Objective: To reveal the molecular mechanism underlying the compatibility of Salvia miltiorrhiza Bge (S. miltiorrhiza, Dan Shen) and C. tinctorius L. (C. tinctorius, Hong Hua) as an herb pair through network pharmacology and subsequent experimental validation. Methods: Network pharmacology was applied to construct an active ingredient-efficacy target-disease protein network to reveal the unique regulation pattern of S. miltiorrhiza and C. tinctorius as herb pair. Molecular docking was used to verify the binding of the components of these herbs and their potential targets. An H9c2 glucose hypoxia model was used to evaluate the efficacy of the components and their synergistic effects, which were evaluated using the combination index. Western blot was performed to detect the protein expression of these targets. Results: Network pharmacology analysis revealed 5 pathways and 8 core targets of S. miltiorrhiza and C. tinctorius in myocardial protection. Five of the core targets were enriched in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. S. miltiorrhiza-C. tinctorius achieved vascular tone mainly by regulating the target genes of the HIF-1 pathway. As an upstream gene of the HIF-1 pathway, STAT3 can be activated by the active ingredients cryptotanshinone (Ctan), salvianolic acid B (Sal. B), and myricetin (Myric). Cell experiments revealed that Myric, Sal. B, and Ctan also exhibited synergistic myocardial protective activity. Molecular docking verified the strong binding of Myric, Sal. B, and Ctan to STAT3. Western blot further showed that the active ingredients synergistically upregulated the protein expression of STAT3. Conclusion The pharmacodynamic transmission analysis revealed that the active ingredients of S. miltiorrhiza and C. tinctorius can synergistically resist ischemia through various targets and pathways. This study provides a methodological reference for interpreting traditional Chinese medicine compatibility.

Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.

Objective·To study the relationship between evolution and the developmental process from the perspective of DNA sequence conservation,and explore their inherent principles.Methods·First,conservation rate(CR)was established by analyzing the conservation of amino acid sequences of coding genes in 100 species to quantify the evolutionary conservation of genes.The relationship between CR and developmental potential was verified by using the feature genes involved in embryonic stem cells pathways.Secondly,cell type-specific genes and their characteristics in conservation were studied by analyzing the RNA sequencing(RNA-seq)data of the three early germ layers(ectoderm,mesoderm and endoderm)and their corresponding mature organs(brain,heart,liver,etc).Then,chromatin immunoprecipitation sequencing(ChIP-seq)data of enhancer histone H3 acetylated at lysine 27(H3K27ac)from early germ layers and mature organs were collected to search for enhancer sites and identify super enhancers in various cells and tissues by using the ROSE procedure.Functional enrichment and signaling pathway analysis of genes was used to examine the identity correlation between SEs-regulated genes and the corresponding cell characteristics,to clarify whether the SEs identified in this study were consistent with the characteristics reported in previous studies.Finally,PhastCons program was used to calculate the DNA conservation score(CS)of non-coding regulatory regions to study their relationship with developmental potential.Results·In the coding region of DNA,CR was successfully established to quantify the conservation of genes.The gene expression data of early germ layers and mature organs showed that the genes with higher conservation rate were more relevant to the stemness and early developmental process,and the differences between the tissues from early and late development could be distinguished by using CR.In the non-coding regions of DNA,it was found that the conservation of regulatory regions was also correlated with development.The CS of the SE sequences in the early developmental germ layers was significantly higher than that of the SE sequences in the corresponding mature organs.However,cell-specific typical enhancers(TEs)did not show such a trend.Conclusion·During the developmental process,CR of genes expressed in the coding region decreases,and CS of super-enhancer DNA in the non-coding region decreases.

