Polyhydroxylated Spirostanol Saponins from the Rhizomes of Paris dulongensis.

Three new polyhydroxylated spirostanol steroidal saponins, dulongenosides B-D (2-4), along with 14 known compounds, dulongenoside A (1), padelaoside B (5), parisyunnanoside G (6), polyphyllin D (7), ophiopogonin C' (8), formosanin C (9), dioscin (10), paris saponin VII (11), paris H (12), parisyunnanoside I (13), protodioscin (14), proprotogracillin (15), crustecdysone (16), and stigmasterol-3-O-ß-d-glucopyranoside (17), were isolated from the rhizomes of Paris dulongensis (Melanthiaceae). Their chemical structures were elucidated based on extensive analyses of NMR and MS data and acidic hydrolyses. The isolates were evaluated for their cytotoxicity to five human cancer cell lines (HL-60, SW480, MDA-MB-231, A549, and A549/Taxol) and the normal human bronchial epithelial cell line BEAS-2B by the MTS test. Compounds 7-12 and 14 showed cytotoxic activity, with IC50 values ranging from 0.20 to 4.35 µM. Proprotogracillin selectively inhibited A549 (IC50=0.58 µM) and A549/Taxol (IC50=0.74 µM) cells, with no significant cytotoxic activity against HL-60, SW480, MDA-MB-231, or BEAS-2B cells, with IC50 values greater than 40 µM.

Combining features selection strategy and features fusion strategy for SPAD estimation of winter wheat based on UAV multispectral imagery.

The Soil Plant Analysis Development (SPAD) is a vital index for evaluating crop nutritional status and serves as an essential parameter characterizing the reproductive growth status of winter wheat. Non-destructive and accurate monitorin3g of winter wheat SPAD plays a crucial role in guiding precise management of crop nutrition. In recent years, the spectral saturation problem occurring in the later stage of crop growth has become a major factor restricting the accuracy of SPAD estimation. Therefore, the purpose of this study is to use features selection strategy to optimize sensitive remote sensing information, combined with features fusion strategy to integrate multiple characteristic features, in order to improve the accuracy of estimating wheat SPAD. This study conducted field experiments of winter wheat with different varieties and nitrogen treatments, utilized UAV multispectral sensors to obtain canopy images of winter wheat during the heading, flowering, and late filling stages, extracted spectral features and texture features from multispectral images, and employed features selection strategy (Boruta and Recursive Feature Elimination) to prioritize sensitive remote sensing features. The features fusion strategy and the Support Vector Machine Regression algorithm are applied to construct the SPAD estimation model for winter wheat. The results showed that the spectral features of NIR band combined with other bands can fully capture the spectral differences of winter wheat SPAD during the reproductive growth stage, and texture features of the red and NIR band are more sensitive to SPAD. During the heading, flowering, and late filling stages, the stability and estimation accuracy of the SPAD model constructed using both features selection strategy and features fusion strategy are superior to models using only a single feature strategy or no strategy. The enhancement of model accuracy by this method becomes more significant, with the greatest improvement observed during the late filling stage, with R2 increasing by 0.092-0.202, root mean squared error (RMSE) decreasing by 0.076-4.916, and ratio of performance to deviation (RPD) increasing by 0.237-0.960. In conclusion, this method has excellent application potential in estimating SPAD during the later stages of crop growth, providing theoretical basis and technical support for precision nutrient management of field crops.

Honokiol and magnolol: A review of structure-activity relationships of their derivatives.

Honokiol (HK) and magnolol (MAG) are typical representatives of neolignans possessing a wide range of biological activities and are employed as traditional medicines in Asia. In the past few decades, HK and MAG have been proven to be promising chemical scaffolds for the development of novel neolignan drugs. This review focuses on recent advances in the medicinal chemistry of HK and MAG derivatives, especially their structure-activity relationships. In addition, it also presents a comprehensive summary of the pharmacology, biosynthetic pathways, and metabolic characteristics of HK and MAG. This review can provide pharmaceutical chemists deeper insights into medicinal research on HK and MAG, and a reference for the rational design of HK and MAG derivatives.

