Effect Of Allyl Glycoside On Functional Change Of Schwann Cells Under The Circumstances Of High Glucose

Objective1. To establish Schwann cell model having the features of diabetic lesion ; this cell model can act as an experimental tool and a test system of screening effective drugs and its mechanism research.2. To make further observations on the functional change features of Schwann cells to provide a direct experimental basis for clarifying the mechanism of diabetic neuropathy.3. To test and verify if allyl glycoside, which is extracted from traditional Chinese medicine, has the effect of improving experimental diabetic lesion of Schwann cells.4. To research the possible mechanism of action of allyl glycoside to accumulate experimental data for modernization of traditional Chinese medicine and bring forth new ideas of theory of traditional Chinese medicine.Methods1. Isolation, purification, culture, and identification of Schwann cells:Adopting Brokes modification, Schwann cells were isolated from the sciatic nerves of newborn Wistar sucking rats and purified. They were cultured in DMEM culture solution, containing 10% fetus bovine serum and 0. 1% GS. Identification method: the cells were adhered to the slide and fixed; the immune cytochemical staining of S100 antibody and MBP antibody (ABC method) and hematoxylin counter staining were made in order to calculate the positive rate of immunostaining positive cells.2. Effect of culture with high glucose on activity and proliferation of Schwann cells:After strapping-wall culture of Schwann cells, the culture solution was thrown aside. They were divided into the normal group, the high glucose group, and the high glucose and hyperosmotic group. The culture solution, containing normal glucose (5.0 mmol/L GS), high glucose (30. 0 mmol/L GS), high and hyperosmotic glucose (30. 0 mmol/L GS +100 mmol/L NaCl), was cultured for 48 hours. XTT method to measure the activity of Schwann cells under three different cultural circumstances was adopted. 50μl XTT/PMS applied solution was added to each pore of the cells of each group; after the cells were protected from light, shaken, and cultured for 4 hours, 450-500 mm wavelength on the enzyme labelling apparatus was adopted to measure 0D value of each pore. By the use of ~3H-TdR incorporative method, the proliferative ability of Schwann's cells which were cultured under different cultural circumstances in the three groups was measured. ~3H-TdR of 0. 5 uci/10μl was added to each pore of cells of the three groups; these cells were continually cultured for 16 hours. After cell-cleavage disposal, 100μl of lytic solution of cell-cleavage were added to a test bottle which contains 4 ml of scintillation liquid to measure CPM reading on the scintillation counter.3. Effect of allyl glycoside on the activity and change of proliferative ability of Schwann cells cultured by high glucose:After strapping-wall culture of Schwann cells, the culture solution was thrown aside. They were divided into the normal group (LG), the high glucose group (HG), the allyl glycoside group, and the radix ophiopogonis group, normal glucose (5. 0 mmol/L GS), high glucose (30. 0 mmol/L GS), high glucose plus allyl glycoside (30. 0 mmol/L GS+8mg/ml AG) , and high glucose plus radix ophiopogonis(30.0 mmol/L GS+10mg/ml RO) were added to the culture solution to be cultured. After 48 hours in the four groups the activity and proliferative ability of Schwann cells were measured using XTT method and ~3H-TdR incorporative method, and the test results were recorded.4. Synchronous observations on the effect of allyl glycoside on the proliferative ability NGF synthesis, and expressive change of Schwann cells cultured by high glucose:After strapping-wall culture of Schwann cells for 12 hours, the culture solution was thrown aside. They were divided into the normal group, the high glucose group, the allyl glycoside group, and the radix ophiopogonis group. normal glucose (5. 0 mmol/L GS), high glucose (30.0 mmol/L GS), high glucose plus allyl glycoside (30. 0 mmol/L GS +8mg/ml mgAG), and high glucose plus radix ophiopogonis (30. 0 mmol/L GS +10mg/ml R0) were respectively added to the culture solution for culture. After 48 hours, the supernatant fluid was collected. OD values of reading on the enzyme labelling in all group were measured with Elisa method. Schwann's cells were collected and their proliferative ability was measured by ~3H-TdR in- corporative method. The general RNA was prepared; the expressive levels of NGFmRNA in all groups were measured by the real-time fluorescent quantitative PCR method.Results1. Isolation, purification, culture, and identification of Schwann cells:Schwann's cells were isolated from the sciatic nerves of Wistar sucking rats; about 1×10~6 cells were obtained from each rat; under high power lens observations were made, the cellular body was less; the cell was spindle in shape. The specimen was taken and stained by ABC method; in the staining cells of anti-S100 and anti-MBP, the positive rates were respectively 92±2% and 93±3% (N=10). When the positive rates of two methods were compared, there was no significant difference.2. Effect of high glucose culture on activity and proliferative ability of Schwann cells: The activity of Schwann cells measured with XTT method. As compared with the normal group, the cellular activity in the high glucose group was markedly attenuated and there was a very marked difference (P<0.01). As compared with the normal group, the cellular activity in the high glucose and hyperosmotic group was markedly attenuated, and there was a very obvious difference (P<0. 