S-3’-hydroxy-7’,2’,4’-trimethoxyisoflavane Study On The Mechanism Of Inducing Ferroptosis In Nsclc Cells By Inhibiting Nrf2/HO-1 Pathway

Background:Lung cancer is still one of the malignant tumors threatening human life and health all over the world.Its incidence rate and mortality rate remain high all over the world and are on the rise year by year.It is of great significance to find drugs that can effectively induce the death of NSCLC cells and understand the molecular mechanism of their effects for the treatment of lung cancer.In recent years,with the continuous research of traditional Chinese medicine,natural compounds have shown irreplaceable advantages in tumor treatment,and have good development and application value due to their advantages such as strong effectiveness and small side effects.S-3’-hydroxy 7’,2’,4’-trimethoxyisoxanthane（ShtIX）is a new isoflavane compound isolated and structurally identified from the ethyl acetate extract of the ethanol extract of Lignum Dalbergiae odoratum,which has antitumor activity,but its specific molecular mechanism is unclear.ferroptosis was first reported by Scott J.Dixon et al.in 2012 as an iron dependent mode of cell death distinct from apoptosis and necrosis.As a new regulatory cell death mode,ferroptosis plays an important regulatory role in the occurrence and development of tumor cells.Inducing ferroptosis in tumor cells may provide new ideas and strategies for the treatment of cancer.Nuclear Factor E2 Related Factor 2（Nrf2）is a defensive transduction pathway for the body to resist internal and external oxidation and chemical stimulation.Abnormal expression of Nrf2 is closely related to tumor development,chemotherapy resistance,and poor prognosis.Heme oxygenase-1（HO-1）is the main downstream target protein of Nrf2.In the process of cell ferroptosis,almost all genes that regulate ferroptosis are related to Nrf2,and the level of Nrf2 expression is closely related to the sensitivity to ferroptosis.Currently,there is increasing evidence that certain natural compounds have the effect of promoting ferroptosis and can exert anti-tumor effects in the form of ferroptosis,but their specific molecular mechanisms need to be further studied and confirmed.This study aims to explore the anti-tumor activity of ShtIX and its specific molecular mechanism of inducing NSCLC cell death,providing new candidate drugs and strategies for the treatment of NSCLC.Objective:Through in vitro and in vivo experiments,it is clear that ShtIX has anti-tumor activity against NSCLC and can induce NSCLC cell death in the form of ferroptosis,revealing the mechanism of Nrf2/HO-1 pathway in ShtIX induced ferroptosis in NSCLC cells.Method:1.MTT and CCK-8 methods were used to detect the IC₅₀value and cell viability of ShtIX on selected tumor cells;The effects of ShtIX on the proliferation of NSCLC cells were detected by EDU and cell cloning experiments;Trypan blue exclusion test and LDH lactate dehydrogenase cytotoxicity test were used to detect NSCLC cell death;Cell cycle was detected by flow cytometry.2.Fluorescence microscopy was used to detect the effect of ShtIX on the morphology of apoptosis;The effect of ShtIX on the apoptosis of NSCLC cells was detected by flow cytometry;Western blot was used to detect the expression of apoptosis related proteins.The above methods were used to detect the effects of inhibitors with different death modes on cell viability and death,and to determine the manner in which ShtIX induces NSCLC cell death.3.Observe whether there are morphological characteristics of ferroptosis by electron microscopy;The content of Fe²⁺,MDA,and GSH in NSCLC cells treated with ShtIX was detected with a kit;Stream detection of the generation of ROS and Lipid ROS;Western blot was used to detect the expression level of ferroptosis related proteins;The effects of ferroptosis inhibitors on NSCLC cell viability,proliferation,and ferroptosis were detected using the above methods.4.NSCLC cells before and after ShtIX treatment were sent to the company for high-throughput RNA sequencing,and genes Nrf2 and HO-1 with significant differences in gene expression were selected;q RT-PCR and Western blot were used to verify gene expression;The above methods were used to detect the effects of interference or overexpression of Nrf2 on NSCLC cell viability and ferroptosis.5.Establish an animal model,measure the tumorigenic volume and tumor weight of mice,detect ferroptosis related indicators and the expression of Nrf2/HO-1,and determine whether ShtIX induces ferroptosis in NSCLC cells by inhibiting the Nrf2/HO-1 pathway in vivo.Result:1.ShtIX has the strongest cytotoxic effect on NSCLC cells.Compared with other selected tumor cell lines,ShtIX has the strongest cytotoxic effect on NSCLC cell line（A549）cells.2.ShtIX can inhibit the proliferation of NSCLC cells and induce their death.ShtIX significantly inhibits the activity and proliferation of NSCLC cells,induces NSCLC cell death,and inhibits the cell cycle in G0/G1 phase.3.Apoptosis is not the main mode of inducing NSCLC cell death.Detection of apoptosis-related indicators found that ShtIX can only lead to apoptosis at high concentrations.Experimental results of different cell death inhibitors showed that ferroptosis inhibitors can significantly reverse the cell viability of NSCLC cells.Further,it was found that ShtIX treatment can increase the production of Fe²⁺in NSCLC cells,downregulate the expression of FTH,thereby breaking the iron homeostasis.4.ShtIX can induce ferroptosis in NSCLC cells.After ShtIX treatment,NSCLC cells showed typical morphological characteristics of ferroptosis,resulting in the accumulation of ROS and lipid ROS,an increase in MDA levels,and a decrease in GSH and GPX4 levels,which can be reversed by Fer-1.5.ShtIX induces NSCLC cell genesis by inhibiting Nrf2/HO-1 signaling pathway.RNA high-throughput sequencing results showed that ShtIX significantly downregulated the expression of Nrf2 and HO-1 genes,consistent with the results of q RT-PCR and Western blot detection.RNA interference with Nrf2 significantly enhanced ShtIX induced ferroptosis in NSCLC cells,while the results of overexpression vectors were contrary to this,demonstrating the molecular mechanism of ShtIX induced ferroptosis in NSCLC cells from both sides.6.Construct an animal model to detect the antitumor activity and molecular mechanism of ShtIX in vivo.Compared with the control group and the combined group of ferroptosis inhibitors,the tumor volume and weight of mice in the ShtIX treatment group were significantly reduced.ShtIX treatment promoted the occurrence of ferroptosis,downregulated the expression of Nrf2 and HO-1,and the results were consistent with in vitro experiments.Conclusion:1.ShtIX can inhibit the vitality and proliferation of NSCLC cells and induce NSCLC cell death.2.ShtIX can induce ferroptosis in NSCLC cells.3.ShtIX can induce ferroptosis in NSCLC cells by inhibiting the Nrf2/HO-1 pathway.4.ShtIX can inhibit tumor growth,induce ferroptosis,and inhibit the expression of Nrf2 and HO-1 in vivo.