Estimation of the rice aboveground biomass based on the first derivative spectrum and Boruta algorithm.

Aboveground biomass (AGB) is regarded as a critical variable in monitoring crop growth and yield. The use of hyperspectral remote sensing has emerged as a viable method for the rapid and precise monitoring of AGB. Due to the extensive dimensionality and volume of hyperspectral data, it is crucial to effectively reduce data dimensionality and select sensitive spectral features to enhance the accuracy of rice AGB estimation models. At present, derivative transform and feature selection algorithms have become important means to solve this problem. However, few studies have systematically evaluated the impact of derivative spectrum combined with feature selection algorithm on rice AGB estimation. To this end, at the Xiaogang Village (Chuzhou City, China) Experimental Base in 2020, this study used an ASD FieldSpec handheld 2 ground spectrometer (Analytical Spectroscopy Devices, Boulder, Colorado, USA) to obtain canopy spectral data at the critical growth stage (tillering, jointing, booting, heading, and maturity stages) of rice, and evaluated the performance of the recursive feature elimination (RFE) and Boruta feature selection algorithm through partial least squares regression (PLSR), principal component regression (PCR), support vector machine (SVM) and ridge regression (RR). Moreover, we analyzed the importance of the optimal derivative spectrum. The findings indicate that (1) as the growth stage progresses, the correlation between rice canopy spectrum and AGB shows a trend from high to low, among which the first derivative spectrum (FD) has the strongest correlation with AGB. (2) The number of feature bands selected by the Boruta algorithm is 19~35, which has a good dimensionality reduction effect. (3) The combination of FD-Boruta-PCR (FB-PCR) demonstrated the best performance in estimating rice AGB, with an increase in R² of approximately 10% ~ 20% and a decrease in RMSE of approximately 0.08% ~ 14%. (4) The best estimation stage is the booting stage, with R2 values between 0.60 and 0.74 and RMSE values between 1288.23 and 1554.82 kg/hm2. This study confirms the accuracy of hyperspectral remote sensing in estimating vegetation biomass and further explores the theoretical foundation and future direction for monitoring rice growth dynamics.

Proteomic Analysis of Preeclampsia Amniotic Fluid Based on a Novel Solid-State Preservation Method.

Objective: Amniotic fluid (AF) plays a crucial role in diagnosing and predicting perinatal diseases, specifically preeclampsia (PE). Adequate preservation of AF samples is essential for advancing the development of PE-related biomarkers and understanding the disease's mechanisms. Materials and Methods: This study presents a method for preserving proteins in AF on a solid medium, specifically a nitrocellulose membrane, which is referred to as an AF membrane. Samples were collected from normotensive subjects and PE patients and treated with direct freezing and the AF membrane methods, respectively. Protein quality was assessed through sodium dodecyl sulfate-page and capillary electrophoresis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) with data-independent acquisition was employed for proteomic analysis. Bioinformatics analysis identified differentially expressed proteins and pathways distinguishing normotensive subjects from PE patients. Results: Comparison of the AF membrane method to the direct freezing method showed no significant impact on the protein content in the AF. The preservation methods employed did not result in evident protein differences or degradation in the AF obtained from both normotensive subjects and PE patients. Analysis based on Gene Ontology and HALLMARK gene sets revealed the upregulation of pathways associated with angiotensin, reactive oxygen species, and coagulation in PE patients. Furthermore, several biomarkers previously reported to be increased in PE serum, namely ENG, ERN1, FLT1, GDF15, HSPA5, LGALS3, PAPPA, PTX3, and SERPINE1, were significantly elevated in the AF. Conclusion: The AF membrane method proved to be highly effective, reliable, and durable for preserving proteins in AF samples. Preserving AF samples in a solid state holds significant value in discovering novel protein biomarkers and investigating the underlying mechanisms of PE.

Progress in the relationship between mechanical ventilation parameters and ventilator-related complications during perioperative anesthesia.

