Based On Metabolomics Analysis And Validation Of The Effect Of Propofol On Glioma Resistance And Related Pathway Mechanism

Background:Glioma is a primary malignant tumor of the central nervous system.It is highly invasive,so it is the most common and most aggressive brain tumor in adults.Because it is easy to relapse,the median survival period is deficient,only 12-15 months after the initial diagnosis.Compared with malignant tumors in other systems,glioma is relatively rare.Its incidence rate is about 5 per 100000 people every year,but it still accounts for about 70% of all adult malignant brain tumors.Due to the powerful proliferative ability of gliomas,they exhibit infiltrative growth in relatively small cranial cavities and have unclear boundaries with normal brain tissue,making it difficult to achieve complete resection even under a microscope.Even with extended resection of normal brain tissue at the edge of the tumor,there is still a possibility of recurrence in most gliomas.The survival benefits of Nitroso Uracil-based chemotherapy for glioma patients have been established for more than 30 years.Later,surgical resection,radiation therapy,and chemotherapy became routine treatment methods for newly diagnosed glioma patients.In recent years,the treatment of glioma also includes immunotherapy,Targeted therapy,photodynamic therapy,Chinese medicine,etc.The treatment status and surgical prognosis are not optimistic.Not only has it brought tremendous pain to patients,but it has also placed a heavy burden on society.In recent years,research on glioma has been a hot topic in medical research.Since Stupp et al.established Temozolomide concurrent chemoradiotherapy followed by Temozolomide adjuvant chemotherapy for the treatment of glioma patients in the critical phase III clinical trial in 2005,the chemotherapy programs developed in the later clinical trials have not been significantly improved.As the first choice of chemotherapy drugs,Temozolomide plays an essential role in treating glioma.However,glioma’s insensitivity and drug resistance to Temozolomide are necessary factors affecting the therapeutic effect.Improving the sensitivity of the tumors to Temozolomide and improving the drug resistance of the tumors to Temozolomide is the focus of enhancing the comprehensive treatment effect of glioma.Research has found that propofol plays an anticancer role in many cancers through different molecular mechanisms,such as liver,lung,esophageal squamous cell carcinoma,and gastric cancer treatment.New research has also confirmed the protective effect of propofol on the development of glioma.Propofol significantly inhibits tumor oxidative stress and glioma growth and can also inhibit the invasion and migration of glioma cells.This effect is mainly achieved by blocking the PI3K/AKT pathway through the mi R-206/ROCK1 axis.Metabolomics is an emerging scientific field that has rapidly developed in recent years.Metabolomics provides a deeper understanding of the metabolic processes in the body by measuring the concentration of small molecule metabolites present in cells,tissues,and body fluids.Due to the ability of metabolomics to detect changes in metabolic products in the body and to identify biological markers with guiding significance in the occurrence and development of diseases,it has become a hot research topic both domestically and internationally.Objective:The effects of Temozolomide alone and in combination with propofol on blood metabolites of glioma-bearing animals were studied by metabonomics.Through the analysis of metabonomics data to enrich the differences of related pathways and in vitro cell experiments,it was verified that propofol could improve the sensitivity of Temozolomide to glioma by affecting the metabolites of glioma.Method:1.Animal models were used to detect the effects of temozolomide alone and in combination with propofol on glioma-bearing animals: human astrocytoma-resistant temozolomide cells(U251MG/TMZ cells)were collected and cultured in vitro,and when the cell growth reached about 70-80% of the fusion degree,they were digested with trypsin,and the cell suspension concentration was adjusted to 5 × 10~7/ml.Inject 0.1ml of cell suspension subcutaneously on the ipsilateral back of nude mice,feed the mice in the usual manner,and observe the growth of the nude mice daily.Use a caliper to measure the size of the tumor in two orthogonal directions and weigh the weight of nude mice.Record the importance of each mouse and the longest diameter(L)of cancer,and its maximum transverse diameter(S)in the vertical direction.Calculate the tumor volume(mm~3)according to the standard formula: tumor volume=0.5 × L × S~2.When the tumor grows to about 150 mm~3(2 weeks after cell injection is expected),mice are randomly divided into the control group,temozolomide administration group,and temozolomide+propofol administration group to start administration.Animal tumor and body weight measurement: After starting administration and administration,the tumor volume and body weight of nude mice were measured every five days.The administration period was 30 days(once every day,15 times).After four weeks of administration,the mice were euthanized,and serum samples were obtained for serum metabolomics testing.2.Metabonomics methods were used to study the effects of temozolomide alone and in combination with propofol on the metabolites in the blood of glioma-bearing animals: mouse blood samples were unfrozen,centrifuged,and operated on the computer,and the models were non-targeted metabonomics studied using liquid chromatographymass spectrometry(LC-MS)technology.By screening differential metabolites,valuable biomarkers were found for further bioinformatics analysis.3.Cell experiment verified that propofol could improve the sensitivity of temozolomide to glioma by influencing the metabolites of glioma and its related mechanism: after passage,U251MG/TMZ cells in logarithmic phase were selected for parallel planking and divided into four groups,namely,control group,temozolomide group,temozolomide+propofol group,temozolomide+nitroso L-arginine methyl ester group.Cell viability was detected through CCK8;Detection of cell apoptosis level by flow cytometry;Detect the invasiveness of cells through transwell experiments;Detect the migration ability of cells through scratch experiments;Detect