| Objective:By utilizing in cell experiments,a heat symptom glioma animal model,and transcriptomics technology,we have provided preliminary insights into the molecular mechanisms,biological processes,and signaling pathways influenced by the compound Neferine found in the cold-natured Chinese herb,Lotus Heart.We have also elucidated its efficacy and mode of action in the intervention of the heat symptom glioma animal model.Our findings have significant implications for the development of novel medications that employ traditional Chinese medicinal ingredients,in accordance with clinical practices.Methods:In the in vitro experiments,the efficacy of Neferine in the treatment of glioma was evaluated through MTT assay,EDU-488/Hoechst33342 fluorescence staining,flow cytometry,and Transwell chamber assay.For the in vivo experiments,BALB/c nude mice were used,and a new U87 MG nude mouse heat symptom glioma model was established,wherein mouse signs were observed and evaluated after model establishment.Treatment was then administered to the respective groups,and tumor inhibition rates and relative tumor growth rates（T/C%）were calculated based on tumor weight and volume.The histopathological changes in the tumor tissue and damages to major organs in the nude mice were observed through HE staining,while the liver and kidney markers of the animals were analyzed through an automatic biochemistry analyzer after treatment with Neferine.Following the investigation of the mechanisms underlying the inhibitory effect of Neferine on the growth of human glioma U87 MG cells using RNA-Seq sequencing,the mRNA and protein expression levels of key genes in the tumor tissue of U87 MG tumor-bearing nude mice were detected using real-time quantitative PCR and Western blot techniques.Results:In the in vitro experiments,the efficacy of Neferine in the treatment of glioma was evaluated through multiple assays.MTT assay showed that Neferine had inhibitory effects on A172 and U87 MG glioma cells,with IC50 values of 6.89±1.14μM and18.39±3.22μM,respectively,while having no significant effect on normal liver cells at concentrations below 25μM.Therefore,concentrations of 6.25μM,12.5μM,and 25μM were chosen as low,middle,and high doses,respectively,and a 48-hour exposure time was used for subsequent studies.（1）EDU-488/Hoechst33342 fluorescence staining showed that the number of green granular particles and the ratio of EDU-positive cells significantly decreased with increasing concentrations of Neferine,indicating that the drug inhibited the proliferation of A172 and U87 MG cells in a dose-dependent manner.（2）Cell cycle analysis showed that treatment with Neferine increased the percentage of cells in the G0/G1phase in both A172 and U87 MG cells,with the low,middle,and high dose groups showing significant increases compared to the control group.（3）Apoptosis analysis showed that Neferine induced dose-dependent apoptosis in both A172 and U87 MG cells,with the high dose group showing a significant increase in the percentage of apoptotic cells compared to the control group.（4）Transwell chamber assay showed that the number of cells that passed through the chamber significantly decreased with increasing concentrations of Neferine,indicating that the drug inhibited the migration ability of A172 and U87 MG cells in a dose-dependent manner.The results of the in vivo experiments showed that:（1）Neferine had no significant effect on the body weight of U87 MG tumor-bearing nude mice,indicating that the dose of30 mg/kg was safe for tumor-bearing nude mice.（2）The tumour inhibition rate was 92.13%,53.31%,127.31%,98.27%,65.65%and 68.97%in the Glioma neferine group,Glioma pinocembrin group,Glioma temozolomide group,Heat syndrome glioma neferine group,Heat syndrome glioma pinocembrin group and Heat syndrome glioma temozolomide group,respectively.The T/C%based on tumour volume was 54.29%,15.48%,89.48%,73.11%,40.48%and 43.81%in each group respectively.According to the results of the two indexes,it was found that the antitumor efficacy of Neferine was second only to temozolomide in the glioma treatment group,and the best efficacy of Neferine in the heat syndrome glioma neferine group,and the antitumor efficacy of Neferine in the heat syndrome glioma group was significantly better than that in the non-heat syndrome glioma group.（3）According to the results of HE staining of the main organs and liver and kidney indexes of nude mice,Neferine had no obvious toxic side effects on the heart,liver,spleen,lung and kidney of tumour-bearing nude mice at the experimental dose.（4）According to the results of HE sections of tumour tissues of nude mice,Neferine had damaging effects on the tumour tissues of both the heat-syndrome glioma group and the non-heat-syndrome group,and the efficacy of the tumour tissues of the heat-syndrome group was significantly better than that of the non-heat-syndrome group.Using RNA-Seq sequencing technology,we investigated the mechanism of Neferine in inhibiting U87 MG cell proliferation.The results showed that Neferine could regulate the expression of 618 genes to approach normal levels.We intersected the genes regulated by Neferine with glioma disease targets and identified 49 common targets,which are the core genes for Neferine treatment of glioma.We then performed KEGG pathway enrichment analysis on the core targets and found three highly significant signaling pathways:Ras signaling pathway,HIF-1 signaling pathway,and PI3K-Akt signaling pathway.These findings provide valuable insights into the molecular mechanisms underlying the anti-glioma effects of Neferine.The mRNA and protein expression levels of key genes in the tumor tissues of U87 MG tumor-bearing nude mice were detected using real-time quantitative PCR with Western blot method.The results showed that Neferine could significantly down-regulate the mRNA and protein expression levels of P-PI3K,P-AKT,Ras and HIF-1αin tumor tissues.The above results suggest that Neferine may exert its antitumor efficacy by inhibiting the Ras-PI3K-Akt-HIF-1αpathway,and its nature-effect relationship may be due to the upregulation of genes P-AKT and Ras,and the upregulation of genes P-PI3K and HIF-1α.The results suggest that the nature-effect relationship may be related to the up-regulation of P-AKT,Ras,P-PI3K and HIF-1α.Conclusion:Neferine,a constituent of the cold Chinese herb Lotus heart,showed good anti-tumour effects in a combined model of heat syndrome glioma in U87 MG nude mice,and was superior to the control group and the non-heat-syndrome group,indicating that there is a certain nature-effect relationship between neferine and its anti-heat syndrome glioma efficacy,which is in line with the principles and methods of"treating heat syndrome with cold natured drugs"in traditional Chinese medicine.The cellular pharmacodynamic assay and transcriptome sequencing revealed that Neferine may exert its anti-glioma efficacy by inhibiting the Ras-PI3K-Akt-HIF-1αsignaling pathway,and its nature-effect relationship may be related to the upregulation of P-PI3K,P-AKT,Ras,and HIF-1αgenes,and the pathway may be the pathway through which Neferine induces apoptosis,cycle arrest and inhibits proliferation and migration of U87 MG cells. |
