To Explore The Autophagy Formation Mechanism Of Lung Cancer Cells Induced By Qingzao Jiufei Decoction Based On AMPK Signaling Pathway

Objective:The effect of Qingzao Jiufei Decoction on autophagy formation related protein expression in Lewis-bearing mice lung cancer cells was observed after adenylate activated protein kinase（AMPK）inhibitor（compound C）.Method:Fifty male C57BL/6J mice were random Ly divided into model group,cyclophosphamide（CTX）group(50mg·kg^-1),Qingzao Jiefei Decoction group(11g·kg^-1),AMPK inhibitor group(10 mg·kg^-1),Qingzao Jiufei Decoction combine with AMPK inhibitor group（Qingzao combine with inhibitor group）(11 g·kg^-1+10 mg·kg^-1).24 h after modeling,CTX group was given intraperitoneal injection,once every other day,7 times in total;Compound C was injected intraperitoneally in AMPK inhibitor group and Qingzao combine with inhibitor group once a day for 14 days;Qingzao Jiufei Decoction group and Qingzao combine with inhibitor group were given traditional Chinese medicine at set dosage by intragastric administration 14 days before and after modeling.At the end of the experiment,the mice in each group were sacrificed and the tumor bearing tissues were collected;Hematoxylin-eosin（HE）staining was used to observe the histopathological changes of lung cancer in each group;The autophagic lysosome formation in lung cancer cells of each group was observed by transmission electron microscopy（TEM）;Western blot（WB）was used to detect AMPK,phosphorylated adenylate activated protein kinase（p-AMPK）,mammalian target of rapamycin（m TOR）,phosphorylated mammalian target of rapamycin（p-m TOR）,UNC-51-like kinase 1（ULK1）,phosphorylated UNC-51-like kinase 1（p-ULK1）,autophagy key molecule yeast Atg6 homolog（Beclin-1）,phosphorylated autophagy key molecule yeast Atg6 homolog（p-Beclin-1）,type III phosphatidylinositol 3-kinase（VPS34）,phosphorylated type III phosphatidylinositol 3-kinase（p-VPS34）,autophagy protein microtubule-associated eggs The expression levels of white light chain 3B（LC3B）and ubiquitin binding protein（P62）;The expression levels of ATG9A,ASSC2 and TFE3m RNA were detected by Real-time PCR;immunofluorescence staining（IF）was used to detect protein expression levels of LC3B and P62.Result:1.Effect of Qingzao Jiufei Decoction on tumor quality of Lewis-bearing miceCompared with model group,the tumor mass of Qingzao Jiufei Decoction group and Qingzao combine with inhibitor group decreased（P<0.01）.2.Effect of Qingzao Jiufei Decoction on pathological changes of lung cancer tissue in Lewis-bearing miceThe lung cancer tissue in the model group was compact in texture,and no obvious inflammatory cell infiltration and bleeding were observed.Lung cancer cells in CTX group,Qingzao Jiefei Decoction group,AMPK inhibitor group and Qingzao combine with inhibitor group were loose,and necrosis and inflammatory cell infiltration were observed.3.Effect of Qingzao Jiufei Decoction on autophagic lysosome production in Lewis-bearing mice lung cancer cellsAutophagic lysosomes were found in Qingzao Jiufei Decoction group under electron microscope,but not in model group and Qingzao combine with inhibitor group.4.Effect of Qingzao Jiufei Decoction on autophagy formation related protein expression in Lewis-bearing mice lung cancer cells4.1 Effects of Qingzao Jiufei Decoction on autophagy initiation related proteins expression in Lewis-bearing mice lung cancer cellsCompared with model group,the protein expressions of p-AMPK and p-ULK1 and p-AMPK/AMPK and p-ULK1/ULK1 ratio in Qingzao Jiufei