the expression level of cell proliferation and apoptosis-related proteins,invasion and metastasis,NOS-related proteins,and invasion and metastasis-related proteins through Western blot experiments.Results:1.Temozolomide significantly inhibited the tumor volume of U251MG/TMZ cell transplanted mice,Temozolomide significantly reduced the tumor growth of U251MG/TMZ cell transplanted mice,and propofol treatment significantly accelerated the inhibition of Temozolomide on the tumor growth of U251MG/TMZ cell transplanted mice.After receiving Temozolomide,temozolomide+propofol treatment,the final average tumor volume of the temozolomide group and temozolomide+propofol group was smaller than that of the model group.temozolomide can reduce the tumor weight of U251MG/TMZ cell transplant mice.In contrast,after treatment with propofol,temozolomide can significantly reduce the tumor weight of U251MG/TMZ cell transplant mice(P<0.05 vs P<0.001).Propofol effectively overcomes this resistance.After receiving treatment with Temozolomide or temozolomide+propofol,the final average tumor weight in the temozolomide group and temozolomide+propofol group was significantly lower than that in the model group,with a statistically significant difference in weight(P<0.05).2.Temozolomide can reduce the tumor weight of U251MG/TMZ cell transplantation mice.In contrast,Temozolomide can significantly reduce the tumor weight of U251MG/TMZ cell transplantation mice after treatment with propofol,and propofol can effectively overcome this drug resistance.After receiving Temozolomide,Temozolomide+propofol treatment,the final average tumor weight of the temozolomide group and temozolomide+propofol group was lower than that of the model group,and the difference in weight was statistically significant.After using the orthogonal partial leastsquares method and discriminant analysis(OPLS-DA)method to analyze the serum sample data of model control and normal control mice,it can be seen that compared with the temozolomide group,the contour of metabolism in mice in the model control group is significantly separated.The results indicate a significant difference in serum metabolism between the model and temozolomide groups.3.Western blot was used to detect the pathway proteins screened by metabolomics.Compared with the control group,the expression of N-cadherin,NOS1,and ADH1 protein in the temozolomide group increased,and the presentation of DLG4 protein decreased;Compared with the Temozolomide group,the expression of N-cadherin and NOS1 protein in the temozolomide+propofol group decreased,and the manifestation of DLG4 and ADH1 protein increased,with a statistically significant difference.Through hierarchical cluster analysis of different metabolites,the thermogram indicates that there is a considerable difference in the changes of metabolites in serum samples between the model group and the temozolomide group,the temozolomide group,and the temozolomide+propofol group.4.After analyzing the serum sample data of each group of mice using the orthogonal partial least squares discriminant analysis(OPLS-DA)method,it can be concluded that the model established in the experiment is effective and can be used for further future screening of model differential metabolites.5.Venn plot analysis was performed on the differential metabolites identified in the serum of each group.In the positive ion mode,a total of 45 differential metabolites were placed in the serum samples of tumor-bearing mice in each group,of which two or three groups shared 20;In the negative ion mode,a total of 66 differential metabolites were identified in the serum samples of tumor-bearing mice in each group,of which two or three groups shared 38.In the positive ion mode,there are five common differential metabolites among the three groups;In the negative ion mode,there are two common differential metabolites among the three groups.6.The results of metabolic pathway analysis showed that there were six major metabolic pathways with significant changes,namely arginine biosynthesis,Arachidonic acid metabolism,Phenylalanine,tyrosine,Tryptophan biosynthesis,pantothenate and coenzyme A biosynthesis,Pyrimidine metabolism,and Unsaturated fat acid biosynthesis.7.The CCK8 test detected cell viability.The cell viability was significantly reduced after the combination of propofol and Temozolomide;a Clonal formation assay was used to detect the clonal formation ability of glioma cells in each group.The colony formation of glioma cells was significantly reduced after the combination of propofol and Temozolomide;The apoptosis level was detected by flow cytometry.The apoptosis rate was significantly increased after propofol was combined with Temozolomide;the Transwell test showed that the number of cells invading from the upper chamber to the lower section was substantially less after the combination of propofol and Temozolomide;The ability of Cell migration was tested by scratch test.After the combination of propofol and Temozolomide,the width of the cell scratch wound was significantly more significant;a Western blot was used to detect the expression level of apoptosis-related proteins.After propofol was combined with Temozolomide,the expression of cleaved-caspase-3 and bax proteins was significantly up-regulated;Western blot was used to detect the invasion and metastasis and the manifestation of NOS-related proteins.After propofol and Temozolomide were combined,the expression of NOS1,E-cadherin,and N-cadherin protein decreased,and the presentation of MMP2 and MMP9 protein decreased.Conclusion:1.Temozolomide has a specific inhibitory effect on Temozolomide-resistant tumorbearing mice,and the combination of propofol can further inhibit the growth of transplanted tumors.2.The serum of glioma-bearing mice was detected by metabonomics.The results showed that N-cadherin,NOS1,ADH1,and DLG4 significantly changed when Temozolomide was used alone or in combination with propofol.3.Cell experiment results showed that Temozolomide could inhibit cell proliferation,and propofol could further inhibit cell proliferation after the intervention.Propofol had a more significant effect than NOS inhibitor Nitroso L-arginine methyl ester intervention.4.It is preliminarily predicted that propofol may reduce the Temozolomide resistance of Temozolomide-resistant cells through NOS-related pathways.