Study On The Inhibitory Effect And Mechanism Of Bai Lu Tang On The Proliferation Of Human Lung Adenocarcinoma A549 Cells

Background:Lung cancer is the most common malignant tumor on the global scale,leading to the death of a large number of cancer patients?about7 million each year globally?.The incidence of lung cancer is high and the survival time is short.The most common type of lung cancer is Non-small cell lung cancer?NSCLC?.In the traditional books of Chinese medicine,there are descriptions related to"lung cancer"diseases,such as"pulmonary accumulation"and"pulmonary sputum".Focusing on the"integrated dialectic"thinking,the medical doctors combined with the syndrome type of lung cancer patients to flexibly use the Chinese medicine compound to fully play its role,to inhibit cancer cell proliferation,to induce cancer cell apoptosis,and to enhance the body immunity of lung cancer patients.The Bailu Decoction in this study is a self-made Chinese medicine compound in the clinical application,which is suitable for lung cancer patients with Qi and Yin deficiency and phlegm and blood statis in the lung.Objective:To study the inhibitory effect of Bailu Decoction on the proliferation of human lung adenocarcinoma A549 and its synergistic effect with cisplatin,and to explore its effect on the Warburg effect of human lung adenocarcinoma A549 and its regulatory mechanism,and provide an experimental basis for the clinical application of Bailu Decoction.Research methods:Part I:Inhibition of proliferation and mechanism of human lung adenocarcinoma A549 cells by Bailu DecoctionHuman lung adenocarcinoma A549 cell was cultured by three-dimensional?3D?culture method,and serum-pharmacological method was used to prepare SD rats Bailu Decoction-containing serum.They were randomly divided into 8 groups:blank group?group A?,Bailu decoction drug-containingserumlow-dosegroup?groupB?,Bailu decoction-containing serum group?group C?,Bailu decoction-containing serum high-dose group?group D?,cisplatin group?group E?,cisplatin plus Bailu decoction drug-containing low-dose group?group F?,cisplatin plus white dew soup containing serum group?group G?and cisplatin plus white dew soup containing serum High dose group?group H?.After being cultured for 72 hours undre 3D under normoxic conditions,A549 cells were cultured for 48 hour according to the group containing the corresponding drug-containing serum or drugs.The pH values of the cell culture supernatants of each group were detected.The cell viability of each group was detected by CCK8 method.The changes of lactic acid?LD?and adenosine triphosphate?ATP?levels were detected by microplate reader.The cell apoptosis was detected by flowing cytometry;Western-Blot and RT-PCR were used to detect glucose transporter-1?GLUT1?,hexokinase-2?HK2?,pyruvate kinase-2?PKM2?,lactate dehydrogenase-A?LDH-?A)and expression of hypoxia-inducible factor-1??HIF-1??protein and gene.Part II:Inhibitory effect and mechanism of Bailu Decoction on human lung adenocarcinoma A549 xenografted in nude miceClean male BALB/c-nu nude mice,human lung adenocarcinoma A549cells were inoculated into the right side of the skin,and A549 nude mice xenograft model was prepared.After screening,they were randomly divided into 8 groups:blank group?group A?,Bailu decoction low dose group?group B?,Bailu decoction medium dose group?group C?,Bailu decoction high dose group?group D?,and cisplatin group?group E?,cisplatin plus Bailu Decoction low dose group?group F?,cisplatin plus Bailu decoction medium dose group?group G?and cisplatin plus Bailu decoction high dose group?group H?.Except the blank group and the cisplatin group,the other groups were intragastrically administered with the corresponding concentration of Bailu Decoction,once a day for 3 weeks.Cisplatin group and cisplatin plus Bailu decoction were injected intraperitoneally with cisplatin?2 mg/kg?in low,medium and high dose groups,once every 3 days.The blank group was intragastrically administered with an equal volume of physiological saline.The tumor growth curve and tumor inhibition rate were observed;HE staining was used to detect tumor pathological changes;colorimetric method was used to detect LDH,Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP levels in tumor tissues;TUNEL method was used to detect apoptosis;immunohistochemistry method for expressions of apoptosis-related proteins Bcl-2,Bax and Caspase-3 were detected.Western-Blot and RT-PCR were used to detect glucose transporter-1?GLUT1?,hexokinase-2?HK2?and pyruvate.Expression of kinase-2?PKM2?,lactate dehydrogenase A?LDH-A?and hypoxia inducible factor 1a?HIF-1??proteins and genes.Findings:Part I:Inhibition of proliferation and mechanism of human lung adenocarcinoma A549 cells by Bailu Decoction1.Compared with the blank group,the CCK8 activity of each drug group was decreased to some extent,and there was a significant difference P<0.05.Compared with the cisplatin group,the cisplatin plus Bailu decoction drug-containing serum low-dose group,cisplatin plus The activity of CCK8 in the high-dose group containing Bailu Decoction and the high-dose group containing cisplatin plus Bailu Decoction decreased to some extent,and the activity of CCK8 decreased with the increase of serum concentration of Bailu Decoction.2.Compared with the blank group,the lactic acid and ATP levels of the cells in each administration group were