To investigate the effects of almonertinib on the proliferation, invasion, epithelial-mesenchymal transition (EMT), and stemness of non-small-cell lung cancer (NSCLC) cells by regulating microRNA-22 (miR-22) targeting ubiquitin-conjugating enzyme E2O (UBE2O).

Human lung adenocarcinoma cell line NCI-H1975 was randomly divided into control group (normal culture with no treatment), medium-dose group (4 μmol·L^-1 almonertinib), inhibitor negative control (NC inhibitor) group (4 μmol·L^-1 almonertinib + miR-22 inhibition negative control), miR-22 inhibitor group (4 μmol·L^-1 almonertinib + miR-22 inhibition), miR-22 inhibitor+short hairpin negative control (sh-NC) group (4 μmol·L^-1 almonertinib + miR-22 inhibition + short hairpin negative control), and miR-22 inhibitor+short hairpin UBE2O (sh-UBE2O) group (4 μmol·L^-1 almonertinib + miR-22 inhibition + short hairpin UBE2O). The expression of miR-22 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) ; Western blot analysis was performed to detect the expression of octamer-binding transcription factor 4 (OCT4), SRY-Box transcription factor 2 (SOX2), and Nanog; the cell proliferation rate was assessed using the 5-ethynyl-2-deoxyuridine (EdU) assay; and Transwell assays were conducted to evaluate cell invasion ability.

The expression levels of miR-22 in the control group, medium-dose group were 1.00±0.16, 2.07±0.34. The expression levels of OCT4 in the control group, medium-dose group, NC inhibitor group, and miR-22 inhibitor group were 1.00±0.16, 0.63±0.09, 0.67±0.08 and 0.94±0.12, respectively; SOX2 expression levels were 1.00±0.14, 0.48±0.06, 0.54±0.07 and 0.81±0.11; and Nanog expression levels were 1.00±0.18, 0.45±0.08, 0.52±0.11 and 0.89±0.15. The cell proliferation rates in the control group, medium-dose group, NC inhibitor group, miR-22 inhibitor group, miR-22 inhibitor+sh-NC group, and miR-22 inhibitor+sh-UBE2O group were (87.29±10.73)%, (41.65±7.82)%, (48.47±6.54)%, (79.31±10.26)%, (84.50±11.98)% and (36.82±6.79)%, respectively. The numbers of invading cells were (156.92±24.81), (72.53±9.06), (64.41±10.75), (143.27±26.19), (137.58±21.46) and (75.23±11.94), respectively. Compared between the control group and medium-dose group, NC inhibitor and miR-22 inhibitor groups, as well as miR-22 inhibitor+sh-NC and miR-22 inhibitor+sh-UBE2O groups showed statistically significant differences (all P<0.05).

Almonertinib can inhibit the proliferation and invasion of NSCLC NCI-H1975 cells, likely by regulating miR-22 to target UBE2O.

To investigate the regulatory effects of aloperin on bladder cancer cell growth and endoplasmic reticulum stress-dependent mitochondrial pathway.

253J cells were randomly divided into control group, experimental-L group (25.0 μmoL·L^-1 aloperin), experimental-M group (50.0 μmoL·L^-1 aloperin), experimental-H group (100.0 μmoL·L^-1 aloperin), 4-PBA group [100.0 μmoL·L^-1 aloperin+20 μmoL·L^-1 4-phenylbutyric acid (4-PBA)], si-NC (transfected with si-NC+100.0 μmoL·L^-1 aloperin), si-CHOP group [transfected with si-CCAAT-enhancer-binding protein homologous protein (CHOP)+100.0 μmoL·L^-1 aloperin]. Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine (EdU) assay; protein expression was detected by Western blot assay; apoptosis was detected by flow cytometry; mitochondrial membrane potential (MMP) was detected by JC-1 assay.

The EdU positive cell rates in control group, experimental-L group, experimental-M group and experimental-H group were (37.51±2.98)%, (29.52±2.19)%, (23.10±2.58)% and (12.94±2.52)%, respectively; the expression levels of C/EBP homologous protein (CHOP) were 0.29±0.04, 0.53±0.05, 0.71±0.08, 0.90±0.12, respectively. The apoptosis rates in control group, experimental-H group and 4-PBA group were (4.79±0.62)%, (23.01±2.03)% and (13.75±1.14)%, respectively. The expressions of CHOP protein in control group, experimental-H, si-NC and si-CHOP groups were 0.30±0.03, 0.92±0.08, 0.91±0.11 and 0.20±0.02, respectively; the apoptosis rates were (4.39±0.30)%, (22.11±1.82)%, (22.65±2.40)% and (15.32±1.19)%, respectively; mitochondrial cytochrome C (Cyto-C) protein levels were 0.78±0.07, 0.43±0.05, 0.42±0.05 and 0.65±0.06; cytoplasmic Cyto-C protein levels were 0.51±0.06, 0.91±0.10, 0.88±0.05 and 0.61±0.04; MMP levels were (100.00±2.79)%, (57.49±3.34)%, (54.09±4.32)% and (69.91±6.81)%, respectively. The above indexes in the experimental-L, -M, -H groups were compared with the control group, the above indexes in the 4-PBA group were compared with the experimental-H group, and the above indexes in the si-CHOP group were compared with the si-NC group, the differences were statistically significant (all P<0.01).

