The Effect And Mechanism Of YZZ4’s Anticancer On Non-Small Cell Lung Cancer (NSCLC) In Vitro And In Vivo

Objective:Yuzhizi is a commonly used medicine in the clinical prevention of cancer by promoting qi and promoting blood circulation.In addition,it has certain curative effects on lung cancer,esophageal cancer,stomach cancer,liver cancer,intestinal cancer,breast cancer,ovarian cyst,etc.The compound triterpene saponins YZZ4 isolated from Predictor has significant anti-tumor effects.However,70%to 80%of current cancer chemotherapy regimens are platinum-based or platinum-based drugs participate in the compatibility,which has obvious toxic side effects.So far,the global incidence and mortality of lung cancer ranks first among malignant tumors.For patients with early non-small cell lung cancer,surgery should be the first choice for patients with surgical indications.But even after complete resection,many still have the risk of lung cancer recurrence.In order to explore better anti-tumor drugs,this study selected the more effective triterpene saponin YZZ4.The study found that YZZ4 can activate caspase-3in NSCLC cells（A549,H1299）,and activated caspase-3 can cleave Gasdermin family members（Gasdermin E）produces an activated form of N-terminal fragment（GSDME-NT）,which can produce a pore-forming effect on the cell membrane,thereby inducing secondary cell necrosis/pyroptosis of the cell.This article explores the mechanism of YZZ4 inducing NSCLC cell pyroptosis,aiming to clarify the in vivo and in vitro effects and mechanisms of YZZ4 against non-small cell lung cancer（NSCLC）and provide a reliable theoretical basis for clinical medication.Methods:（1）MTS detects the cytotoxicity of YZZ4 to determine the time and concentration of YZZ4 on NSCLC cells;（2）Annexin V/7-AAD staining method,propidium iodide（PI）/Hochest 33342 staining method,bright field electron microscope observation and transmission electron microscopy to explore the death mode and morphological characteristics of cells treated with YZZ4;（3）LDH reagent and detection of the effect of YZZ4on LDH release;（4）Confocal laser observation of HMGB1 transfer;（5）Protein immunity Blotting method was used to detect the expression level of pyroptosis-related proteins in cell lysates after YZZ4 treatment;（6）DCFH₂-DA kit to detect the change of intracellular ROS after YZZ4treatment;（7）JC-1 kit to detect YZZ4 on cell mitochondria The influence of membrane potential;（8）Pretreat cells with ROS inhibitor NAC,caspase inhibitor z-VAD-fmk,and JNK inhibitor SP600125 to explore whether it can inhibit YZZ4-induced pyroptosis;（9）Target GSDME/JNK transfected cells with si RNA,knocked down the constitutive expression of GSDME/JNK cells,and tested whether it can inhibit YZZ4-induced pyroptosis;（10）Tumor transplantation was performed on C57BL/6 mice,and intraperitoneal injections of different concentrations were carried out for 13 consecutive days.YZZ4,measure and record the body weight and tumor volume of the mice;（11）The mice were sacrificed on the 14th day,and various indexes were detected and analyzed.Results:（1）The cytotoxicity of YZZ4 was detected by MTS,and the concentration of A549 and H1299 was determined to be 10,15,and 20μM.The action time of A549 was 2 h,and the action time of H1299 was 1.5 h;（2）Through Annexin V/7-AAD staining method,propidium iodide（PI）/Hochest33342 staining method,bright field electron microscopy,transmission electron microscopy,LDH detection,and laser confocal observation of HMGB1 metastasis preliminarily determined that YZZ4-induced NSCLC cell death is pyroptosis（3）Detect the expression level of pyroptosis-related proteins in cell lysates after YZZ4 treated cells by western blotting,and determine that the method of NSCLC cell death induced by YZZ4 is pyroptosis;（4）Use ROS inhibitors NAC and caspase inhibitors z-VAD-fmk and JNK inhibitor SP600125 pretreated cells and found that it can inhibit YZZ4-induced pyroptosis,the increase of ROS and the decrease of mitochondrial membrane potential;（5）si RNA transfected cells targeting GSDME/JNK,It was found that the pyroptosis induced by YZZ4 was inhibited;（6）Tumor transplantation was performed on C57BL/6 mice,and different concentrations of YZZ4 were injected intraperitoneally for 13consecutive days.Measurements showed that the weight of the mice did not decrease significantly,but the tumor volume in the YZZ4 treatment group increased.It was significantly inhibited;（7）The detection of various indicators of mouse tumors found that YZZ4 significantly inhibited the increase/decrease of various indicators of tumors.Conclusion:YZZ4 through JNK-caspase-3-GSDME pathway to induce pyroptosis in NSCLC cells.