| Objective: Lung cancer is the malignant tumor with the highest morbidity and mortality in my country.Non-small cell lung cancer(NSCLC)is the most common pathological type of lung cancer,and the overall treatment effect is not ideal.In this study,A549,H1299 cells and subcutaneously transplanted tumor models in nude mice were used to verify that Shenqigujin Decoction mediates apoptosis and necroptosis by inducing endoplasmic reticulum(ER)stress at the cellular and animal levels.It provides a new idea for the treatment of NSCLC,and also provides a theoretical basis for expanding the clinical application of Shenqigujin Decoction.Methods: 1.In vitro inhibitory effect of Shenqigujin Decoction on NSCLC:(1)The quality and stability of different batches of Shenqigujin Decoction were detected by high performance liquid chromatography.(2)The inhibition rate of Shenqigujin Decoction on NSCLC cells was detected by CCK8 assay.(3)A549 and H1299 cells were treated with low concentration(0.2 mg/m L)and high concentration(0.4 mg/m L)Shenqigujin Decoction respectively,and the effect of Shenqigujin Decoction on the proliferation of NSCLC cells was detected by clone formation and Ed U assay;(4)The effect of Shenqigujin Decoction on the migration ability of NSCLC cells was detected by Transwell assay;(5)The effects of different concentrations of Shenqigujin Decoction on cell cycle and apoptosis were detected by flow cytometry;(6)Western blot was used to detect expression of Bax,cleaved caspase 3 and cleaved caspase 9,p-MLKL,p-RIPK3 and p-RIPK1.2.In vivo efficacy and safety evaluation of Shenqigujin Decoction on NSCLC:(1)In the subcutaneous tumor model of NSCLC,400mg/kg and800mg/kg Shenqigujin Decoction was used by gavage for 4 weeks.The tumor volume was recorded every 5 days.(2)Immunohistochemical staining was used to evaluate the positive rate of Ki67,cleaved caspase 3 and p-MLKL in each group;AST and ALT levels and HE staining of heart,liver,lung and kidney tissue sections were used to evaluate the safety of Shenqigujin Decoction.3.Shenqigujin Decoction regulates apoptosis and necroptosis of NSCLC cells through endoplasmic reticulum stress mediated by PERK/ATF4/CHOP axis:(1)Second-generation sequencing technology detects the changes in the transcriptome of NSCLC cells after treatment of Shenqigujin Decoction;(2)Detection of PERK,ATF4 and CHOP RNA and protein expression in Shenqigujin Decoction-treated A549 cells;Detection of ATF4,CHOP,Caspase 3.Caspase 9,p-RIPK1,p-MLKL protein expression between each group;CCK8 is used to detect cell viability,Ed U is used to detect changes in cell proliferation between Salubrinal+Shenqigujin Decoction and Shenqigujin Decoction.4.Chromatin immunoprecipitation-sequencing(Ch IP-seq)technology combined with functional experiments were used to study the direct molecular mechanism between PERK/ATF4/CHOP axis-mediated endoplasmic reticulum stress and apoptosis and necroptosis in NSCLC cells :(1)Ch IP-seq was used to compare the overall changes in the binding of CHOP to gene promoters after Shenqigujin Decoction treatment;(2)q RTPCR and Western blot were used to detect the RNA and protein of USP9 X in A549 cells treated with Shenqigujin Decoction Expression;USP9X expression,Western blotting and immunofluorescence were used to detect the expression levels of key proteins of apoptosis and necroptosis.Results: 1.Shenqigujin Decoction can inhibit the proliferation and migration of NSCLC cells,and play a role in the treatment of NSCLC cells in vitro by inducing apoptosis and necroptosis.The inhibition rate of Shenqigujin decoction on NSCLC cells was significantly increased.(2)The results of colony formation assay and Ed U assay indicated that Shenqigujin Decoction could decrease the number of clones and the positive signal of Ed U in A549 and H1299 cells in a concentration-dependent manner.(3)Shenqigujin Decoction can block the G0/G1 cell cycle of NSCLC cells in a concentration-dependent manner.(4)Transwell experiments confirmed that the migration ability of A549 and H1299 cells decreased significantly with the increase of drug concentration.(5)Western blot experiments confirmed that compared with the control group,Shenqigujin Decoction could increase the protein expression of Bad,cleaved caspase 3,cleaved caspase 9 and pMLKL,p-RIPK3 and p-RIPK1.2.Shenqigujin Decoction can inhibit the growth of NSCLC tumor in vivo,promote the apoptosis and necroptosis of NSCLC,in a safe and reliable way:(1)The tumor volume after the intervention of Shenqigujin Decoction was significantly decreased;(2)Immunohistochemical results showed that Shenqigujin Decoction decreased the expression of Ki67 in tumor tissue,and the positive expression of cleaved caspase 3 and p-MLKL was significantly increased;(3)The nude mice showed no obvious changes in body weight and no major organ damage after the intervention of Shenqigujin Decoction.3.Shenqigujin Decoction mediates ER stress in NSCLC cells through the PERK/ATF4/CHOP axis,and then induces apoptosis and necroptosis:(1)Clustering heat map and volcano plot showed that after the intervention of Shenqigujin Decoction,the gene expressions were significantly changed;GSEA enrichment analysis showed that Shenqigujin Decoction was significantly associated with ER stressrelated pathways,and suggested that ATF4 and CHOP were the main contributing genes of this gene set.(2)Shenqigujin Decoction promotes the protein expression of p-PERK,ATF4 and CHOP of A549 and H1299 cells;(3)Salubrinal can inhibit the expression of ATF4 and CHOP protein in NSCLC cells;(4)Using CCK8 to detect cell activity,it is found that Salubrinal combined with Shenqigujin Decoction significantly reduced the killing ability of Shenqigujin Decoction in A549 cells,and inhibited the apoptosis and necroptosis.4.ER stress promotes apoptosis and necroptosis through transcriptional regulation of USP9 X expression:(1)Ch IP-seq confirmed that Shenqigujin Decoction can cause changes in the binding of CHOP to thousands of promoter sequences;(2)q RT-PCR and Western assays indicated that the RNA and protein expressions of USP9 X were significantly decreased in A549 cells after treatment of Shenqigujin Decoction;(3)CCK8results suggested that overexpression of USP9 X could reduce the killing effect of Shenqigujin Decoction;(4)Overexpression of USP9 X could inhibit the apoptosis and necroptosis induced by Shenqigujin Decoction.Conclusion: Shenqigujin Decoction inhibits the transcription of USP9 X through the PERK/ATF4/CHOP axis,which induces apoptosis and necroptosis in NSCLC cells,and plays an essential role in the treatment of NSCLC. |
