| Objective:In this study,we investigated the NLRP3 related mechanisms of Jinfukang Oral Liquid(JFK)on proliferation and growth of non-small cell lung cancer A549 cell.Method:1.The effect of NLRP3 inflammasome on the proliferation of A549MTT assay was used to detect the effect of knocking down NLRP3 and NF-?B inhibitor BAY 11-7082 on the proliferation of A549 in vitro;QRT-PCR was used to detect the gene expression of NLRP3 after knocking down NLRP3;Western blotting was used to detect the protein expression of NLRP3 and ?-catenin,Cyclin D1,p21 and STAT3 and NF-?B p65 after knocking down NLRP3,and the protein expression of NLRP3,phosphorylation of STAT3 and NF-?B p65 after the NF-?B inhibitor BAY11-7082 intervened.Interleukin1?(IL-1?)ELISA kit was used to detect the change of IL-1? content in supernatant after treatment of A549 cells with BAY 11-7082.2.The effect of Jinfukang Oral Liquid(JFK)on orthotopic xenograft models on nude miceWe established orthotopic xenograft models on nude mice using A549-GFP-Luc cell line.These nude mice were randomly divided into three groups: model group,low dose of JFK(30 g crude drug/kg/d),high dose of JFK(45 g crude drug/kg/d).Small animal imaging technique was used to detect the growth of tumor.The gene expression of NLRP3 was detected by QRT-PCR..Western blotting was used to detect the protein expression of NLRP3.The expression of mi R-155 in lung tumor tissues was detected by Taqman micro RNA RT-PCR.The pathological changes of lung tumors were detected by HE staining.The number of Ki67 and NLRP3 positive cells in lung tumor tissues were detected by immunohistochemistry.3.The effect of JFK on the proliferation of non-small cell lung cancer cell line A549MTT assay was used to investigate the effect of JFK on the proliferation of A549 cells in vitro.The plate colony formation assay and flow cytometry were used toobserve the effect of JFK on the colony formation and apoptosis of A549 cells respectively.The protein expression of ?-catenin,Cyclin D1,Cleaved-PARP,actived-Caspase 3,NLRP3 related pathways and activation of AKT,STAT3 and NF-?B signaling pathways were investigated by western blotting.The IL-1? ELISA kit was used to detect the accumulation of IL-1? in supernatant.The expression of mi R-155 was detected by Taqman micro RNA RT-PCR after intervention of JFK in A549 cells.4.Quality test of JFK and intervention of icariin on A549 cellsThe compositions of JFK were analyzed and identified by high resolution mass spectrometry combined with high performance liquid chromatography.MTT assay was used to detect the effect of icariin on proliferation of A549.The protein expression of NLRP3,?-catenin and p21 in A549 cells were detected by Western blotting.Result:1.Knocking down NLRP3 in A549 cells suppressed the gene and protein expression of NLRP3.Knocking down NLRP3 inhibited the proliferation of A549 cells in vitro,down-regulated ?-catenin,Cyclin D1,STAT3 protein expression and phosphorylation levels of NF-?B and improved p21 protein expression.NF-?B inhibitor BAY 11-7082 significantly inhibited A549 proliferation in vitro,down-regulated NLRP3 protein expression,phosphorylation levels of STAT3 and NF-?B and IL-1? content in the supernatant of A549.2.In animal experiments,JFK(30 g crude drug/kg/d,45 g crude drug/kg/d)was administered 28 days after the intervention,compared with the model group,the lung bioluminescence values of the A549-GFP-luc lung cancer in situ nude mice model were significantly inhibited..JFK significantly inhibits the growth of lung tumors.JFK inhibited the gene and protein expression of NLRP3 in lung tumor tissues while increased the expression of mi R-155.The tumors of JFK-treated group were smaller even could not be found by HE staining.The immunohistochemistry result showed JFK inhibited the number of Ki67 and NLRP3 positive cells in lung tumor tissues.3.JFK inhibited the proliferation and colony formation of A549 cells.JFK induced apoptosis,up-regulated the protein expressions of cleaved-PARP and activated caspase3,and down-regulated the protein expressions of ?-catenin and cyclin D1,partly through inactivating Akt signaling pathways.4.JFK inhibited the protein expression of NLRP3 and its downstreaminflammatory factors,decreased the content of IL-1? in the supernatant,inhibited the protein expression of NF-?B,and down-regulated the phosphorylation level of STAT3 signaling pathways at the T705 and S727 sites,while increased the expression of mi R-155.5.JFK contains astragaloside IV,icariin and calycosin7-O-?-D-glucopyranoside by high-resolution mass spectrometry-high performance liquid chromatography analysis.According to the new drug standard(1.0 mg/10 m L),the content of icariin in JFK is above the standard(3.0 mg/10 m L).6.Icariin inhibited the proliferation of A549 cells in vitro,inhibited the protein expression of NLRP3 and ?-catenin in A549 cells,and increased the protein expression of p21.Conclusion:1.Knocking down NLRP3 or using NF-?B inhibitors both inhibited the proliferation of A549 cells.NLRP3 may play a role in the proliferation of A549 cells.2.JFK inhibited tumor growth on A549-GFP-luc orthotopic xenograft models in vivo.Immunohistochemistry result showed the number of NLRP3 and Ki67 positive cells decreased in the JFK treated group.JFK inhibited the proliferation,colony formation and induced apoptosis of A549 cells in vitro,partly by inactivating the PI3K/Akt signaling pathway.3.JFK inhibited the proliferation of A549 by increasing the expression of mi R-155,inhibiting the expression of NF-?B,down-regulating the phosphorylation of STAT3,and inhibiting the expression of NLRP3 and its downstream inflammatory factors.JFK partially down-regulated NLRP3 by regulating the mi R-155-NF-?B/STAT3 signaling pathway to inhibit the proliferation and growth of A549.4.Icariin inhibited the proliferation of A549 in vitro,decreased the protein expression of NLRP3 and increased the protein expression of p21,which maybe is one of the active compounds for the prevention of non-small cell lung cancer by Jinfukang Oral Liquid. |
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