ObjectiveTo explore the mechanism of Shenmai injection in improving cisplatin resistance in non-small cell lung cancer （NSCLC） based on the endoplasmic reticulum stress through protein kinase R-like endoplasmic reticulum kinase （PERK）/activated transcription factor 4 （ATF4）/C/EBP homologous protein （CHOP） signaling pathway. MethodsBALB/c nude mice bearing cisplatin-resistant human lung cancer cell line （A549/cisplatin） were randomly divided into four groups： Blank control group （0.9% sodium chloride）， cisplatin group （5 µg·g^-1cisplatin）， Shenmai injection group （5.2 mg·g^-1 Shenmai injection）， and combination therapy group （5.2 mg·g^-1 Shenmai injection +5 µg·g^-1cisplatin）. The drug intervention lasted for 4 weeks， and the changes in body weight and tumor volume were monitored. Hematoxylin-eosin （HE） staining was performed to observe tumor tissue pathology. Transmission electron microscopy （TEM） was used to assess the morphology of the endoplasmic reticulum. Immunohistochemical assay was conducted to measure the positive expressions of PERK， ATF4， and CHOP in tumor tissues. Western blot quantified the protein expression of immunoglobulin heavy chain binding protein （BIP）， PERK， phosphorylated PERK （p-PERK）， eukaryotic translation initiation factor 2α （eIF2α）， phosphorylated eIF2α （p-eIF2α）， ATF4， CHOP， B-cell lymphoma -2 （Bcl-2）， and Bcl-2 Associated X protein （Bax）. A549/cis cells were divided into blank group： Blank control group （normal culture medium）， cisplatin group （23.3 µmol·L^-1 cisplatin）， Shenmai Injection group （20 g·L^-1 Shenmai injection）， and combination therapy group （20 g·L^-1 Shenmai injection+23.3 µmol·L^-1 cisplatin）. Cell counting kit-8 （CCK-8） method was used to detect cell viability， TEM was used to observe the morphology of endoplasmic reticulum， and Western blot was used to detect endoplasmic reticulum stress and apoptosis-related proteins. ResultsCompared with the cisplatin group， the combination therapy group showed increased body weight （P<0.05）， decreased tumor volume （P<0.05）， and expanded endoplasmic reticulum in tumor cells. The positive expressions of PERK， ATF4， and CHOP increased （P<0.05）. Western blot revealed elevated protein expression levels of BIP， p-PERK/PERK， p-eIF2α/eIF2α， ATF4， CHOP， and Bax （P<0.05）， while Bcl-2 expression decreased （P<0.05）. As shown in the in vitro experiment， compared with the cisplatin group， the combination therapy group exhibited a reduced cell survival rate （P<0.05）. TEM revealed increased endoplasmic reticulum dilation and vesicular degeneration. Western blotting showed increased protein levels of BIP， p-PERK/PERK， p-eIF2α/eIF2α， ATF4， CHOP and Bax （P<0.05）， with decreased Bcl-2 expression （P<0.05）. ConclusionShenmai injection combined with cisplatin has a synergistic antitumor effect in NSCLC， which may be attributed to the activation of endoplasmic reticulum stress response mediated by the PERK/eIF2α/ATF4/CHOP signaling pathway and the induction of tumor cell apoptosis.