BACKGROUND: Mechanical ventilation, as an important respiratory support, plays an important role in general anesthesia and it is the cornerstone of intraoperative management of surgical patients. Different from spontaneous respiration, intraoperative mechanical ventilation can lead to postoperative lung injury, and its impact on surgical mortality cannot be ignored. Postoperative lung injury increases hospital stay and is related to preoperative conditions, anesthesia time, and intraoperative ventilation settings. METHOD: Through reading literature and research reports, the relationship between perioperative input parameters and output parameters related to mechanical ventilation and ventilator-related complications was reviewed, providing reference for the subsequent setting of input parameters of mechanical ventilation and new ventilation strategies. RESULTS: The parameters of inspiratory pressure rise time and inspiratory time can change the gas distribution, gas flow rate and airway pressure into the lungs, but there are few clinical studies on them. It can be used as a prospective intervention to study the effect of specific protective ventilation strategies on pulmonary complications after perioperative anesthesia. CONCLUSION: There are many factors affecting lung function after perioperative mechanical ventilation. Due to the difference of human body, the ventilation parameters suitable for each patient are different, and the deviation of each ventilation parameter can lead to postoperative pulmonary complications. Inspiratory pressure rise time and inspiratory time will be used as the new ventilation strategy.

Design, synthesis and biological evaluation of tanshinone IIA derivatives as NLRP3 inflammasome inhibitors.

Natural product structures have long provided valuable pharmacophores and even candidates for drug discovery. Tanshinone scaffold showed moderately inhibitory activity in NLRP3 inflammasome/IL-1ß pathway. Herein, we designed a series of derivatives on different regions of Tanshinone IIA (TNA) scaffold. The biological evaluation identified compound T10, a scaffold hybrid of TNA and salicylic acid, as a potent NLRP3 inflammasome inhibitor. Mechanistically, T10 inhibits the production of ROS and prevents NLRP3 inflammasome-dependent IL-1ß production. In addition, treatment with T10 significantly attenuated inflammatory response in DSS-induced peritonitis. Our work describes a potential tanshinone-based derivative, which needs to be further structurally optimized as NLRP3 inflammasome inhibitors for treating inflammatory disorders.

Antagonizing interleukin-5 receptor ameliorates dextran sulfate sodium-induced experimental colitis in mice through reducing NLRP3 inflammasome activation.

Inflammatory bowel disease (IBD) is a condition characterized by inflammation in the gastrointestinal tract. Reducing intestinal inflammation is a promising approach for treating IBD. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, a critical component of the innate immune system, is implicated in the pathogenesis of IBD. Therefore, inhibiting NLRP3 inflammasome activation is a potential therapeutic strategy for IBD. In this study, we investigated the effects of the interleukin-5 (IL-5) receptor antagonist YM-90709 on dextran sulfate sodium-induced experimental colitis in mice. We found that YM-90709 reduced the expressions of IL-1ß and caspase-1 p20 in the colon and ameliorated colitis. Furthermore, we identified YM-90709 as an effective agent for inhibiting NLRP3 inflammasome activation. Knockdown of IL-5 receptor or using an inhibitor of STAT5, a key transcription factor downstream of the IL-5/IL-5 receptor signal pathway, also reduced NLRP3 inflammasome-dependent IL-1ß release and ASC speck formation. Our study is the first to demonstrate that the NLRP3 inflammasome may be a downstream signal of IL-5/IL-5 receptor and that YM-90709 protects against IBD by inhibiting IL-5 receptor. These findings suggest a new strategy for regulating intestinal inflammation and managing IBD.

Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum / 药学学报

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

A rare tetrahydroimidazopyridine from the marine-derived fungus Paraconiothyrium sp. YK-03.

A new tetrahydroimidazopyridine named butyl (5R,6R,7S,8S)-5,6,7,8-tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-carboxylate(1), together with eight known compounds (2-9), were isolated from the fermentation broth of a marine-derived fungus Paraconiothyrium sp. YK-03. Their chemical structures were elucidated by extensive analysis of one-dimensional and two-dimensional NMR spectroscopy, HR-ESIMS and optical rotation. Among these compounds, compound 1 represented a rare tetrahydroimidazopyridine, and compounds 2-7 were isolated from the Paraconiothyrium species for the first time. A plausible biosynthetic pathway for compound 1 was proposed.

Statins Accelerate Coronary Calcification and Reduce the Risk of Cardiovascular Events.

