Study On The Effect Of Ascorbate Combined With Cold Atmospheric Plasma On Glioma

Glioma is the most common primary intracranial malignant tumor with high mortality.In recent decades,glioma treatment has made rapid progress,but glioma patients’prognosis has not improved significantly.Therefore,it is an urgent clinical need to explore new adjuvant therapy strategies for glioma.Ascorbate is an adjuvant drug for cancer treatment,which has been proven many times in clinical trials.It exerts a mechanism of promoting oxidation in the physiological concentration to treat cancer,and its anti-cancer activity in combination with radiotherapy and chemotherapy is significantly higher than that of ascorbate alone.Cold atmospheric plasma（CAP）is a kind of physical device used to treat cancer.It is a gas containing Reactive Nitrogen Oxides（RNOS）produced by spraying.It has a selective killing effect on tumor cells.In this study,based on the can produce reactive oxygen species or reduce the activity of antioxidant system in order to break the cancer cells within the fragile redox equilibrium anti-cancer therapy,the high-dose ascorbate combined with cold temperature plasma-activated medium（CAP–activated medium,PAM）treatment glioma cell lines（U251,U87）and normal cell line mice primary astrocytes（Astroglia,AS）.In vitro,cells were treated with 4m M ascorbate for 1h and then treated with complete medium exposure with CAP for 3min or 6min for 2h.Cell Titer-Blue staining method used to detect cell viability.We found that pretreatment with ascorbate increased the sensitivity of glioma cell lines U251 and U87 to PAM,and compared with normal cell line AS,high-dose ascorbate combined with PAM selectively inhibit the cell viability of U251 and U87.Using the drug synergy software Compu Syn analysis,this inhibition is synergistic.The Calcein-AM/PI live cell/dead cell double staining kit was used to detect cell death,and it also verified that the combined use of ascorbate and PAM significantly induced the death of U251 and U87.To further study the inhibition of ascorbate and PAM on U251 and U87 cells,we used a hemocytometer to determine cell proliferation rates,used Annexin-FITC/PI double-staining and flow cytometry to detect apoptosis,and verified signal pathways caused by ascorbate combined with PAM on western blotting.The results showed that the combination of ascorbate and PAM inhibited cell proliferation by inhibiting the activation of the AKT/m TOR signaling pathway,and at the same time,induced cell apoptosis by activating caspase-3 and cleaving PARP.Because high-dose ascorbate auto-oxidizes to produce H₂O₂ and PAM is rich in ROS produced by CAP.Therefore,next,we explored the ascorbate and ROS role produced by PAM in the combined use.ROS probe and GSH/GSSG detection kit used to detect the redox state in the cell.The results showed that after co-treatment with ascorbate and PAM,the ROS levels in U251 and U87 cells increased,and the ratio of GSH/GSSG decreased,indicating that oxidative stress occurred in the cells.The H₂O₂ detection kit found that the combined use of ascorbate and PAM increased the H₂O₂ content in U251 and U87cells,which was higher than the sum of the H₂O₂ content in the cells treated with ascorbate or PAM alone.According to the transcriptome sequencing results,combined with q PCR and western blot,we found that ascorbate treatment promoted the expression of aquaporin AQP3 on the cell membrane,thereby promoting the diffusion of H₂O₂ across the membrane to enter the cell.Based on the sequencing results,we used the GO database to annotate the function of all up-regulated genes in the combination group,and the up-regulated genes were found to be enriched in the MAPK signaling pathway.Eleven genes were randomly selected from all the up-regulated genes in the combination group,and the sequencing results were verified by fluorescence quantitative PCR.It was found that the combination of ascorbate and PAM up-regulated the m RNA levels of C-FOS,C-JUN and IL-6.Because C-JUN is a downstream protein of the JNK signaling pathway,JNK is a subfamily of the MAPK family,and ROS can activate the JNK/MAPK pathway.We used western blot to detect the effect of ascorbate and PAM on the JNK signaling pathway in U251 and U87 cells and found that the combination of ascorbate and PAM activated the JNK signaling pathway.The JNK inhibitor SP600125 partially reversed the cell death induced by the combination.Through this study,the following conclusions are drawn:Compared with normal cells,the combined action of ascorbate and PAM selectively inhibits the cell viability of tumor cells and induces cell death,and the inhibitory effect is synergistic.The pretreatment of ascorbic acid induces the expansion of the expression of the transport membrane protein AQP3 in tumor cells,promotes the transport of H₂O₂ by AQP3 into the cell,causes the increase of intracellular ROS,and induces oxidative stress,thereby achieving the synergistic inhibitory effect of ascorbic acid and PAM.Furthermore,the H₂O₂ produced by auto-oxidation of high-dose ascorbic acid and the rich H₂O₂ in PAM provides conditions.Besides,the increase of ROS in tumor cells activates the JNK/c-Jun signaling pathway and induces apoptosis.In summary,this study confirmed the anti-cancer potential of the combined use of ascorbate and PAM,and expected to provide fresh ideas and methods for glioma treatment.