Aloperin can inhibit the proliferation of 253J cells by up-regulating the endoplasmic reticulum stress-dependent mitochondrial apoptosis pathway mediated by CHOP.

To establish an ultra-high performance liquid chromatography tandem high resolution mass spectrometry (UHPLC-HRMS) method for the determination of serum demthylzeylasteral concentration in diabetic nephropathy patients, which treated by tripterygium wilfordii polyglycosides tablets (TGTs), and to apply this method in the exploratory study of clinical pharmacodynamic marker.

Serum samples and ¹⁸O-demethylzeylasteral which was used as the internal standard were processed by solid phase extraction method and took the supernatant for the determination. Chromatographic separation of the analytes was accomplished using a BEH C₁₈ column (2.1 mm×50.0 mm, 1.7 μm) under gradient elution, where the mobile phase consisted of a mixture of 0.1% formic acid aqueous solution and acetonitrile, as well as 0.1% formic acid aqueous solution. The flow rate was set at 0.3 mL·min^-1 and the column temperature at 30 ℃. The mass spectrometer was equipped with an electrospray ionization source with positive multiple reaction monitoring mode. The specificity, standard curve and lower limit of quantification, precision and recovery rate, matrix effect and stability were investigated. This method was applied to determine the serum concentration of demethylzeylasteral from 15 patients with oral TGTs. Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were utilized to explore the correlation between serum concentration of demethylzeylasteral and efficacy in treating diabetic nephropathy.

The calibration curve of demethylezylasteral was linear over the range of 1-200 ng·mL^-1, and the lower limit of quantification was 1 ng·mL^-1. The precision and accuracy relative standard deviation of intra-day and inter-day was less than 10% and 15%, respectively. The average absolute recovery was more than 89%, no matrix effect has been affected and all stability tests met the acceptance criteria. The AUC of the efficacy of TGTs predicted by serum demethylzeylasteral quantitation was 0.66 (95% confidence interval was 0.53-0.78), the cut-off value was 61.73 ng·mL^-1, the sensitivity was 83.3% and the specificity was 46.7% (P<0.05).

The UHPLC-HRMS method established in this study has the advantages of simple operation, high sensitivity, accurate and reliable results, which can be suitable for determining serum concentration of demethylzsylasteral and studying clinical drug efficacy markers of TGTs.

Acute lung injury (ALI)/acute respiratory distress syndrome is a life-threatening acute diffuse inflammatory lung injury characterised by increased pulmonary vascular permeability, inflammatory cell infiltration, pulmonary oedema, diffuse alveolar damage and reduced respiratory output. Its pathogenesis is complex, and oxidative stress is a key mechanism leading to ALI exacerbated by alveolar epithelial cell dysfunction. The Kelch-like ECH associated protein 1 /nuclear factor -E2-related factor 2 (Keap1/Nrf2) signalling pathway is an important signalling pathway of traditional Chinese medicine in the treatment of ALI by regulating oxidative stress. This paper analyses and summarizes the relevant Chinese medicine formulas and active ingredients for the treatment of ALI, and finds that a large number of antioxidant active ingredients can regulate oxidative stress through the Keap1/Nrf2 signaling pathway and play a role in improving ALI, which also provides a basis for the prevention and treatment of ALI disease and further drug development.

To establish a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method for the determination of nicorandil concentrations in human plasma and for pharmacokinetic studies in healthy subjects.

Human plasma samples were pretreated with acetonitrile extraction. Chromatographic cdumn: Acquity HPLC BEH C₁₈ (1.7 μm, 2.1 mm×50.0 mm), mobile phase: methanol (0.1% formic acid)-5 mmoL· L^-1 ammonium acetate (with 0.1% formic acid). Flow rate: 0.2 mL·min^-1, column temperature: 40 ℃, injection volume: 5 μL. Gradient elution, electrospray ion source, multiple reaction monitoring, positive ion scanning, nicorandil-d4 isotope as internal standard. The specificity of the method, the standard curve and the lower limit of quantification, the carryover effect and dilution reliability, the precision and recovery, the matrix effect, and the stability of the method were investigated.