Lipid-lowering therapy with statins is well recognized as an effective therapy in reducing adverse cardiovascular events. However, the relationship between statin therapy and progression of coronary artery calcification (CAC) is unclear. A few of studies suggested that statins fail to slow and even accelerate progression of CAC; meanwhile, some researchers demonstrate opposite results. With the purpose of seeking out the effect of statin therapy on CAC, we summarized the existing evidence on statins and undertook meta-analyses of clinical trials assessing the effect of statin therapy on CAC. Fourteen trials were identified suitable for inclusion in the analysis of the effect of statin treatment on CAC, of which 11 were randomized controlled trails, 1 was case-control study, 1 was cross-sectional study, and 1 was observational study. In the meta-analysis of CAC progression, statin therapy seemed to accelerate the progression of CAC. Meanwhile, the analysis revealed a significant correlation between statin treatment and lower risk of cardiovascular events. In conclusion, meta-analyses of the available trials have shown a significant reduction of risk of cardiovascular events. In contrast, statins accelerated CAC. This suggests that statin-mediated atheroma calcification may enhance plaque stability and reduce the risk of plaque rupture.

Recent advances on cyclodepsipeptides: biologically active compounds for drug research.

Cyclodepsipeptides are a large family of peptide-related natural products consisting of hydroxy and amino acids linked by amide and ester bonds. A number of cyclodepsipeptides have been isolated and characterized from fungi and bacteria. Most of them showed antitumor, antifungal, antiviral, antimalarial, and antitrypanosomal properties. Herein, this review summarizes the recent literatures (2010-2022) on the progress of cyclodepsipeptides from fungi and bacteria except for those of marine origin, in order to enrich our knowledge about their structural features and biological sources.

[Chemical constituents from Paris rugosa rhizomes and their antimicrobial activities].

Paris rugosa(Melanthiaceae) only grows in Yunnan province of China at present, and its chemical constituents have not been systematically studied. In this study, nine compounds, including one new compound pariposide G(1) and eight known compounds of cerin(2), stigmast-4-en-3-one(3), ß-ecdysone(4), ophiopogonin C'(5), methyl protogracillin(6), gracillin(7), parissaponin H(8), and parisyunnanoside G(9), were isolated and identified from the ethanol extract of P. rugosa rhizomes by column chromatography methods and semi-preparative high-performance liquid chromatography(HPLC). Compounds 1-9 were isolated from this plant for the first time. The antibacterial and antifungal activities of all the compounds were evaluated. The results showed that ophiopogonin C' had strong inhibitory effects on Candida albicans [MIC_(90)=(4.68±0.01) µmol·L~(-1)] and the fluconazole-resistant strain of C. albicans [MIC_(90)=(4.66±0.02) µmol·L~(-1)].

Lignan glycosides from Fritillaria verticillata Willd. and their anti-inflammatory activities.

Three undescribed lignan glycosides, echiunines E-G (1-3), as well as eight known compounds (4-11) were isolated from Fritillaria verticillata Willd. Among them, compounds 1-3 were a series of lignan glycosides reported for the first time from genus Fritillaria. Their structures were elucidated by analyses of extensive spectroscopic data and comparison of the NMR data with those reported previously, the absolute configuration of compounds were further confirmed by calculated ECD method. The NO release inhibitory effects of compounds were evaluated in LPS-activated RAW264.7 macrophages. Compounds 7-8 showed inhibitory acitivities in a dose-dependent manner.

Santalane-type sesquiterpenoids and isobenzofuranones from cultures of Paraconiothyrium sporulosum YK-03.

Three undescribed santalane-type sesquiterpenoids (parasantalenoic acids A-C) and two undescribed epimeric isobenzofuranones (paraphthalides A and B) were isolated from cultures of the marine mud-associated fungus Paraconiothyrium sporulosum YK-03. Their structures were elucidated by analysis of the extensive spectroscopic and crystal X-ray diffraction data, combined with ECD calculations and comparison. Santalane-type sesquiterpenoids have been firstly found in the Paraconiothyrium species. Parasantalenoic acids A-C represent three rare polyhydroxylated santalane-type sesquiterpenoid carboxylic acids, and parasantalenoic acid A represents the first example of 2-chlorinated santalane-type sesquiterpenoid. A plausible biosynthetic pathway for parasantalenoic acids A-C was proposed. Additionally, the anti-neuroinflammatory activities of parasantalenoic acids A-C were investigated by evaluating their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglia cells. Among them, parasantalenoic acid C showed significant anti-neuroinflammatory activity with an inhibition of 86.45 ± 2.45% at 10 µM.

