| Background Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death in the world.Treatments currently include surgery,chemotherapy,radiotherapy,targeted therapy,and immunotherapy.In the past 40 years,with the improvement of treatment technology,the 1-year survival rate of lung cancer has increased from 34%to 45%.However,gastrointerstinal reaction and myelosuppression caused by treatment(such as:chemotherapy)seriously affect the quality of life of lung cancer patients.Therefore,it is of great significance to further explore the mechanism of lung cancer tumorigenesis and progression and find effective therapy with small side effects.Autophagy is mainly regulated by AMPK and mTOR signaling pathways,and is a "double-edged sword" in the development of lung cancer.On the one hand,autophagy can effectively eliminate toxic products in cells and prevent lung cancer.On the other hand,autophagy can provide lung cancer cells under stress with energy supply and promote the survival of lung cancer cells.Fei-Liu-Ping ointment is an effective prescription for the treatment of lung cancer.It has the functions of nourishing Qi-Yin,reducing phlegm and resolving masses,detoxifying and promoting blood circulation.Previous studies have shown that FLP can inhibit the growth of lung cancer cells and inhibit AKT expression,and AKT-mTOR is a key pathway regulating autophagy.Therefore,we assume that FLP can regulate autophagy remodeling tumor microenvironment affecting lung cancer progression.Objective To clarify the mechanism of FLP affecting lung cancer progression based on regulating AMPK signaling pathway.Methods(1)Human lung adenocarcinoma A549 cell line was inoculated into the right axilla of BALB/C nude mice to establish a mouse xenograft model.Intervention with saline,FLP.Then,measuring the tumor weight and calculating the tumor inhibition rate.Immunohistochemistry(IHC)was employed to examine the expression of Ki-67 related to proliferation in tumor tissues.(2)Intervention with saline,FLP,Metformin(Met),Met+FLP,Chloroquine(CQ)and FLP+CQ for 14d,21d and 35d.Western Blotting(WB)and IHC was used to examine the expression levels of autophagy-related proteins LC3,ULK1,P-ULK1,Beclin 1,ATG3,ATG5,ATG7 and P62 in tumor tissues.(3)Intervention with saline,FLP,Metformin(Met),Met+FLP for 14d,21d and 35d.WB and IHC were employed to examine the expression levels of LKB1,AMPK,P-AMPK,mTOR and P-mTOR in tumor tissues.(4)Intervention with saline,FLP,Metformin(Met),Met+FLP for 14d,21d and 35d.WB and IHC were employed to examine the expression levels of apoptosis-related proteins Cleaved Caspase-3,XIAP,Survivin and Cyclin D1 in tumor tissues.(5)Human acute monocytic leukemia cell line(THP-1)was induced into macrophages.THP-1 macrophages and A549 cells were co-cultured in vitro to simulate tumor inflammatory microenvironment.WB was employed to examine the expression levels of autophagy-related proteins LC3,ULK1,P-ULK1 and ATG3 in co-cultured A549 cells.(6)WB was employed to examine the expression levels of LKB1,AMPK,P-AMPK,mTOR and P-mTOR in co-cultured A549 cells.(7)ELISA methods were used to examine the expression of the inflammatory factors TNF-,IL-1 and IL-6 in cell supernatant.Results(1)FLP can inhibit the growth of tumor tissue in A549 xenograft model,and the tumor inhibition rates increased with the extension of intervention time.35d is higher than 21d,and 21d is higher than 14d.The tumor inhibition rates ranged from high to low were 40.7%,29.3%,and 8.8%.In the expression of Ki-67 related to tumor proliferation:the expression of FLP group was lower than that of Model group,and it was statistically significant at 14d and 35d(P<0.001).(2)At 14d,21d and 35d,WB results showed that the ratio of LC3 Ⅱ/LC3 Ⅰ in the tumor tissues of FLP group was higher than that of Model group.And the expression of ULK1,P-ULK1,Belin 1,ATG3,ATG5 and ATG7 in the FLP group was higher at different extents than the Model group.P62 expression of FLP group is lower than Model group.The expression of these proteins in Met group and FLP+Met group were similar to FLP group.IHC results in the expression of each protein consistent with WB results.The results suggested that FLP could upregulate the level of autophagy in tumor tissues of A549 xenograft model.(3)The WB results:the expression of LKB1,AMPK and P-AMPK protein in FLP group,Met group and FLP+Met group was higher than that in Model group at 14d,21d and 35d.At 14d and 21d,the expression of mTOR and P-mTOR in LFP group was lower than that in Model group.At 35 days,the expression of mTOR and P-mTOR in FLP group was higher than that in Model group.In terms of IHC results,the FLP group was consistent with WB results in terms of LKB1,AMPK,and P-AMPK expression.The expression of P-mTOR in FLP group was lower than that in Model group at 3 time points.The results suggested that FLP could upregulate the activity of AMPK signaling pathway and inhibit the activity of mTOR signaling pathway in tumor tissues of A549 xenograft model.(4)At 14d,21d and 35d,WB results showed that the expression of apoptosis protein Cleaved Caspase-3 in tumor tissues of FLP group was higher than that of Model group.The expression of apoptosis inhibitory proteins XIAP and Survivin was lower than that of Model group.Cyclin D1 in the FLP group is lower than Model.And IHC results are consistent with WB results.Met is AMPK activator,and activated AMPK promotes autophagy-dependent apoptosis.The results of experiment 3 showed that FLP could upregulate AMPK level.Moreover,the results of FLP group and Met group in this experiment can promote cell apoptosis.Thus,the results suggested that FLP might promote cell apoptosis and block cell cycle by upregulating AMPK signaling pathway.(5)In the in vitro co-culture model,WB results showed that the ratio of LC3 Ⅱ/LC3 Ⅰ,P-ULK1 and ATG3 expression in the CO-FLP group were higher than those in CO-NRS group.The results suggested that FLP-containing serum could upregulate the level of autophagy in vitro co-culture model.(6)In the in vitro co-culture model,WB results showed that the expression of LKB1,P-AMPK in CO-FLP group were higher than those in CO-NRS group.Otherwise,the expression of P-mTOR in CO-FLP group were lower than those in CO-NRS group.The results suggested that FLP-containing serum could upregulate the activity of AMPK signaling pathway and inhibit the activity of mTOR signaling pathway in vitro co-culture model.(7)FLP-containing serum could reduce the levels of inflammatory factors TNF-,IL-1 and IL-6 in supernatants of A549 and THP-1 macrophages co-culture models at 24h and 48h.The results suggest that FLP-containing serum can improve the inflammatory state in the tumor microenvironment.Conclusion Under this experimental conditions,we speculate that:(1)FLP can inhibit the growth and proliferation of tumor tissue in A549 xenograft model.(2)FLP may upregulate the autophagy level of tumor tissue in A549 xenograft model by regulating AMPK and mTOR signaling pathways.(3)FLP may promote apoptosis and block tumor cell cycle of A549 xenograft model by upregulating AMPK signaling pathway.(4)FLP may improve the inflammatory state of the tumor microenvironment.These results provide a new theoretical basis for FLP in the prevention and treatment of lung cancer,but more rigorous experimental and clinical trials are still needed. |
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