A real-world study and network pharmacology analysis of EGFR-TKIs combined with ZLJT to delay drug resistance in advanced lung adenocarcinoma.

OBJECTIVE: This study aimed to explore the efficacy and safety of combining epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) with ZiLongJin Tablet (ZLJT) in delaying acquired resistance in advanced EGFR-mutant lung adenocarcinoma (LUAD) patients. Furthermore, we employed network pharmacology and molecular docking techniques to investigate the underlying mechanisms. METHODS: A retrospective comparative study was conducted on stage IIIc/IV LUAD patients treated with EGFR-TKIs alone or in combination with ZLJT at the Second Affiliated Hospital of the Air Force Medical University between January 1, 2017, and May 1, 2023. The study evaluated the onset of TKI resistance, adverse reaction rates, safety indicators (such as aspartate aminotransferase, alanine aminotransferase, and creatinine), and inflammatory markers (neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio) to investigate the impact of EGFR-TKI combined with ZLJT on acquired resistance and prognostic indicators. Additionally, we utilized the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine, PubChem, UniProt, and Swiss Target Prediction databases to identify the active ingredients and targets of ZLJT. We obtained differentially expressed genes related to EGFR-TKI sensitivity and resistance from the Gene Expression Omnibus database using the GSE34228 dataset, which included sensitive (n = 26) and resistant (n = 26) PC9 cell lines. The "limma" package in R software was employed to detect DEGs. Based on this, we constructed a protein‒protein interaction network, performed gene ontology and KEGG enrichment analyses, and conducted pathway network analysis to elucidate the correlation between the active ingredients in ZLJT and signaling pathways. Finally, molecular docking was performed using AutoDockVina, PYMOL 2.2.0, and Discovery Studio Client v19.1.0 software to simulate spatial and energy matching during the recognition process between predicted targets and their corresponding compounds. RESULTS: (1) A total of 89 patients were included, with 40 patients in the EGFR-TKI combined with ZLJT group (combination group) and 49 patients in the EGFR-TKI alone group (monotherapy group). The baseline characteristics of the two groups were comparable. There was a significant difference in the onset of resistance between the combination group and the monotherapy group (P < 0.01). Compared to the monotherapy group, the combination group showed a prolongation of 3.27 months in delayed acquired resistance. There was also a statistically significant difference in the onset of resistance to first-generation TKIs between the two groups (P < 0.05). (2) In terms of safety analysis, the incidence of adverse reactions related to EGFR-TKIs was 12.5% in the combination group and 14.3% in the monotherapy group, but this difference was not statistically significant (P > 0.05). There were no statistically significant differences in serum AST, ALT, CREA, TBIL, ALB and BUN levels between the two groups after medication (P > 0.05). (3) Regarding inflammatory markers, there were no statistically significant differences in the changes in neutrophil-to-lymphocyte Ratio(NLR) and Platelet-to-lymphocyte Ratio(PLR) values before and after treatment between the two groups (P > 0.05). (4) Network pharmacology analysis identified 112 active ingredients and 290 target genes for ZLJT. From the GEO database, 2035 differentially expressed genes related to resistant LUAD were selected, and 39 target genes were obtained by taking the intersection. A "ZLJT-compound-target-disease" network was successfully constructed using Cytoscape 3.7.0. GO enrichment analysis revealed that ZLJT mainly affected biological processes such as adenylate cyclase-modulating G protein-coupled receptor. In terms of cellular components, ZLJT was associated with the cell projection membrane. The molecular function primarily focused on protein heterodimerization activity. KEGG enrichment analysis indicated that ZLJT exerted its antitumor and anti-drug resistance effects through pathways such as the PI3K-Akt pathway. Molecular docking showed that luteolin had good binding activity with FOS (-9.8 kJ/mol), as did tanshinone IIA with FOS (-9.8 kJ/mol) and quercetin with FOS (-8.7 kJ/mol). CONCLUSION: ZLJT has potential antitumor progression effects. For patients with EGFR gene-mutated non-small cell LUAD, combining ZLJT with EGFR-TKI treatment can delay the occurrence of acquired resistance. The underlying mechanisms may involve altering signal transduction pathways, blocking the tumor cell cycle, inhibiting tumor activity, enhancing cellular vitality, and improving the bioavailability of combination therapy. The combination of EGFR-TKI and ZLJT represents an effective approach for the treatment of tumors using both Chinese and Western medicine.

Anti-fibrotic Effects and Mechanism of Shengmai Injection () on Human Hepatic Stellate Cells LX-2.

OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×104 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 µL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 µL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 µL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 µL/mL (P<0.05). CONCLUSION: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 µL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.

Anti-fibrotic effects of Salvia miltiorrhiza and Ligustrazine Injection on LX-2 cells involved with increased N-myc downstream-regulated gene 2 expression.