A new amide from the fruiting bodies of Tricholoma bakamatsutake.

A new amide tricholomine C was isolated from the dried fruiting bodies of Tricholoma bakamatsutake. Its structure was identified by a combination of nuclear magnetic resonance spectroscopic analysis and electronic circular dichroism (ECD) calculations. The ethyl alcohol crude extract and tricholomines A-C from T. bakamatsutake were evaluated for neuroprotective activities. Of these substances, the crude extract showed weak neurite outgrowth-promoting activity in rat pheochromocytoma (PC12) cells, as well as weak inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

Heterodimers with a cucurbitane-type triterpenoid skeleton from the branches of Elaeocarpus dubius.

Four undescribed and two known cucurbitane-type triterpenoids, including two heterodimers, elaeocarpudubins A and B, were isolated from the branches of Elaeocarpus dubius (Elaeocarpaceae). The chemical structures of these undescribed isolates were determined by analyses of 1D and 2D NMR and MS data, electronic circular dichroism (ECD) calculations, and chemical transformation. Biogenetically, elaeocarpudubins A and B might be derived from cucurbitacin F through Michael addition with vitamin C and (-)-catechin, respectively. These six isolates were evaluated for their cytotoxic activities against human leukemia HL-60, human lung adenocarcinoma A549, human hepatoma SMMC-7721, human breast cancer MCF-7, human colon cancer SW480, and paclitaxel-resistant A549 (A549/Taxol) cell lines, for their antioxidant properties using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and for their differentiation effects on nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells. Cucurbitacins F (IC50 of 4.98-38.11 µM) and D (IC50 of 0.03-4.40 µM) showed growth-inhibitory activities against these six cancer cell lines. Elaeocarpudubin B (IC50 of 61.04 µM) and elaeocarpudoside B (IC50 of 6.93 µM) showed antioxidant activities. Elaeocarpudubin B and elaeocarpudoside B also showed neurite outgrowth-promoting activities in PC12 cells at a concentration of 10 µM.

Screening and validation of double allele-specific binding F-primers for the measurement of antihypertensive pharmacogenomics.

Objective: Previous studies have proposed that genetic polymorphisms of CYP2D6*10, ADRB1, NPPA, CYP3A5*3, ACE, CYP2C9*3, and AGTR1 are involved in antihypertensive pharmacogenomics. The purpose of this study is to develop an amplification analysis using double allele-specific (AS) binding primers for accurate measurement of antihypertensive pharmacogenomics. Methods: To establish a quadruplex quantitative PCR (qPCR) analysis for genotyping of CYP2D6*10, ADRB1 (1165 G>C), NPPA (2238 T>C) and CYP3A5*3, and a triplex qPCR analysis for genotyping of ACE (I/D), CYP2C9*3 and AGTR1 (1166 A>C), mismatch AS F-primers were screened by detection of plasmid/gDNA, and were validated by agreement analysis/reproducibility evaluation, in which the ΔCq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutant-type F-primer-based amplification assay) was employed to determine genotypes. Results: Seven pairs of primers were successfully selected through three rounds of F-primers screening. Except for ADRB1, the robustness assessment showed the amplification efficiency ranging from 0.9 to 1.1. In agreement analysis, two specimens in the training set (n = 203) were defined by the triplex analysis rather than NGS as heterozygotes for ACE, which was evidenced by gel electrophoresis. Reproducibility evaluation demonstrated that the coefficient of variation (CV) was <5%. Conclusion: Multiplex amplification analysis using screened AS binding primers is a simple, reliable, and accurate tool to guide drug delivery in antihypertensive personalized treatment.

Application value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma / 肿瘤研究与临床

Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.