The Study About The Stability Of The Oral Liquid Of2,3,5,4’-tetrahydroxystilbene Glucoside And Its Effects On LPS-induced Expression Of INOS In Microglia

Objective Based on the apperance of muddy phenomenon such as precipitation during the storage of the oral liquid of2,3,5,4’-tetrahydroxystilbene glucoside (TSG), we studies the stability of the2,3,5,4’-tetrahydroxystilbene glucoside solution.Methods To investigate the influence of different auxiliary materials such as stabilizer, antioxidant, chelating agent, and solubilizer to the content of TSG in the oral liquid of TSG using the method of HPLC.Results1. TSG solution was suitable for the storage in mildly alkaline environment, and its pH value should be modulated by weak alkaline substances.2. TSG solution was unstable, and low temperature environment did not significantly alter the degradation of TSG.3. In the prescription, PEG-400significantly reduced the degradation of TSG; addition of EDTA-Na2did not protect the active ingredients, but neper gold indeed protected the active ingredients from degradation. 4. There was no significant difference between the effects of PEG-400at the content of15%and20%(P>0.05) respectively. When the content of neper gold was no less than0.03%, it exhibited protective effect on the active ingredients, and this effect was dose-dependent between the content of0.03%and0.07%. There is no further increase in the protective effect when the content was larger than0.07%.5. Although glycerin works as preservative, in the present study TSG still exhibited significant degradation when glycerin was added as a solubilizer. TSG was almost totally degradated at the end of the experiment.Conclusions TSG is unstable. The addition of PEG-400and Neper gold can protect the TSG from degradation. Thus both of them can be used as the auxiliary material of the oral liquid of TSG solution. Objective To study the influences and possible mechanisms of TSG on LPS-induced expression of iNOS in microglia as well as the microglia-derived conditioned medium-induced neuronal injury.Methods We evaluated the influences of TSG to the LPS-induced expression of iNOS and NO release in BV-2cells using Western blot and NO detection kit, respectively. The effects of TSG on BV-2cell-derived conditioned medium-induced neuronal injury were investigated by nucleus staining and LDH assay kit. By using PCR, we detected the effect of TSG on iNOS gene expression. By using Western blot, we also investigated the changes of IκB-α, NF-κB, and p-NF-KB expression in order to confirm the relationship between the effects of TSG and the classical NF-κB signaling pathway, and investigated the changes of p-JNK, p-ERK1/2, and p-p38in MAPK pathway in order to confirm the relationship between the effects of TSG and MAPK signaling pathway. The change of the binding activity of NF-κB to its DNA element was measuted by NF-κB DNA binding activity kit in order to further explore the mechanism of TSG. Finally, we detected the expression of iNOS, the release of NO as well as the binding activity of NF-κB to its DNA element in primary microglia to confirm that TSG performed similar effects in primary microglia.Results1.TSG significantly inhibited the LPS-induced expression of iNOS as well as the release of NO in BV-2cells. The effect of TSG is presented as dose-and time-dependent way.2.TSG inhibited the BV-2cell-derived conditioned medium-induced neuronal injury.3.TSG did not influence the LPS-induced dagradation of IκB-α, the increase in NF-κB phosphorylation and nucleus translocation in BV-2cells.4. TSG had no significant effects on the change of MAPK signal, including p-JNK, p-ERK1/2, and p-p38.5. TSG inhibited the binding of NF-κB to its DNA element in BV-2cells.6. TSG significantly inhibited the expression of iNOS, the release of NO, as well as the binding of NF-κB to its DNA element in primary microglia.Conclusions1.TSG has inhibitory roles in the LPS-induced expression of iNOS as well as the release of NO in microglia;2.TSG has protective roles in the BV-2cell-derived conditioned medium-induced neuronal injury;3.TSG exerts its effects by inhibiting the binding of NF-κB to its DNA element, but not interfering with the MAPK-IκB-α-NF-κB pathway.