Abstract:

Objective To discuss the effect of ligustrazine on growth of the glioma stem cells（GSCs）subcutaneous xenografts in the nude mice， transforming growth factor-β （TGF-β） signaling pathway and epithelial-mesenchymal transition（EMT），and to provide the reference for selecting drugs in the clinical therapy of glioma. Methods The human brain glioma U87 cells were collected and the GSCs were cultured， separated， enriched， and identified. Forty female BALB/c nude mice were used to establish the GSCs subcutaneous xenograft models， which was randomly divided into model group and low， medium， and high doses of ligustrazine groups， and there were 10 mice in each group. The mice in low， medium， and high doses of ligustrazine groups were intraperitoneally injected with ligustrazine once every 3 d， and the dosages were 1.5， 3.0， and 6.0 mg·kg^－1， respectively， while the mice in model group were only given equal volume of saline，and this process continued for 15 d. The transplanted tumor volumes and tumor weights were detected， and the inhibitory rates of tumor weights of the mice was caculated. The tumor growth curve was drawn.HE staining was used to observe the pathomorphology of tumor tissue of the mice；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of TGF-β1 and transforming growth factor β receptor Ⅰ（TβRⅠ） mRNA in tumor tissue of the mice in various groups； Western blotting method was used to detect the expression levels of TGF-β1， TβRⅠ，small mother against decapentaplegic homolog 2/3 （Smad2/3）， phosphorylated Smad2/3 （p-Smad2/3），E-cadherin， TWIST family bHLH transcription factor 1 （TWIST1），Vimentin， and Snail proteins in tumor tissue of the mice in various groups. Results The GSCs were successfully enriched and separated from the U87 cells， and the immunofluorescence staining resutls showed the positive expression of stem cell marker CD133. Compared with model group， the transplanted tumor volumes of the nude mice in low， medium， and high doses of ligustrazine groups were significantly decreased （P<0.05）， in a dose-dependent manner； the tumor weights were also significantly decreased （P<0.05） in a dose-dependent manner as well. The inhibitory rates of tumor weights in low， medium， and high doses of ligustrazine groups were 12.72%， 39.90%， and 55.36%， respectively.The HE staining results showed that the cells in tumor tissue of the mice in model group were gradually increased and exhibited good morphology and tight connection， the growth density was high， and the atypical nuclei was obviously； the tumor tissue structures of the mice in low， medium， and high doses of ligustrazine groups were disordered， the cell density was gradually decreased， the nuclear condensation and karyorrhexis were increased，the necrotic zones were gradually enlarged， and these improvements became more obvious as the increasing of the dose of ligustrazine. The RT-qPCR results showed that compared with model group， the expression levels of TGF-β1 and TβRⅠ mRNA in tumor tissue of the mice in low， medium， and high doses of ligustrazine groups were decreased （P<0.05） in a dose-dependent manner.The Western blotting results showed that compared with model group， the expression levels of E-cadherin protein in tumor tissue of the mice in low， medium， and high doses of ligustrazine groups were increased （P<0.05），and the expression levels of TWIST1，Vimentin，Snail，TGF-β1，TβRⅠ，and p-Smad2/3 proteins were decreased（P<0.05） in a dose-dependent manner. Conclusion Ligustrazine can inhibit the growth of GSCs subcutaneous xenografts in the nude mice， and can interfere the TGF-β1/Smad2/3 signaling activation and EMT induction.

Key words: Ligustrazine, Glioma stem cell, Transforming growth factor-β, Epithelial-mesenchymal transition, Small mother against decapentraplegic homolog 2/3