ABSTRACT
Objective:To observe the effect of Qiyu Sanlong decoction （QYSL） on the expressions of key molecules in signal axis of mammalian rapamycin target protein （mTOR）/yeast Atg6 homologous （Beclin1）/ microtubule-associated protein1 light chain3 （LC3） in A549 cells. Method:With A549 cells as the research object， the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 （CCK-8） method. The effect of QYSL decoction on A549 cell apoptosis， autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） method， transmission electron microscope （TEM）， Real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group， the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased （P<0.01）， and the formation of autophagosome was significantly increased. Compared with the blank serum group， the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased （P<0.01）， while mRNA and protein expressions of Beclin-1， autophagy related genes 5 （ATG5）， autophagy related genes 13 （ATG13） were significantly increased （P<0.01）. Conclusion:QYSL decoction can induce autophagy in A549 cells， and its specific mechanism may be related to the down-regulation of mTOR expression， the up-regulation of Beclin1， ATG5， ATG13 and LC3 expression， and the promotion of LC3Ⅰ conversion to LC3Ⅱ.
ABSTRACT
Objective::To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule, yeast Atg6 homologous (Beclin1), autophagy related genes5 (Atg5), and microtubule-associated protein1 light chain3 (LC3B). Method::Lewis lung carcinoma cells (LLC) were used to reproduce the lung cancer mice transplanted model. After the modeling, the mice were randomly divided into model group, Qiyu Sanlong decoction group, chemotherapy group and combination group, with 18 transplanted mice in each group. In model group, mice were fed with 0.9% saline 20 mL·kg-1 daily. In Qiyu Sanlong decoction group, mice were fed with Qiyu Sanlong decoction 80.48 g·kg-1 daily. The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution (DDP) at the 1st, 3rd and 5th day. The combination group was orally given the drugs at the concentration of 80.48 g·kg-1, and 0.4 mL DDP solution was intraperitoneally injected at the 1st, 3rd and 5th day. After 21 days of continuous treatment, tumor tissue was exfoliated and weighed, and the tumor inhibition rate was calculated. Hematoxylin-eosin (HE) staining was used to observe the histological changes of tumor. The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining. Protein expressions of Beclin1, Atg5, LC3B-Ⅰand LC3B-Ⅱ were determined by Western blot, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. The transcription levels of Beclin1, Atg5 mRNA in tumor tissues were detected by Real-time PCR. Result::Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor, with an inhibitory rate of 31.2%. Under microscope, patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group. Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1, Atg5 and LC3B protein (P<0.01), and promote the conversion from LC3B-Ⅰ into LC3-Ⅱ compared with the model group. Real-time PCR results showed that Qiyu Sanlong decoction could promote the transcription of Beclin1 mRNA and Atg5 mRNA compared with the model group (P<0.01). Conclusion::Qiyu Sanlong decoction has a mild inhibitory effect on lung tumors, and its mechanism may be related to up-regulating the expressions of autophagy key proteins Beclin1, Atg5 and LC3B, and promoting the conversion from LC3B-Ⅰ to LC3B-Ⅱ.