decreased to different degrees,P<0.01 was significantly different.Compared with the cisplatin group,the cisplatin plus Bailu decoction drug-containing serum low-dose group,the levels of LD and ATP in the high dose group of Platinum Plus Bailu Decoction and the high dose of cisplatin plus Bailu Decoction were decreased to varying degrees,and the LD and ATP levels were increased with the concentration of serum containing Bailu Decoction.3.Compared with the blank group,the apoptosis rate of each drug-administered group increased to different degrees,P<0.05.Compared with the cisplatin group,the cisplatin plus Bailu decoction drug-containing serum low-dose group,cisplatin plus white dew The apoptotic rate of the high-dose group containing the serum containing medicinal herbs and the cisplatin plus Bailu decoction group increased in different degrees.With the increase of serum concentration of Bailu Decoction,the apoptosis rate increased.4.Compared with the blank group,the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?protein and gene in each group were decreased to different degrees,P<0.01,which was significantly different;compared with cisplatin group,cisplatin plus Bailu decoction drug-containing low-dose group,cisplatin plus Bailu decoction-containing serum medium-dose group and cisplatin plus Bailu decoction-containing serum high-dose group cells GLUT1,HK2,PKM2,LDH-A and HIF-1?The expression of protein and gene was decreased to some extent.With the increase of serum concentration of Bailu Decoction,the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?protein and gene decreased.Part II:Inhibitory effect and mechanism of Bailu Decoction on human lung adenocarcinoma A549 xenografted in nude mice1.Compared with the blank group,the tumor volume and tumor weight of each drug-administered group were reduced to different degrees,P<0.01;compared with the cisplatin group,cisplatin plus Bailu decoction low-dose group,cisplatin plus white dew soup The tumor volume and tumor weight of the dose group and the high dose group of cisplatin plus Bailu decoction decreased to different extents,and the tumor body and tumor weight decreased with the increase of the concentration of Bailu Decoction.2.HE staining:Compared with the blank group,the number of tumor cells in the tumor tissues of each drug-administered group was significantly reduced,the nucleus was pyknotic,the cytoplasm was swollen or fused,the connective tissue was increased and the tumor cells were separated,and the hair cell blood vessels were significantly reduced.With the increase of the concentration of Bailu Decoction,the above pathological changes are more obvious.3.Compared with the blank group,the levels of lactic acid,Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP in the tumor tissues of each drug-administered group were decreased to different degrees,P<0.01;compared with the cisplatin group,cisplatin plus white dewt levels of lactic acid,Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP in the low-dose group,the cisplatin plus Bailu decoction group and the cisplatin plus Bailu decoction high-dose group were decreased to varying degrees,with the concentration of Bailu Decoction.The increase of LDH,Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP levels decreased.4.Immunohistochemistry:Compared with the blank group,the number of apoptosis,the expression of Bax and Caspase-3 protein in the tumor tissues of each drug-administered group increased to different degrees,and the expression of Bcl-2 protein was significantly decreased,with significant difference P<0.01;Compared with cisplatin group,the number of apoptosis,Bax and Caspase-3 protein expression in tumor tissues of cisplatin plus Bailu decoction low dose group,cisplatin plus Bailu decoction medium dose group and cisplatin plus Bailu decoction high dose group,the expression of Bcl-2 protein was significantly decreased in different degrees,and the above changes were more obvious with the increase of the concentration of Bailu Decoction.5.Compared with the blank group,the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?protein and gene in each group of tumor tissues decreased to different degrees,P<0.01,P<0.05;Compared with the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?protein and gene in the tumor tissue of cisplatin plus Bailu decoction low dose group,cisplatin plus Bailu decoction medium dose and cisplatin plus Bailu decoction high dose group There were different degrees of reduction.With the increase of the concentration of Bailu Decoction,the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?proteins and genes in tissues decreased.Research conclusions:Experimental studies in vitro and in vivo have shown that:1.Bailu Decoction has obvious inhibitory effect on human lung adenocarcinoma A549,and combined with cisplatin its inhibitory effect can be enhanced.2.Bailu Decoction can inhibit the energy metabolism of tumor cells by reducing the levels of LDH and ATP in human lung adenocarcinoma A549.3.Bailu Decoction can promote the occurrence and development of apoptosis of tumor cells by regulating the expression of apoptosis-related proteins Bcl-2,Bax and Caspase-3 in human lung adenocarcinoma A549.4.Bailu Decoction can regulate the glycolytic process of human lung adenocarcinoma A549 by inhibiting the protein and gene expression of GLUT1,HK2,PKM2,LDH-A and HIF-1?.