OBJECTIVE: To investigate the effects of Salvia miltiorrhiza and Ligustrazine Injection (SML) on proliferation and apoptosis of human hepatic stellate cell LX-2 and the expression of N-myc downstreamregulated gene 2 (NDRG2, a tumor suppressor gene). METHODS: HSCs from the LX-2 cell line were cultured in vitro. The proliferative state of different initial LX-2 cell numbers was measured using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LX-2 cells were plated in 96-well plates at an approximate density of 2.50×104 cells/mL and cultured for 24 h followed by the application of different concentrations of SML (1, 2, 4 and 8 µL/mL). Cell proliferation was measured using the MTT assay at 24 and 48 h. Apoptosis was detected by flow cytometry at 24 h. LX-2 cells were treated with different concentrations of SML and extracted with protein lysis buffer. The levels of NDRG2 and ß-catenin were measured by Western blot. RESULTS: With the exception of the 1 and 2 µL/mL concentrations, 4 and 8 µL/mL SML inhibited cell proliferation in a concentration-dependent manner at 24 and 48 h (P<0.05). With the exception of the 1 and 2 µL/mL concentrations, the NDRG2 expression level was greatly increased in a concentration-dependent manner. However, the level of ß-catenin was unaffected. CONCLUSION: SML inhibit LX-2 cell proliferation in a concentration-dependent manner, and the mechanism may be associated with NDRG2 over-expression.

[Effect and mechanism of emodin for regulating aquaporin-2 expression in cultured NRK cells].

OBJECTIVE: To investigate the effect and mechanism of emodin for regulating aquapoin-2 (AQP2) in NRK cells cultured in vitro. METHODS: Experiments on NRK cells cultured with alpha-DMEM medium in vitro were conducted in two steps. (1) Cells were randomly divided into 4 groups: the control group, and the three emodin treated groups treated with different dosages of emodin (5, 10 and 20 mg/L) respectively. After 24 h treatment, the location of AQP2 was decided by indirect immunofluorescene, and the AQP2 protein and mRNA expression levels were detected by Western blot and semiquantive RT-PCR. (2) Cells were randomly divided into 4 groups, the control group, and the three treated groups treated respectively with 10 mg/L 8-Bromo-cAMP, 20 mg/L emodin, and 20 mg/L emodin +10 mg/L 8-Bromo-cAMP. The activity of protein kinase A (PKA) in NRK cells after 24 h treatment was determined with non-radioactive detecting method. RESULTS: AQP2 was located at the cell membrane of NRK cells. Western blot and semiquantitive RT-PCR found that AQP2 protein and mRNA expressions were significantly decreased in NRK cells of groups treated by 10 mg/L and 20 mg/L emodin (P < 0.05). PKA activity determination showed significantly decreased phosphorylation level of PKA in NRK cells of groups treated with 20 mg/L emodin group (P < 0.05). CONCLUSION: Emodin can inhibit the genetic transcription and the translation of AQP2 gene in NRK cells, which demonstrates that the change of AQP2 expression regulated by emodin may be correlated with the diuresis effect of rhubarb, and it is likely that the regulation is going through PKA signal pathway.

[Influence of moxibustion of baihui (GV 20) on hemodynamics of common carotid artery in healthy subjects].

OBJECTIVE: To observe the effect of moxibustion of Baihui (GV 20) on the hemodynamics of internal carotid artery in health subjects so as to study its underlying mechanism of moxibustion in the treatment cerebrovascular disorders. METHODS: Thirty healthy male volunteer students between 20 and 22 years in age were enrolled into this study. Qm (mean blood flow), Vm) (mean velocity of blood flow), Vmax (maximal velocity of blood flow), Vmin (minimal velocity of blood flow), Wv (pulse wave velocity), Zcv (characteristic impedance), Rv (peripheral resistance), DR (dynamic resistance), CP (critical pressure) and DP (difference of pressure) of the right and left common carotid arteries were measured before and after moxibustion of GV20 (5-10 min each time, once daily, 5 times altogether) by using CBA CV-300 Cerebrovascular Hemodynamics Detector. RESULTS: Following moxibustion of GV20, Qm, Vm and Vmin of both right and left common carotid arteries increased significantly (P < 0.01); while Rv and DR of the brain artery lowed evidently (P < 0.05); The rest indexes had no significant changes (P > 0.05). CONCLUSION: Moxibustion of Baihui (GV 20) can significantly raise the velocity of blood flow of the common carotid artery and low the resistance of blood vessels.

[Protection effect of Salvia miltiorrhiza on model of acute lung injury in rats].

OBJECTIVE: To investigate the effect of SM on aquaporin-1 (AQP1) expression in rats with acute lung injury (ALI), and study protection against ALI. METHODS: 48 SD rats were randomly divided into normal group, ALI 7-day group, ALI 8-hour group, SM prevention group, SM 7-day treatment group, and SM 8-hour treatment group. At 8 hours and 7 days after treatment, lung wet-to-dry weight ratio, lung pathology, electron microscope, AQP1 immunohistochemistry, and AQP1 Western blot were performed. RESULTS: The model group rats all appeared ALI symptom. Compared with the 7-day ALI group, the degree of lung edema statement alleviated in SM 7-day treatment group. But the expression of AQP-1 was not significant between the two groups (P < 0.05). Compared with the 8-hour ALI group, the degree of lung edema statement alleviated in SM 8-hour treatment group and SM prevention group. The expression of AQP-1 was significantly expression in the group of SM prevention group (P < 0.01). CONCLUSION: SM may improve the body fluid metabolism and alleviate the degree of the lung edema by regulating the expression of AQP-1. The results suggest SM can regulate the activity of AQP-1 to improve the water metabolism of ALI.