The specificity of the established method was good, nicorandil had a good linear relationship between 0.2-200.0 ng·mL^-1, the standard curve was y=1.08×10^-1x+2.66×10^-2 (r=0.999 7), the lower limit of quantification was 0.2 ng·mL^-1, there was no residual effect, the dilution results were reliable, and the intra-day and inter-day precision of quality control samples were 0.64%-8.70% and 0.91%-8.70%, respectively. The intra-day and inter-day accuracy were 100.00%-115.00% and 98.00%-115.00%, respectively. The extraction recoveries of nicorandil at low, medium and high quality control concentrations were 92.78%-95.35%, with no obvious matrix effect and good stability.

This method is highly specific, accurate, stable, reproducible, and simple to operate, and can be used to determine the concentration of drug in plasma of nicorandil.

To explore the effect and mechanism of piperlongumine on hepatic steatosis induced by sleep deprivation.

Mice were randomly divided into control group (normal feeding), model group (sleep deprivation treatment), low-, middle- and high dose groups (2.5, 5.0 and 10.0 mg·kg^-1 piperlongumine was given on the basis of sleep deprivation), and the mice were given continuous gavage for 14 days. Liver index was detected after 14 days of continuous gavage. Western blot assay was used to detect protein expression in hepatic tissue, lipid levels in hepatic tissue were detected by kit, and reactive oxygen species (ROS) levels in hepatic tissue were detected by dihydroethidium (DHE).

The liver index of control group, model group, and high-dose group were (2.99±0.40)%, (4.32±0.17)% and (3.30±0.37)%, respectively; triglyceride (TG) levels were (0.16±0.011), (0.29±0.02) and (0.17±0.02) mmol·mgprot^-1, respectively; fatty acid synthase (FAS) protein levels were 0.31±0.03, 0.69±0.08 and 0.35±0.03, respectively; hypoxia-inducing factor-1 α (HIF-1α) protein levels were 0.38±0.04, 0.94±0.08 and 0.44±0.04, respectively; the protein levels of adaptor p66Shc (p66Shc) were 0.21±0.03, 0.82±0.07 and 0.37±0.04, respectively; ROS levels were 1.00±0.09, 4.72±0.44 and 1.30±0.07, respectively. The above indexes in the model group were significantly different from those in the control group, and the above indexes in the high-dose groups were significantly different from those in the model group (all P<0.05).

Piperlongumine can improve liver steatosis induced by sleep deprivation in mice, which may be related to the regulation of HIF-1α/p66Shc signaling pathway.

To study the effect of hydroxylsafflower yellow A (HSYA) on epiphyseal injury of tibial in rats through neurotrophic factor-3 (NT-3).

Fifty male SD rats were randomly divided into control group, model group, HSYA group, NC group and sh-NT-3 group, with 10 rats in each group. Except the control group, the injury model of tibial epiphyseal was established in the other 4 groups by high speed dental abrasive drill. The HSYA group was given 10 mg·kg^-1 HSYA by intragastric administration once a day for 6 weeks. NC group and sh-NT-3 group were respectively injected with 1 mg·kg^-1 sh-NC and sh-NT-3 through the tail vein, once every two days for 6 weeks. The control group and model group were given the same amount of normal saline intragastric and caudal intravenous injection. Quantitative real-time polymerase chain reaction was used to detect the expression level of NT-3 mRNA. The expression levels of bone repair markers and Notch receptor 1 (Notch1) signaling pathway related proteins were detected by Western blot.

The mRNA levels of NT-3 in control group, model group and HSYA group were 1.00±0.17, 3.31±0.59 and 5.16±0.84, respectively. The relative expression of runt related transcription factor 2 protein in control group, model group, HSYA group, NC group and sh-NT-3 group were 1.00±0.17, 3.67±0.63, 7.36±1.35, 7.31±1.42, 5.74±1.32, respectively; the relative expressions of osteoblast-specific transcription factor protein were 1.00±0.18, 5.76±1.01, 8.49±1.42, 8.56±1.54, 6.48±1.16, respectively; the relative expressions of Notch1 protein were 1.00±0.18, 1.44±0.23, 1.79±0.34, 1.83±0.35, 1.52±0.27, respectively. The above indexes in the model group were compared with the control group, the HSYA group was compared with the model group, and the sh-NT-3 group was compared with the NC group, and the above indexes were statistically significant (all P<0.05).

HSYA improves tibial epiphyseal injury in rats by promoting NT-3 expression and activating Notch1 signaling pathway.