Study On The Anti-Lung Cancer Effect And Mechanism Of Dendrobium Effective Ingredient Erianin

Objective:The morbidity and mortality of lung cancer have been high and rising all over the world.As the largest developing country in the world,the incidence of lung cancer ranks first all the year round,which poses a great threat to the life and health of the people,and also poses a new challenge to the treatment of lung cancer.Therefore,in addition to the existing treatments,it is of great significance to actively look for new anticancer drugs with unique anti-lung cancer mechanism,so as to improve the cure rate of patients with lung cancer.Relying on the unique resources of traditional Chinese medicine in China,it is self-evident to develop lung cancer therapeutic drugs with independent intellectual property rights,which have both economy and low toxicity,and can be used in the treatment of clinical lung cancer patients.Therefore,this study will explore the anti-tumor efficacy and mechanism of erianin,an effective component mainly derived from the Dendrobium chrysotoxum Lindl,in lung cancer.In order to provide a theoretical basis for the follow-up development of erianin as a new drug for the treatment of lung cancer.Methods:This thesis is mainly studied in two parts.The first part is the research about anti-lung cancer pharmacodynamic effect of erianin,the effective component of Dendrobium,including（1）The study of the effect of erianin on promoting the lung cancer cells death and inhibiting the lung cancer cells proliferation.The inhibitory effect of erianin on lung cancer cells was studied by CCK-8（Cell Counting Kit-8）assay combined with microscope photography.At the same time,the Annexin V-FITC/PI staining was used to detect the effect of erianin on promoting lung cancer cell death,and the inhibition of lung cancer cell colony forming ability and its effect on cell cycle were qualitatively and quantitatively analyzed by colony forming assay and cell cycle experiments.The anti-tumor activity and toxicity of erianin in vivo were studied by subcutaneous tumor model in nude mice.（2）Study on the inhibitory effect of erianin on the migration of lung cancer cells.Wound healing assay and Transwell experiment were used to study the inhibitory effect of erianin on the migration of lung cancer cells H460 and H1299,and Western blot was used to study the inhibitory effect of erianin on the migration of lung cancer cells by affecting migration-related proteins.The second part is the research on the molecular mechanism of the anti-lung cancer effect of erianin,the effective component of Dendrobium.Through the differential m RNA expression heatmap analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis of the results of RNA-sequencing,the possible targets and pathways of erianin were studied.The reversal effects of apoptosis inhibitor Z-VAD-FMK（Z-VAD）,autophagy inhibitor Chloroquine（CQ）,necroptosis inhibitor Necrostatin-1（Nec-1）,antioxidants N-acetyl-L-cysteine（NAC）,Glutathione（GSH）and different ferroptosis inhibitors Ferrostatin-1（Fer-1）and Liproxstatin-1（Lip-1）on erianin-induced ferroptosis in lung cancer cells were studied by CCK-8 assay.The levels of intracellular reactive oxygen species（ROS）and iron ions were detected by fluorescent probe DCFH-DA and Phen green SK.The effects of erianin on lipid peroxidation and GSH in lung cancer cells were detected by Malondialdehyde（MDA）and GSH detection kits.The effect of erianin on mitochondria of lung cancer cells was observed by transmission electron microscope.Mitro-tracker green mitochondrial fluorescence probe staining experiment was used to study the changes of mitochondrial state in lung cancer cells induced by erianin.The online tool Swiss Target Prediction was used to predict the potential action targets of erianin.At the same time,the structural pharmacology software was used to study the structure pharmacology between the predicted potential action targets and erianin to simulate whether they interact with each other.Fluo-3/AM fluorescence probe was used to detect the level of calcium in lung cancer cells.The antagonistic effects of calcium signal inhibitor Ruthenium red and Calmidazolium on ferroptosis induced by erianin in lung cancer cells were studied by CCK-8 assay,and the