared with control groups. The expression of AQP1 protein in SB203580 group was lower (150mmol/L: P<0.05;240mmol/L : P < 0.001 ), but higher than normal control group (P < 0.01).3. The expression of AQP1 protein was significantly increased after Lenti-AQP1 infection (P < 0.001). The protein level of AQP1 was significantly decreased after 12 hours of acetazolamide intervention (10-5mol/L:P<0.001,10-6mol/L:P<0.01),in a concentration-dependent manner(10-5:10-6mol/L:P <0.05).4. The phosphorylation level of p-ERK was the strongest at 6 h (P <0.001) after 12 hours of acetazolamide intervention. ERK inhibitor U0126 could significantly prevent the expression of AQP1 in acetazolamide (P < 0.05).5. MYH14-shRNA sequence was correct. After transfecting Schwann cells, MYH14 was significantly decreased (P <0.01) and without influencing cells' viability.6. After acetazolamide intervention, the downstream cytoskeletal MYH14 interacts with AQP1, activate the MAPK signal pathway, translocates from the cytoplasm to the cell membrane and reaches peak at 1h (P <0.01). After MYH14-shRNA plasmid transfection, AQP1 expression increased and AZA inhibition of AQP1 disappeared(P<0.05).7. After cell pretreatment with MG132, ubiquitin proteasome pathway was inhibited,AZA inhibition of AQP1 disappeared(P <0.001). It proved that AQP1 was degraded by ubiquitin proteasome pathway.8. Observation of facial nerve injury showed that facial nerve function score was correlated with facial palsy. The facial nucleus neuron count was significantly lower than control side (P<0.05) . IHC and WB both proved that AQP1 expression reached peak at 7 d after facial axotomy (P<0.001).9. ERX/P38 signal pathway was activated after facial nerve injury. p-ERK and p-p38 increased at 3th day post-operation (P<0.001) . In ERK inhibitor U0126 intervention group, AQP1 decreased (P<0.001) , showed no difference with control group. In p38 inhibitor SB203580 group, AQP1 decreased compared with operation group (P<0.001), which was similar to cellular change. All the results showed that MAPK signal pathway might participate in AQP1 modulation after facial nerve edema.[Conclusions]1. AQP1 is the main factor in the formation of Schwann cells edema.2. MAPK involved in the regulation of hypotonic cell edema formation is through AQP1 expression regulation.3. Acetazolamide in Schwann cells works through the transfer mechanism and ubiquitin proteasome pathway to complete the regulation of AQPl expression.4. MAPK is crucially involved in the expression of AQP1 in rat facial nerve edema.