| Inflammation is inseparable from the occurrence and development of tumors.The inflammatory process is the inducement of tumor occurrence and development.When the inflammatory response becomes uncontrollable and develops into a chronic process,it may induce malignant growth of surrounding tissues and form tumors.Immune response can promote a protective response against tumors,but it also participates in tumor angiogenesis and tumor cell growth,invasion and metastasis through direct or indirect effects.Nuclear transcription factor NF-?B plays a key role in cellular inflammatory response and immune response.Misregulation of NF-?B is related to the occurrence of chronic inflammation and a variety of malignant tumors.At the same time,NF-?B pathway activation is related to activation.Isoliquiritigenin(ILG)is a flavonoid compound extracted from the rhizome of the traditional Chinese medicine Glycyrrhiza,which has a variety of biological and pharmacological effects.Studies have shown that ILG can resist inflammation and oxidative stress,and can inhibit the proliferation and migration of tumor cells,and promote tumor cell apoptosis,but its mechanism of action and whether it has the same effect on lung inflammation and lung malignant tumors is not clear In addition,whether NF-?B is the anti-inflammatory and anti-tumor effect pathway of ILG remains to be explored.Objective:The relationship between inflammation and tumor occurrence and development has attracted more and more attention,and lung cancer with inflammation has made clinical treatment more difficult.This study aims to observe whether ILG can bidirectionally regulate chronic lung inflammation and lung malignant tumors,clarify the regulatory role of NF-?B and PI3K/AKT and Nrf2/HO-1 signaling pathway-related genes in it,and clarify ILG through the NF-?B pathway Play the dual role of anti-inflammatory and anti-tumor.Provide scientific basis for the clinical treatment of lung inflammation and lung cancer and the development of new drugs.Methods:Part ?: Study on the anti-inflammatory mechanism of IsoliquiritigeninUsing a self-made smoke exposure device,C57BL/6 mice were given daily smoke exposure treatment for 1 hour.After 12 weeks of smoke treatment,a COPD mouse model was constructed,and 10,20,and 30 mg/kg ILG were administered,respectively,using HE staining method and mice.Pulmonary wetness/dryness ratio to observe the inflammation and edema of the lungs of mice;by measuring the activity of myeloperoxidase(MPO)and the content of malondialdehyde(MDA)in the lung tissues of mice,the total cells in the bronchoalveolar lavage fluid of the mice were counted,neutrophils and macrophages,and detection of inflammatory factors TNF-? and IL-1? in bronchoalveolar lavage fluid,to understand the improvement of anti-oxidative stress and inflammatory response of ILG in COPD model mice;Real time-PCR method and Western Blot method were used to detect the expression levels related to NF-?B and Nrf2/HO-1 signaling pathway in lung tissue,and to clarify the protective effect of NF-?B and Nrf2/HO-1 signaling pathway in ILG on COPD Regulatory role.Part 2: Study on the anti-tumor mechanism of IsoliquiritigeninIn vitro experiments: A549 cells were cultured using RPMI 1640 medium and given different doses of ILG.MTT method was used to determine the viability of A549 cells;flow cytometry was used to detect the apoptosis rate,Real time-PCR and Western Blot method were used to determine the apoptosis-related proteins Bcl-2,Bax,Caspase 3,Caspase 8,and Caspase 9 of A549 cells.Transwell method was used to determine the migration ability of A549 cells,and Real time-PCR and Western Blot methods were used to detect the expression levels of inflammatory and migration-related MMP-2,MMP-9 and COX-2 in A549 cells,and the ILG to A549 was clarified apoptosis,migration and inflammatory response.Western Blot method was used to detect the expression levels of NF-?B and PI3K/AKT signaling pathway-related proteins in A549 cells,and to elucidate the regulatory role of NF-?B and PI3K/AKT signaling pathways in ILG inhibition of A549 cells.In vivo experiments: BALB/c nude mice were used to construct A549 transplanted tumor models,which were randomly divided into control group(NC),tumor model group(TC),ILG low dose group(ILG-L),ILG high dose group(ILGH),during the analysis of tumor size,weight and organ coefficients to analyze the inhibitory effect of ILG on A549 transplanted mice;ELISA method was used to detect the levels of tumor markers CEA,NSE,CA125 and SCC in tumor-bearing mice;Real time-PCR and Western Blot method were used to detect the mRNA and protein expression levels of apoptosis-related genes Bcl-2,Bax,Caspase 3,Caspase 8 and Caspase 9 in tumor tissues,to observe the apoptosis of transplanted tumor cells in tumor-bearing mice influences.Real time-PCR and Western Blot method were used to determine the expression levels of MMP-2,MMP-9 and COX-2 mRNA and protein related to inflammatory and migration genes in mouse tumor tissues,and to determine the migration and inflammatory response of ILG to tumor cells in tumor-bearing mice The influence of NF-?B and PI3K/AKT signaling pathway related protein expression levels was detected by Western Blot method,which further verified the regulatory role of NF-?B and PI3K/AKT signaling pathway in ILG inhibition of A549 cells.Results:Part ?: Study on the anti-inflammatory mechanism of Isoliquiritigenin1.COPD mouse model was caused by smoking and 10,20,30 mg/kg ILG were gaven to different groups of mice.The lung wetness/dryness ratio measurement results of the mice showing that the degree of pulmonary edema of the COPD model mice is significantly lower than that of the control group(P <0.05).Lung edema of mice with ILG dose was significantly