effects of Ruthenium red and Calmidazolium on CAM and ferroptosis related proteins induced by erianin were studied by Western blot experiment.Results:The research results of this thesis are mainly divided into two parts.In the first part,the results of the study on the anti-lung cancer effect of the effective component of Dendrobium in vivo and in vitro are as follows:（1）Study on the effect of erianin on promoting lung cancer cell death and inhibiting lung cancer cell proliferation.Through the experiment of in vitro,it is proved that erianin（12.5 n M,25 n M,50 n M,100 n M）has a concentration-dependent anti-lung cancer effect on lung cancer cells H460 and H1299.It was proved by Annexin V-FITC/PI staining that erianin could significantly induce the death of lung cancer cells.In addition,erianin has a significant inhibitory effect on the colony forming ability of lung cancer cells and has the effect of inducing lung cancer cell G2/M phase arrest.At the same time,the significant anti-lung cancer effect of erianin was further verified by subcutaneous lung tumor models in nude mice.It was also confirmed by Hematoxylin-eosin（HE）staining that erianin has high-efficient and low-toxic anti-tumor effect.（2）Study on the inhibitory effect of erianin on the migration of lung cancer cells.The results showed that erianin could significantly inhibit the migration of lung cancer cells by down-regulating the expression of migration-related proteins N-cadherin,Vimentin,MMP9,Slug and Snail,and promoting the expression of E-cadherin protein.In the second part,the research results of the molecular mechanism of the anti-lung cancer effect of erianin,an effective component of Dendrobium,are as follows:through the analysis of m RNA differential expression and KEGG pathway enrichment in transcriptome data,it was found that there were significant changes in ferroptosis related genes and ferroptosis pathway in lung cancer cells treated with erianin.Further studies showed that the death of lung cancer cells H460 and H1299induced by erianin was accompanied by ROS accumulation,disappearance of mitochondrial cristae,lipid peroxidation and GSH depletion,as well as significant down-regulation of ferroptosis-related negative regulatory proteins GPX4（Glutathione peroxidase 4）,SLC7A11（Solute carrier family 7 member 11）,Glutaminase（Kidney-type glutaminase）,SLC40A1（Solute carrier family 40 member1）.After treatment of lung cancer cells with Fer-1 and Lip-1,the ferroptosis induced by erianin was significantly reversed.However,administration of Z-VAD,CQ or Nec-1 could not reverse the lung cancer cell death induced by erianin,suggesting that ferroptosis is an important way for erianin to induce lung cancer cell death.Through the study on the specific mechanism of ferroptosis in lung cancer cells induced by erianin,it was found that erianin could specifically target calmodulin（CAM）and could significantly up-regulate the expression of CAM in lung cancer cells,accompanied by a significant increase in the levels of calcium and iron ions.Pretreatment with Ruthenium red and Calmidazolium could significantly antagonize the ferroptosis induced by erianin in lung cancer cells.Therefore,it is proved that erianin may induce ferroptosis in lung cancer cells by activating Ca²⁺/CAM signal pathway.Conclusion:In this study,we confirmed that erianin,an effective component of Dendrobium,can induce the death of lung cancer cells H460 and H1299 and inhibit the proliferation and migration of lung cancer cells H460 and H1299 in vitro.And its anti-lung cancer effect in vitro was verified by subcutaneous tumor model in nude mice.The results show that erianin also has significant anti-lung cancer effect and low therapeutic toxicity in vivo.At the same time,through this study,we confirmed that erianin can induce ferroptosis in lung cancer cells H460 and H1299,which is a new type of cell death,to exert its anti-lung cancer effect.In addition,we also found that Ca²⁺/CAM signal pathway is an important mechanism of ferroptosis induced by erianin in lung cancer cells.Therefore,this study can provide a potential new therapeutic drug with high efficiency and low toxicity for the treatment of patients with lung cancer,and provide a new theoretical basis for the anti-lung cancer effect of erianin.