ObjectiveTaking Aurantii Fructus Immaturus juice（AFI）-processed Atractylodis Macrocephalae Rhizoma（AMR） as an example， this study aims to systematically compare the volatile and non-volatile components of AMR and its processed products， investigate the key differential components， evaluate their anti-inflammatory activities， and elucidate the synergistic mechanism of processing. MethodsThe chemical compositions of volatile and non-volatile components in AMR and AFI-processed AMR were systematically characterized using gas chromatography-mass spectrometry（GC-MS） and ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， with relative mass fractions and response values determined separately. Volatile components were identified through searches in the National Institute of Standards and Technology（NIST）17 database， comparison with retention index（RI） and fragmentation pattern matching. Non-volatile components were identified by searching Waters Traditional Chinese Medicine （TCM） spectral library， in conjunction with PubChem and MassBank， characteristic fragmentation patterns and response values were also used to support identification. Differential components were screened using principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA）， with variable importance in the projection（VIP） value >1. Components with high log₂fold change（FC） among major differential groups were selected as those exhibiting significant changes before and after processing. The anti-inflammatory activity of the differential compounds was evaluated by assessing their effects on nitric oxide（NO） production in a lipopolysaccharide（LPS）-induced RAW264.7 macrophage model. Enzyme-linked immunosorbent assay（ELISA） was used to detect the effects of the differential components on tumor necrosis factor（TNF）-α， interleukin（IL）-1β， IL-6， and monocyte chemotactic protein（MCP）-1 levels， and immunofluorescence（IF） was employed to assess their effects on nuclear transcription factor（NF）-κB p65 translocation， thereby elucidating the underlying molecular mechanisms. ResultsA total of 36 compounds were identified in the volatile components of AMR and AFI-processed AMR， among which， sesquiterpenes and monoterpenes were significantly increased after processing. In the non-volatile components， 36 compounds were identified， and the main differential components were flavonoids， sesquiterpenoids， and triterpenoids. Flavonoids were the primary differential components distinguishing AMR from its processed products， representing compounds directly introduced during processing. Five compounds， including atractylenolide Ⅲ， tangeritin， nobiletin， hesperidin and narirutin， were selected as representatives of three classes based on their most prominent differential expression among different compound types for subsequent anti-inflammatory activity studies. The results showed that 100 μmol·L^-1 tangerine and narirutin could significantly inhibit LPS-induced NO production（P<0.01） in a concentration-dependent manner. Tangeritin was able to significantly inhibit the levels of TNF-α and MCP-1 secreted by RAW264.7（P<0.05）， while narirutin significantly inhibited the levels of TNF-α， IL-1β， MCP-1 and IL-6（P<0.01）. IF revealed that both tangeritin and narirutin significantly blocked the translocation of NF-κB p65 from the cytoplasm to the nucleus. ConclusionAFI-processed AMR significantly alters the chemical composition profile of AMR， and the newly introduced flavonoid components during processing may be key to its enhanced anti-inflammatory effects.

Objective:To develop and validate a liquid chromatography-tandem mass spectrometry method for determining levofloxacin in plasma sample from pediatric patients.Method:This is a prospective, observational study. The clinical residual plasma samples from healthy individuals for physical examination in Children's Hospital Affiliated to Zhengzhou University were collected as blank matrix. Plasma samples from five pediatric patients who did not receive levofloxacin or ciprofloxacin in the department of Respiration were collected for methodological evaluation. In addition, 34 clinical plasma samples from 22 pediatric patients (9 males and 13 females; mean age (8.1±3.7) years) using levofloxacin was collected, and their plasma concentrations were determined. Using ciprofloxacin as the internal standard, levofloxacin in plasma samples was quantified by liquid chromatography-tandem mass spectrometry following protein precipitation using acetonitrile. A C18 column (Shim-pac GIST-HP C18, 2.1 mm×100 mm, 3 μm) and mobile phase composed of water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid) with gradient elution at a flow rate of 0.4 ml/min were used to separate levofloxacin. The column temperature was 40 ℃, injection volume was 1 μl and the total analysis time was 9 min. Levofloxacin and ciprofloxacin were ionized with an ESI source in positive ion mode and detected in multiple reaction monitoring (MRM) mode. The detected ions of levofloxacin and ciprofloxacin were m/z 362.10→318.1 and 332.15→231.05, respectively. The method′s specificity, sensitivity, linearity, precision, accuracy, recovery rate, stability, matrix effect, and carry-over were validated. All statistical analyses were performed with SPSS statistical software (version 17.0). The normality of the data was detected by the K-S test. A P<0.05 was considered statistically significant for two tailed tests. Results:The LC-MS/MS method showed a good linearity within the range of 0.062 5-20 mg/L, with the lower detection limit of levofloxacin of 0.062 5 mg/L. The calibration curve for levofloxacin was Y=0.093X+0.010 ( R2>0.99). Under different quality control levels, the accuracy ranged from 92.57% to 104.39%, and the intra-day and inter-day imprecision ranged from 2.32% to 9.35%. These values were not affected by the normal matrix, 5% hemolysis matrix and 15% hyperlipidemia matrix. Furthermore, the levofloxacin plasma samples were stable in the short term. A total of 34 plasma samples from 22 patients were collected and analyzed. Only 2 plasma samples were below the lower limit of quantification, while the other plasma concentrations of levofloxacin were ranged from 0.091 to 6.755 mg/L. Cmax was (5.52 ± 1.09) mg/L. Conclusion:The LC-MS/MS method meets the requirements of the reference method and requires a small sample size (50 μl), making it suitable for the determination of levofloxacin in plasma from pediatric patients.

