Effects Of Tongluotangtai Decoction On Schwann Cells Apoptosis Induced By High Glucose

Objective: The Objective of this research were to study the influence of Tongluotangtai decoction on primary cultured Schwann cell apoptosis induced by high glucose and PI3K/AKT signal pathway,and explore the molecular mechanism of Tongluotangtaidecoction on treating diabetic peripherl neuropthy(DPN),so as to provide certain evidence for the mechanism of preventing and treating DPN with Tongluotangtai decoction.Methods:Sciatic nerve trunk was obtained from newborn SD rats for primary cultured Schwann cells and subcultured.Schwann cells of the third generation with sound growing state were taken as the research objectives.50 m M high glucose was adopted for culture for 72 h to induce DPN Schwann cell models.Serum containing Tongluotangtaidecoction(four concentrations)were added separately for culture and were divided into six groups.The rate of apoptosis was detected by Annexin-FITC /PI double staining flow cytometry of the Schwann cells that cultured in different medium for 72 h.Transmission electron microscope was applied to observe the changes of cell ultrastructure.The Western Blot method was applicated to detect the expression of active Caspase-3,AKT,andp-AKT in high-glucose-cultured Schwann cell for 24 h,48h,and 72 h.the expression of the apoptosis regulator Bax,Bcl-2,and Bcl-x L m RNA by RT-PCR method.To observe the effects of medicated sera of Tongluotangtai decoction on apoptosis of Schwann cells induced by high glucose.By studying its influence on PI3K/AKT signal pathway,the molecular biological mechanism of Tongluotangtai decoction on treating DPN was studied from the perspective of inhibiting Schwann cells apoptosis.Results: 1.The detection of apoptosis rate with Annexin-FITC/PI double staining flow cytometry proved that 50 m M high glucose culture can lead to marked apoptosis of Schwann cells.Compared to the control group under 72 h of high glucose culture,the apoptosis rate of high glucose group increased markedly(p<0.01);compared to the high glucose group,the apoptosis rate of 5% and 10% medicated sera of Tongluotangtaidecoctiongroup declined markedly(p<0.01),and the 2.5%,20% medicated sera of Tongluotangtai decoction group showed declined apoptosis rate(p<0.05).2.When observing the ultrastructure with transmission electron microscope and with 72 h of culture,the apoptotic Schwann cells of high glucose increased.Aggregated nuclear chromatin was observed.There were a large number of vacuoles in the cytoplasm.Markedly swollen mitochondrion,disappeared mitochondrial cristae,and expanded rough endoplasmic reticulum were observed;the Schwann cells damage of medicated sera of Tongluotangtai decoction group were improved at varying degrees.3.Western Blot results prompted that compared with the control group,the expression of active Caspase-3 of high glucose group under 24 h,48h and 72 h of culture increased markedly,and the difference was of significance(P<0.01);compared with the high glucose group,the expression of active Caspase-3 of 5% and 10% medicated sera of Tongluotangtai decoction group under 24 h,48h and 72 h of culture declined markedly(P<0.01).Compared with the control group,the p-AKT expression of high glucose group was declined significantly(P<0.01);compared with the high glucose group,the expression of p-AKT protein of 2.5%,5% and 10% medicated sera of Tongluotangtai decoction under 24 h,48h and 72 h of culture rose up and the difference was of significance(P<0.01);4.RT-PCR method analysis results prompted that compared with the control group,the expression of bcl-2m RNA and Bcl-x Lm RNA of high glucose group under 24 h,48h and 72 h of culture declined markedly(p<0.01),while the expression of Baxm RNA of high glucose group under 24 h,48h and 72 of cultured increased markedly(p<0.01);compared with the high glucose group,the expression of bcl-2m RNA and Bcl-x Lm RNA of 5% and 10% drug serum group under 24 h,48h,72 h of culture increased markedly(P<0.01~0.05),while the expression of Baxm RNA of 5% and 10% medicated sera of Tongluotangtai decoction group under 48 h and 72 h of culture declined markedly(p<0.01).Conclusion: 1.50 m M highglucose culture may induce Schwann cell apoptosis,and may lead to marked cell apoptosis after 72 h of intervention.The Schwann cells after 7h of high glucose culture can be regarded as Schwann cells model to study DPN.2.The medicated sera of Tongluotangtaidecoction can efficiently activate the PI3K/AKT signal pathway of high glucose cultured Schwann cells.3.The medicated sera of Tongluotangtai decoction rescues hyperglycemia-induced apoptosis may relate to activation of the PI3K/AKT pathway.