The Mechanism Of Ophiopogonin B Against Drug-Resistance Non-small Cell Lung Cancer By Capsase-1/GSDMD Pathway

[Objective]The purpose of this study is to compare the effects of Ophiopogon B(OP-B)on non-small cell lung cancer(NSCLC)cells and drug-resistant NSCLC cells,which are A549,H460,A549/DDP and A549/PTX cells,and choose the most sensitive cell line to OP-B.On this basis,the mechanism of OP-B acting on the most sensitive cell line has been explored,which will be good for seeking new research ideas and directions in clinical therapy.[Methods]In the first part,CCK8 assay was used to detect the inhibition of OP-B on H460,A549,A549/DDP and A549/PTX cells.And then,the most sensitive cell line under the action of OP-B was chose.The second part is mainly divided into toxicity and efficacy experiment of OP-B.In the toxicity section,the toxicity of OP-B on the heart,liver,spleen,lung and kidney of BALB/c male nude mice was evaluated by calculating the organ index,detecting the activity rate of GPT,GOT and CRE in liver and kidney,and H&E staining technology.In efficacy part,tumor inhibition rate was calculated,in order to evaluate the inhibitory effect of OP-B on A549/DDP xenograft nude mice.In the third part,after treated with OP-B on A549,H460 and A549/DDP cells,the cell morphology,was observed by electron microscopy,the protein expression lever of Cox2 and GSDMD-N were detected by immunofluorescence and Wetern blot,the mitochondrial membrane potential was detected by flow cytometry,and the gene expression of pro-Caspase-1,Caspase-1,IL-1β,GSDMD and GSDMS-N were detected by qRT-PCR,which explained the reason why A549/DDP cells were more sensitive to OP-B.[Results]In the first part,it was confirmed that A549/DDP and A549/PTX cell lines indeed had drug resistance,the times were 30.6 and 13.93 respectively.Secondly,among A549,H460,A549/DDP and A549/PTX cell lines,A549/DDP was confirmed to be more sensitive to OP-B.Finally,it was found that OP-B combined with DDP could enhance the sensitivity of A549/DDP cells to DDP(this study will not discuss in this point).In conclusion,A549/DDP cells were selected as the most sensitive cell line to OP-B.In the second part,it was confirmed that OP-B had no significant side effects on the heart,liver,spleen,lung and kidney of BALB/c male nude mice at the dose of 1.5mg/kg and 3mg/kg through toxicity experiment.Moreover,it was confirmed that OP-B had more significant inhibition on A549/DDP subcutaneous transplanted tumor than A549 through efficacy section(consistent with the conclusion of cell experiment in the first part).In the third part,it was confirmed by electron microscopy that OP-B induced pyroptosis in H460,A549 and A549/DDP cells,and the degree of pyroptosis in A549/DDP cells was most significant.Microscopically,it was observed that the cells were swollen in varying degrees,accompanied with typical morphological features of pyroptosis,such as rupture of plasma membrane and formation of bubble like protrusion.Secondly,because pyroptosis is an inflammatory death mode,the inflammatory indexes such as Cox2,mitochondrial membrane potential change and LDH release rate were detected by immunofluorescence and flow cytometry assay.It was found that among A549,H460 and A549/DDP cells,Cox2 protein expression,mitochondrial membrane potential reduction and LDH release rate were most significant in A549/DDP cells.It is further confirmed that OP-B can induce more significant pyroptosis of A549/DDP cells.Finally,qRT-PCR and Western blot experiments further confirmed that OP-B can induce more significant pyroptosis of A549/DDP cells through Caspase-1/GSDMD classic pyroptosis pathway than A549 cells.[Conclusions]①OP-B can significantly inhibit the growth of A549/DDP cells/transplanted tumors;②The molecular mechanism of OP-B is that it can activate caspase-1/GSDMD classical pyroptosis pathway in A549/DDP cells,and then induce A549/DDP cells to produce significant pyroptosis.