Decoction group were significantly increased（P<0.05,P<0.01）,while the protein expression of p-m TOR and p-m TOR/m TOR ratio were significantly decreased（P<0.05）;Compared with Qingzao Jiufei Decoction group,the protein expressions of p-AMPK and p-ULK1 and the ratios of p-AMPK/AMPK and p-ULK1/ULK1 in Qingzao combine with inhibitor group were significantly decreased（P<0.05,P<0.01）.4.2 Effects of Qingzao Jiufei Decoction on the expression of proteins related to autophagic membrane formation in Lewis-bearing mice lung cancer cellsCompared with model group,the protein expressions of Beclin-1,p-Beclin-1,VPS34 and p-VPS34 and the ratios of p-Beclin-1/Beclin-1 and p-VPS34/VPS34 in Qingzao Jiefei decoction group were significantly increased（P<0.05,P<0.01）.ATG9A m RNA expression level increased（P<0.01）;Compared with Qingzao Jiefei Decoction group,the protein expressions of p-Beclin-1,VPS34 and p-VPS34 and the ratios of p-Beclin-1/Beclin-1 and p-VPS34/VPS34 in Qingzao combine inhibitor group were significantly decreased（P<0.05,P<0.01）.ATG9A m RNA expression level decreased（P<0.01）.4.3 Effect of Qingzao Jiufei Decoction on autophagosome related protein expression in Lewis-bearing mice lung cancer cellsCompared with model group,the protein expressions of LC3B-Ⅱ,LC3B and the ratio of LC3B-Ⅱ/LC3B-Ⅰin Qingzao Jiufei Decoction group were significantly increased（P<0.05,P<0.01）,while the protein expression of P62 was decreased（P<0.01）.Compared with Qingzao Jiefei Decoction group,the protein expressions of LC3B-Ⅱ,LC3B and the ratio of LC3B-Ⅱ/LC3B-Ⅰin Qingzao plus inhibitor group were significantly decreased（P<0.05,P<0.01）,while the protein expression of P62 was increased（P<0.01）.Compared with model group,LC3B protein fluorescence intensity expression in Qingzao Jiufei decoction group was increased（P<0.01）,while P62 protein fluorescence intensity expression was decreased（P<0.01）;Compared with Qingzao Jiufei Decoction group,the protein fluorescence intensity expression of LC3B in Qingzao combine with inhibitor group was decreased（P<0.01）,while the protein fluorescence intensity expression of P62 was increased（P<0.05）.4.4 Effects of Qingzao Jiufei Decoction on lysosome generation related protein gene expression in Lewis-bearing mice lung cancer cellsCompared with model group,the m RNA expression of ASSC2 in Qingzao Jiufei decoction group was increased（P<0.05）,while the m RNA expression of TFE3 was decreased（P<0.01）;Compared with Qingzao Jiefei decoction group,the m RNA expression level of ACSS2 in Qingzao combine with inhibitor group was decreased（P<0.05）,and the m RNA expression of TFE3 was increased（P<0.01）.Conclusion:1.Qingzao Jiufei Decoction can effectively inhibit the proliferation in Lewis-bearing mice lung cancer cells;2.Qingzao Jiufei Decoction can induce autophagic lysosome formation in Lewis-bearing mice lung cancer cells;3.The autophagy mechanism in Lewis-bearing mice lung cancer cells induced by Qingzao Jiufei Decoction may be realized through the following ways.（1）Autophagy initiation mechanism:activation of AMPK and direct phosphory-lation of ULK1 protein;（2）Mechanism of autophagosome membrane formation and extension:Beclin-1and VPS34 proteins were activated,and ATG9A m RNA expression level was increased;（3）Autophagosome formation mechanism:increased the protein expression of LC3B,LC3B-Ⅱand the ratio of LC3B-Ⅱ/LC3B-Ⅰ,decreased the protein expression of P62;（4）Lysosome formation mechanism:increased the expression level of ASSC2m RNA,decreased the expression level of TFE3 m RNA.