ObjectiveTo investigate the role of endoplasmic reticulum stress genes in the prognosis of pancreatic cancer， and to establish a prognostic prediction model based on the prognostic markers for pancreatic cancer. MethodsTranscriptome sequencing data were downloaded from TCGA and GTEx databases， and MsigDB website was used to obtain endoplasmic reticulum stress genes. A univariate Cox regression analysis was performed to obtain the genes associated with the prognosis of pancreatic cancer， and a consensus clustering analysis was used to construct the molecular typing of pancreatic cancer， while the differentially expressed genes between the two subgroups were obtained. A Lasso regression analysis was used to obtain the core genes associated with the prognosis of pancreatic cancer， which were used to construct a prognostic prediction model for pancreatic cancer. Related datasets were obtained from the GEO database to validate the predictive performance of the model. The CIBERSORT analysis was used to investigate the correlation between risk score and immune infiltration. Quantitative real-time PCR was used to measure the expression of genes in pancreatic cancer tissue and cell lines. The independent-samples t test was used for comparison of continuous data between two groups， and the chi-square test was used for comparison of categorical data between groups. Survival was compared using Log-rank test. The predictive value of the model was evaluated by evaluating the area under the ROC curve. ResultsThe endoplasmic reticulum stress genes CEBPB， MARCKS， PMAIP1， and UBXN10 were independent risk factors for the prognosis of pancreatic cancer， and based on the expression characteristics of these genes， the TCGA pancreatic cancer cohort was divided into two subgroups， i.e.， cluster A and cluster B， while the cluster A patients had a significantly shorter overall survival time than the cluster B patients （P<0.01）. The Lasso regression analysis obtained 5 core genes from the differentially expressed genes affecting the prognosis of pancreatic cancer， and the risk scoring system was established as risk score=0.156×CDA+0.135×AHNAK2+0.020×RHOV+0.095×LY6D+0.054×SPRR1B. The ROC curve analysis showed that this model had good overall predictive performance， with the area under the ROC curve of 0.731 at 1 year， 0.712 at 3 years， and 0.686 at 5 years， and the low-risk group based on this model had a significantly longer overall survival time than the high-risk group （χ²=11.733， P=0.001）. The model showed good predictive performance in the external dataset GSE57495. Quantitative real-time PCR results showed that the expression levels of CDA， AHNAK2， RHOV， LY6D， and SPRR1B in 40 pancreatic cancer tissue samples were significantly upregulated compared with those in normal adjacent tissue samples （t=2.529， 2.458， 3.314， 3.583， and 5.082， all P<0.05）. ConclusionThe expression characteristics of CDA， AHNAK2， RHOV， LY6D， and SPRR1B can be used to predict the prognosis of pancreatic cancer， and the high expression levels of these genes are associated with the poor prognosis of pancreatic cancer patients.

OBJECTIVE: To investigate the effect of vitamin D3 on polarization of monocyte macrophages induced by serum from patients with ankylosing spondylitis (AS). METHODS: Twenty AS naïve patients and 20 healthy controls from Wenzhou People's Hospital during January 2016 and December 2017 were enrolled. The macrophages were differentiated from THP1 cells induced by phorbol 12-myristate 13-acetate (PMA), and then co-cultured with the serum from healthy subjects (control group) or AS patients. Vitamin D3 was added in the medium mixed with serum from AS patients. Flow cytometry was used to analyze the ratio of CD68 and CD206 positive cells, and RT-PCR was performed to detect the mRNA expression of inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). RESULTS: THP1 cells could be polarized into mononuclear-macrophages with the induction of PMA. The proportion of CD206 positive cells in AS-serum group was lower than that in the control group (=9.434, <0.05), while the proportion of CD68 positive cells was higher than that in the control group (=43.920, <0.05). The proportion of CD206 positive cells in vitamin D3 group was higher than that in AS-serum group (=8.895, <0.05), while the proportion of CD68 positive cells was lower than that in AS-serum group (=9.089, <0.05). mRNA expression of Arg-1 in AS-serum group was lower than that in the control group (=8.899, <0.05), while mRNA expression of iNOS was higher than that in the control group (=3.656, <0.05). mRNA expression of Arg-1 in vitamin D3 group was higher than that in AS-serum group (=6.219, <0.05), while mRNA expression of iNOS was lower than that in AS-serum group (=5.876, <0.05). CONCLUSIONS Vitamin D3 can regulate the polarization of mononuclear macrophages for immunoregulation in patients with AS.
Adjuvants, Immunologic ; pharmacology ; Cell Differentiation ; drug effects ; Cholecalciferol ; pharmacology ; Humans ; Monocytes ; drug effects ; Spondylitis, Ankylosing ; blood ; physiopathology