Effect Of Tongluo Tangtai Prescription On Differentiation Of SCs In SD Rats Under High Glucose Culture And Research On Culture Method Of DRGn In SD Rats Under High Glucose And Hypoxia Conditions

Objective:To confirm the efficacy of Tongluo Tangtai prescription in promoting the expression of myelin regulatory factor and improving the occurrence and development of diabetic peripheral neuropathy?DPN?by observation of the effect of Tongluo Tangtai prescription in the medullization of Schwann cells?SCs?in SD rats under high glucose culture;as well as research the optimum culture condition of dorsal root ganglion neuron?DRGn?under high glucose and hypoxia conditions,thereby provide a certain scientific theoretical basis for the basic research of DPN.Methods:1.The SCs collected from sciatic nerve of newborn 3-5 day-old rats were carried out primary culture,and the third generation well-grown SCs were selected as research object.The DPN SCs model was established by culturing SCs in high glucose medium?50mM?for 72h.The expression of myelin regulatory factor P0in SCs under high glucose condition at three time points of 24h,48g and 72h after intervention with Tongluo Tangtai drug serum were detected by using Western blot method on the basis of previous research.2.The dorsal root ganglion neurons collected from dorsal root ganglion of newborn 3 day-old rats were performed primary culture,and the well-grown neurons were selected as research object.In order to establish DPN dorsal root ganglion neuron model,the cell morphology was observed under the light microscope,the ROS expression was detected,as well as the damage of DRGn induced by high glucose with various concentrations and hypoxia at time points of 0,6h and 24h were determined with CCK-8 method.Results:1.The purity of SCs identified by S-100 immunofluorescence method reached 90%or more,which could be used for performing the next experiment.Western blot detection results suggested that,compared with control group,the expression of P0 protein in SCs at 48h and 72h in culture of high glucose group were decreased,the decrease degree of which were more remarkable with the increase of time,and the differences were statistically significant?P<0.05??P<0.01?;compared with high glucose group,the expression of P0 protein in SCs at 48h and 72h after intervention with 10%Tongluo Kangtai drug serum were increased,the increase degree of which were more significant with the increase of time,and the differences were statistically significant?P<0.05??P<0.01?;moreover,the expression of P0protein in SCs after the intervention of Tongluo Kangtai drug serum with concentrations of 5%and 20%were only increased at the time point of 72h,and the differences were statistically significant?P<0.05?;the difference in the expression level of P0 protein in SCs at 24h in culture of high glucose group was not statistically significant compared with that of control group?P>0.05?;the difference in the expression level of P0 protein in SCs of high glucose group after the intervention of Tongluo Kangtai drug serum with various concentrations were not statistically significant compared with that of high glucose group?P>0.05?;the difference in the expression level of P0 protein in SCs of high glucose group after intervention with2.5%Tongluo Kangtai drug serum was not statistically significant compared with that of high glucose group?P>0.05?.2.Well-grown DRGn was obtained,and its cell purity identified by?-tubulin?immunofluorescence method reached 90%or more.The optimum concentration of high glucose,the optimum concentration of hypoxia,as well as the optimum concentration of high glucose combined hypoxia explored by cell morphology observation,ROS expression detection and CCK-8 method was 50mM high glucose,hypoxia300uM,and high glucose and hypoxia 50mM+300uM,respectively,and the optimum culture time obtained under the same methods was 24h.Conclusion:1.The SCs of neonatal rats was cultured with double enzyme digestion method and purified by cytarabine+differential adhesion method.The purity of SCs was above 90%by S-100 immunofluorescence,and the primary culture scheme was feasible.2.The expression of myelin regulatory factor P0 protein in SCs can be inhibited by high glucose,which has correlation with time.3.The inhibition effect of high glucose on the expression of myelin regulatory factor P0 protein can be improved by Tongluo Tangtai prescription through up-regulating protein expression and promoting medullization of SCs,thereby improving the pathological characteristics of remyelination disorder in DPN,followed by preventing and treating DPN.4.Using a modified double enzymatic digestion DRGn cultured neonatal rats,purified cytarabine,?-tubulin?immunofluorescence cell purity of 90%or more,the success of the culture DRGn.5.The pathological changes of DRGn phenotype,ROS expression and its activity inhibition can be induced by 50mM high glucose,300mM CoCl₂ as well as 50 mM high glucose+300mM CoCl₂,and the high glucose and hypoxia at this concentration are capable of inducing DPN DRGn model.