Effect And Mechanism Of Pulsatilla Saponin B3 On Non-small Cell Lung Cancer

At present,lung cancer is one of the common malignant tumors,and non-small cell lung cancer accounts for 80%of the total number of lung cancers.Surgery,radiotherapy,and chemotherapy are the main methods of cancer treatment today,but they have large adverse reactions and are prone to drug resistance.When they act on cancer cells,they can also kill normal body cells,resulting in reduced immune function,bone marrow suppression,and organs.Injury and other toxic side effects,and poor treatment effects for advanced and metastatic patients have greatly restricted its application.Traditional Chinese medicine has the characteristics of multiple components and multiple pharmacological effects,but also has the characteristics of low toxic and side effects and not easy to form drug resistance.Therefore,the active ingredients in traditional Chinese medicine have become a hot spot in current research.Pulsatilla Chinensis（Bunge）Regel is a plant of the Ranunculaceae family,which is widely used in the treatment of dysentery,bacterial and fungal infections,and tumors.Pulsatilla saponin B3 is an important active ingredient extracted from Pulsatilla.There are few reports on its biological activity.There are reports in the literature that it has significant anti-cancer activity.However,the mechanism of B3 in the treatment of non-small cell lung cancer has not been elucidated.This article evaluates the anti-lung cancer activity of B3 through cell experiments in vitro and transplanted tumor models of different cells in vivo and discusses related possible mechanisms.Objective:B3 is a compound extracted from Traditional Chinese Medicine Radix Pulsatilla,which has a good effect in treating liver cancer and breast cancer.However,its mechanism for the treatment of non-small cell lung cancer is still unclear.In this study,the effects of B3 on non-small cell lung cancer and its mechanism were studied in vitro and in vivo.Methods:The effect of B3 on the proliferation inhibition of human lung adenocarcinoma A549 cells and H1299 cells in vitro was detected by MTS assay.The effect of B3 on ROS and Ca²⁺in lung cancer A549 cells was detected by flow cytometry.The expressions of NLRP3,Caspase-1,and autophagy pathway-related proteins LC3B and P62 in A549 cells were detected by western blotting.The Lewis lung cancer xenograft model and human A549 lung cancer xenograft model in nude mice were established to investigate the anti-lung cancer effect of B3 in vivo.Blood samples were collected for hematological analysis of the changes of leukocytes,lymphocytes,and neutrophils in peripheral blood.Hematoxylin-eosin staining（HE）was used to observe the effect of the drug on the viscera.The result of B3 on tumor necrosis factorα（TNF-α）was determined by enzyme-linked immunosorbent assay（ELISA）.The anti-lung cancer activity of B3 in vivo and its possible mechanism were investigated by Western blotting to detect related protein expression.Results:MTS results showed that B3（20μM～30.00μM）could significantly inhibit the proliferation of A549 cells,and B3（10μM～20.00μM）could significantly inhibit the proliferation of H1299 cells.Inhibitors vx765 and 3MA reversed B3-induced cell death.Western blotting results showed that B3 could significantly regulate the expressions of NLRP3,Caspase-1 Gasdermin D,and LC3B and P62 in A549 cells.Flow cytometry showed that B3 induced ROS and Ca²⁺production in A549 cells.Animal experiment results showed that B3(2.50,5.00,10.00mg·kg^-1)and B3(10,20,40.00 mg·kg^-1)could inhibit the growth of Lewis lung cancer xenograft in C57BL/6 mice and human A549lung cancer xenograft in nude mice.Blood samples were collected for hematological analysis,and hematoxylin and eosin staining showed no significant organ toxicity.Conclusion:B3 has an excellent anti-lung cancer effect in vivo and in vitro.In vitro,B3 can induce autophagy and pyroptosis of NSCLC cells and induce the production of large amounts of reactive oxygen species and calcium.In vivo,B3 has no obvious toxicity to organs.Its mechanism of action is related to the regulation of TNF-αinflammatory cytokines and related to the regulation of NLRP3,Caspase-1 Gasdermin D,and LC3B and P62,which regulate the autophagy pathway in cells.