improved(P <0.05).The pathological results of the lungs of mice showed that smoking can induce the typical inflammatory changes of COPD in the lungs of mice,and with the increase of the dose of ILG,the inflammatory changes in the lungs of mice were significantly reduced.2.The detection results of MPO and MDA content in the lung tissues of mice show that smoking may cause a significant increase in the content of MPO and MDA in the lung tissues of the mice.The content of MPO and MDA in the lung tissues of the COPD model mice is significantly higher than that of the control group(P <0.05),with the increase of ILG dose,the content of MPO and MDA decreased(P <0.05).3.The number of total cells,neutrophils and macrophages in the BALF of COPD mice increased significantly(P <0.05),the use of ILG may reduce the total cells,neutrophils and macrophages in BALF of mice The number of cells decreased significantly with the increase of ILG dose(P <0.05).The content of inflammatory factors TNF-? and IL-1? in BALF of COPD mice increased significantly(P <0.05).ILG significantly reduced the content of two inflammatory factors,and the expression level gradually decreased with the increase of the concentration of ILG(P <0.05).4.The detection results of NF-?B-related gene expressions in inflammatory and oxidative stress-related pathways showed that COPD mice lung tissue p-p65 and p-I?B were activated,and the expression was significantly higher than that in the control group(P <0.05),while ILG may significantly inhibit the expression of p-p65 and p-I?B protein,and block the NF-?B pathway.With the increase of ILG dose,the inhibitory effect is enhanced(P <0.05).5.Nrf2 is a key protein that inhibits NF-?B.The expression levels of Nrf2 and HO-1 in lung tissue of COPD mice are significantly increased.The use of ILG increases the expression levels of Nrf2 and HO-1 mRNA and protein(P <0.05).With the increase of ILG dose,the expression of these genes and proteins increased significantly(P <0.05).ILG may regulate the inflammatory response and oxidative stress response in COPD mice by affecting the NF-?B and Nrf2/HO-1 pathway.The Nrf2/HO-1 pathway may help increase the inhibitory effect of ILG on NF-?B.Part 2: Study on the anti-tumor mechanism of Isoliquiritigenin(1)With the increase of the concentration of ILG and the extension of the action time,the survival rate of A549 cells gradually decreased(P <0.05).At 24 h and 48 h,the IC50 of ILG on A549 cells was 16.7 ?M and 14.6 ?M,respectively.Therefore,subsequent experiments used drug concentrations of 4 ?M,8 ?M,and 16 ?M.(2)After A549 cells were treated with different concentrations of ILG for 24 h,with the increase in the concentration of ILG,the apoptosis rate of A549 cells increased significantly(P <0.05);the expression levels of pro-apoptotic genes Bax mRNA and protein increased significantly(P < 0.05),the expression level of anti-apoptotic protein Bcl-2 mRNA and protein decreased significantly(P<0.05);the activation degree of Caspase 3,Caspase 8 and Caspase 9 gradually increased,and the mRNA expression level increased significantly(P<0.05).(3)After the A549 cells were treated with different concentrations of ILG for 24 h,as the concentration of ILG was increased,the migration ability of A549 cells decreased significantly(P <0.05);MMP-2,MMP-9 and COX-2 mRNA and protein expression were significantly lower than that of the control group(P <0.05),and with the increase of the dosage,the expression levels of MMP-2,MMP-9 and COX-2 mRNA and protein gradually decreased.(4)After A549 cells were treated with 8?M and 16?M ILG for 24 h,the NF-?B signaling pathway and PI3K/AKT signaling pathway were inhibited in a concentration-dependent manner.2.In vivo experiments(1)The tumor volume of mice in different concentrations of ILG drug treatment group was smaller than that of TC group,and the tumor volume of ILG high-dose group was significantly smaller than that of low-dose group(P <0.05).(2)Compared with the tumor model group,the protein and mRNA expression levels of Caspase 3,Caspase 8 and Caspase 9 were significantly increased in the ILG administration group(P <0.05).(3)The mRNA and protein expressions of MMP-2,MMP-9 and COX-2 in the ILG administration group were significantly lower than those in the TC group(P <0.05),and high-dose ILG on MMP-2,MMP-9 and COX-2 The inhibitory effect of expression is stronger.(4)Compared with the TC group,ILG reduced the expression of p-I?B and p-p65 in mouse tumor tissues and inhibited the activation of the NF-?B pathway;meanwhile,with the increase of the dose of ILG,the inhibitory effect was obviously increased(P <0.05).The use of ILG also reduced the phosphorylation levels of AKT and PI3 K in mouse tumor tissues,and was concentration-dependent(P <0.05).(5)Serum tumor markers NSE and CA125 in the TC group were significantly higher than those in the NC group(P <0.05),but after different doses of ILG,serum NSE and CA125 levels were significantly lower than those in the TC group(P <0.05).Conclusions:1.Isoliquiritin may play an anti-inflammatory effect in the lung inflammation model of COPD mice by regulating NF-?B and Nrf2/HO-1.2.Isoliquiritin may exert anti-tumor effects through NF-?B and PI3K/AKT pathways.3.The target of isoliquiritin may be NF-?B-related molecules,which may play a dual role of anti-inflammatory and inhibiting tumor cell survival,but its downstream mechanisms are different.Although the downstream mechanism is initially found to be different,the specific mechanism needs further study.In summary,the experimental results further support the possibility that isoliquiritin,which is an extract of Chinese medicine,may be used as an anti-tumor drug or auxiliary drug. |
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