Effects Of Phytoplex Renew On The Secretion Of BDNF And The Expression Of AQP1 In Schwann Cells After Hypoxia

Aquaporins?AQPs?,were discovered in the isolation and purification of Rh blood cell antigens by Agre in 1988.It is a class of highly conserved membrane proteins that mediate cell transmembrane transport,and there are very similar protein sequences and three-dimensional structures between the various subtypes.Among them,aquaporin 1?AQP1?is the first aquaporin to be found in many glands and osmotic active tissues.Previous studies have shown that AQPs are expressed in the central nervous system,and few studies have shown that AQPs are expressed in the peripheral nervous system and glial cells.In recent years,studies have shown that AQP1,AQP2 and AQP4 are expressed in the peripheral nerve and glial cells.It has been confirmed that AQP1 is the most abundant aquaporin in the peripheral nervous system and found that AQP1 is located in the peripheral nervous system of Schwann cells.AQP1 is expressed in peripheral nervous system glial cells like AQP4 in the central nervous system Glial cells within the same expression.But the correlation between peripheral nerve edema and the expression of AQP1 is unclear.Neuronal edema and hypoxia exacerbation,increased expression of hypoxia-inducible factor 1?HIF-1??after peripheral nerve injury.However,the expression of AQP1 and HIF-1? in Schwann cells after peripheral nerve injury is not clear.Because Schwann cells are peripheral nerve-specific glial cells,so Schwann cells as the basis for cell damage model of the production,First,the expression of AQP1 and HIF-1? in damaged nerve cells and the correlation between AQP1 and HIF-1? were clarified.Secondly,the recent reports on the repair of peripheral nerve injury were less and the effect of treatment was not satisfactory.In this study,we investigated the effects of PhytoPlex Renew extract on AQP1 and HIF-1? after Schwann cell hypoxia injury,and to study the repair of nerve damage.Methods: 1.Extract the original Schwann cells from SD neonatal facial nerve.SD ratswere sacrificed and the male and female were not cultured for 3-5 days.The primary culture of Schwann cells was carried out by enzymatic digestion,and purified by differential labeling method.in order to prove the presence of AQP1 in the peripheral nerve,the immunofluorescence staining was used to identify s-100 and AQP1 Protein expression.2.The production of Schwann cell hypoxia model.The adherent cells were incubated with different concentrations of CoCl₂?2ul / m,4lul / ml,6ul / ml,8ul / ml,10 ul / ml?and observed for 24 hours.The morphological changes were observed under microscope and the cells were collected.To determine the optimal CoCl₂ concentration for subsequent testing,western blot was used to detect the expression of AQP1 and quantitative analysis of AQP1 content by GraphPad Prime5.3.To determine the concentration of PhytoPlex Renew.The adherent SCs were incubated with different concentrations of resuscitation extract?0.5 ul / ml,1ul / ml,2.5ul/ ml,5ul / ml,10 ul / ml?and tested for CCK-8 after 24 hours.ODs value analysis of the proliferation of Schwann cells and draw the table and the line chart for analysis.4.To explore the repair effect of Euphorbia pulcherrima extract on injured Schwann cells.The optimum concentration of CoCl₂ and PhytoPlex Renew were selected and divided into three groups: blank control group,hypoxia model group and PhytoPlex Renew group.Take the completely adherent cells to carry out scratches test,observe 24 h and 48 h cell migration and take pictures.Three groups of cells were observed for 7 days and western blot were detected for 7 days in AQP1 and HIF-1?.Results: 1.Primary Schwann cells were successfully cultured.Fluorescence staining showed that AQP1 was expressed on the Schwann cell membrane.2.Selecting the concentration of CoCl₂ is 6ul / ml for the optimal hypoxia.The Schwann cell morphology was rounded,axonal shrinkage after 24 hours.And Western blot showed the highest expression of AQP1 protein.3.Selecting the concentration of PhytoPlex Renew is 2.5ul / ml.CCK-8 method wasused to detect the cell proliferation.The absorbance value at 450 nm was determined by the microplate reader.The statistical analysis was used to analyze the OD value of the PhytoPlex Renew group and the blank control group,P> 0.05,the difference was not statistically significant Significance,that is,after adding PhytoPlex Renew,the cell proliferation is not obvious.5-7 days OD value analysis,P <0.05 difference was statistically significant,that is,adding the resuscitation extract after cell proliferation significantly.4.Selecting the concentration of PhytoPlex Renew is 2.5ul / ml and concentration of CoCl₂ is 6ul / ml.The experiment was divided into three groups: blank control group,hypoxia model group,PhytoPlex Renew repair group.Cell scratches were used to observe cell migration to promote cell proliferation.The results showed that compared with the blank control group and the hypoxia group,P <0.05,the resuscitation grass extract could promote the migration of the cells significantly.There was no statistically significant difference between the hypoxia group and the blank control group?P> 0.05?.The expression of AQP1 and HIF-1? was detected by Western blotting.The expression of AQP1 and HIF-1? was significantly decreased in the cell injury.The results showed that the expression of AQP1 and HIF-1? was down-regulated.Conclusion: 1.AQP1 is an important factor to regulate the water transport of Schwann cells.2.AQP1 expression is an important factor in facial nerve hypoxia edema.3.The production of CoCl₂ hypoxia model and the increased expression of HIF-1?,thus induced AQP1 expression increased.4.PhytoPlex Renew to promote cell proliferation and migration,and can down-regulate HIF-1? and AQP1 expression.