01). When the high glucose group was compared with the high glucose and hyperosmotic group, there was also a very obvious difference. It showed that high glucose had a marked inhibitory effect on the activity of Schwann cells, but was less than in the presence of hyperosmotic state.The proliferative ability of Schwann cells was measured with ~3H-TdR incorporative method. As compared with the normal group, the cellular proliferative ability in the high glucose group was obviously inhibited, and there was a very marked difference (P<0.01), as compared with the normal group, the cellular proliferative ability in the high glucose and hyperosmotic group was also markedly inhibited, and there was a very obvious differenct (P<0. 01). When the high glucose group was compared with the high glucose and hyperosmotic group, there was a significant meaning. It showed that the culture with high glucose had an inhibitory effect on the function of Schwann cells, but the degree was less in the presence of hyperosmotic state.3. Effect of allyl glycoside on the change of the activity and proliferative ability of Schwann cells cultured by high glucose: The activity of Schwann cells was measured with XTT method. As compared with the normal group, the cellular activity in the high glucose group was markedly attenuated, and there was a very marked difference (P<0. 01). As compared with the high glucose group, the cellular activity in the allyl glycoside group was not obviously improved, and there was no obvious difference (P>0.05). As compared with the high glucose group, the cellular activity in the radix ophiopogonis group was also not markedly improved, and there was no significant difference. It showed that there was no improving effect when the lowering of the cellular activity caused by high glucose was treated by allyl glycoside and radix ophiopogonis.The proliferative ability of Schwann cells was measured with ~3H-TdR incorporative method. As compared with the normal group, the cellular proliferative ability in the high glucose group was markedly lowered, and there was a significant difference (P<0. 01). As compared with the high glucose group, the cellular proliferative ability in the allyl glycoside group was obviously improved, and there was an obvious difference (P<0. 01). When the radix ophiopogonis group was compared with the high glucose group, there was no significant difference (P<0. 05). It showed that the inhibitory change of the proliferative ability of Schwann cells caused by high glucose was improved by allyl glycoside.4. Effect of allyl glycoside on the proliferative ability, NGF expression and synthesis change of Schwann cells in the circumstances of high glucose:As stated above, allyl glycoside had an obvious improving effect on the inhibitory change of the proliferative ability of Schwann cells under the circumstances of high glucose, but radix ophiopogonis had no marked influence on the inhibitory action. As compared with the normal group, NGF synthesis in the high glucose group was obviously reduced (P<0. 01). It showed that high glucose had an inhibitory effect on the proliferative ability and NGF synthesis of Schwann cells. As compared with the high glucose group, the function of NGF synthesis in the allyl glycoside group was markedly improved (P<0.01).At the same time the expression of NGFmRNA was obviously raised (P<0.01). It showed that allyl glycoside had an improving effect on the proliferation, NGF espression , and synthesis lesion of Schwann cells. As compared with the high glucose group, in the radix ophiopogonis group there was no significant difference (P>0. 05). It showed that there was no marked influence on the proliferation, NGF expression, and synthesis of Schwann cells treated with radix ophiopogonis. Conclusion1. Adopting Brockes modification, Schwann cells were isolated from the bilateral sciatic nerves of newborn Wistar sucking rats. The cell number isolated from sucking rats amounts to 1×10~6 cells. They are immunized, stained, and identified with ABC method of S100 antibody and MBP antiblody. The positive rate accounts for over 90%. It shows that this method isolating Schwann cells can meet the needs of experimental research either in efficiency or in purity.2. The culture of high glucose has marked inhibitory effect on both the activity or proliferative ability of Schwann cells. It shows that the modelling condition that Schwann cells cultured by high glucose serve as the lesion model of experimental diabetic neuropathy has been possessed. Further study of the charactiristics of pathologic changes can serve as a experimental tool and test system for helping us to know the explanation of diabetic neuropathic pathologic mechanism from one aspectand screening effective drugs.3. Although allyl glycoside does not play a role in activity change of Schwann cells cultured by high glucose, its inhibitory change of the proliferative ability has a marked improving effect. The control-treatment drug radix ophiopogonis has not such effect.4. The synchronous observative results of the proliferation, NGF synthesis, and influence of expressive change of Schwann cells cultured by high glucose show that (1) As the proliferative ability of expression Schwann cells cultured by high glucose develops marked changes, their NGF and synthesis also develop obvious changes. It shows that NGFmRNA expression goes down and NGF synthesis drops. The pathologic significance is apparent. (2) Allyl glycoside can markedly improve the inhibitory change of proliferative ability. NGFmRNA expression, NGF synthesis of Schwann cells cultured by high glucose. Because the relay way of the signals mediated by NGF is closely related to proliferation of Schwann cells, there is a reason for guessing that allyl glycoside may influence a given link of NGF synthesis andthrough of intracellular cascade mechanism reverse the lowering of proliferative ability of Schwann cells cultured by high glucose. It will greatly help to develop the latent energy of saving others and themselves of Schwann's in the pathologic conditions.