ObjectiveTo study the relationship between the exposure to two kinds of phthalate esters （PAEs） ［Di-N-butyl phthalate，（DBP） and Di-（2-ethylhexyl）phthalate （DEHP）］ and estrogen homeostasis in pregnant women. MethodsIn 2021， we classified the Jiading District of Shanghai into five geographical areas， east， west， south， north and central. A total of 151 pregnant women from each area were selected for questionnaire survey， with random urine samples during first， second， and third trimesters collected. A DBP metabolite ［Mono-N-butyl phthalate （MBP）］ and two DEHP metabolites ［Mono（2-ethylhexyl） phthalate （MEHP）， Mono（2-ethyl5-oxohexyl） phthalate， （MEOHP）］ and three estrogens ［estrone （E₁）， 17β -estradiol （E₂）， and estriol （E₃）］ in urine were determined by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry. After a natural logarithmic transformation of PAEs metabolite levels and estrogen concentration， multivariable linear regression was used to control potential confounders and determine the relationship between PAEs metabolite levels and estrogen concentration. ResultsThe detection rates of three PAEs metabolites in urine of pregnant women were more than 98%. The median corrected concentrations of MBP， MEHP and MEOHP were 5.18， 0.59 and 4.23 mg·kg^-1， respectively. During the whole pregnancy， MEOHP was positively correlated with E₁ （β=0.450， 95%CI： 0.057‒0.844）， and MBP was positively correlated with E₃ （β=0.250， 95%CI： 0.034‒0.465）. Stratified by trimesters， MBP was positively correlated with E₃ in the first trimester （β=0.428， 95%CI： 0.103‒0.752）. MEOHP was positively correlated with E₁ in the second trimester （β=0.734， 95%CI： 0.130‒0.752）， and had a possitive trend with E₁ in the third trimester （β=0.744， 95%CI： -0.140‒1.629）. In addition， MEHP had a negative correlation with E₁ in the second trimester （β=-0.498， 95%CI： -1.063‒0.066）. MEOHP had a positive correlation trend with E₂ （β=0.628， 95%CI： -0.101‒1.356） in the third trimester. ConclusionPAEs exposure may interfere with estrogen homeostasis during pregnancy and differs by trimesters. Given the cross-sectional nature of this study， it warrants further study to validate the findings.

The importance of structural variants(SVs)for human phenotypes and diseases is now recognized.Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed,few benchmarking procedures are available to confidently assess their performances in biological and clinical research.To facilitate the validation and application of these SV detection approaches,we established an Asian reference material by characterizing the genome of an Epstein-Barr virus(EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls.We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers,including 109x Pacific Biosciences(PacBio)continuous long reads(CLRs),22 x PacBio circular consensus sequencing(CCS)reads,104x Oxford Nanopore Technologies(ONT)long reads,and 114×Bionano optical mapping plat-form,and one de novo assembly-based SV caller using CCS reads.A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing,demonstrating the robustness of our SV calls.Combining trio-binning-based haplotype assemblies,we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions(CHCRs),which covered 1.46 gigabase pairs(Gb)and 6882 SVs supported by at least one diploid haplotype assembly.Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology,disease,and clinical research.

Objective: To determine the in vitro and in vivo absorption properties of active ingredients of the Chinese medicine, baicalein, to enrich mechanistic understanding of oral drug absorption. Methods: The Biopharmaceutic Classification System (BCS) category was determined using equilibrium solubility, intrinsic dissolution rate, and intestinal permeability to evaluate intestinal absorption mech-anisms of baicalein in rats in vitro. Physiologically based pharmacokinetic (PBPK) model commercial software GastroPlus?was used to predict oral absorption of baicalein in vivo. Results: Based on equilibrium solubility, intrinsic dissolution rate, and permeability values of main absorptive segments in the duodenum, jejunum, and ileum, baicalein was classified as a drug with low solubility and high permeability. Intestinal perfusion with venous sampling (IPVS) revealed that baicalein was extensively metabolized in the body, which corresponded to the low bioavailability predicted by the PBPK model. Further, the PBPK model predicted the key indicators of BCS, leading to reclassification as BCS-II. Predicted values of peak plasma concentration of the drug (Cmax) and area under the curve (AUC) fell within two times of the error of the measured results, highlighting the superior prediction of ab-sorption of baicalein in rats, beagles, and humans. The PBPK model supported in vitro and in vivo evi-dence and provided excellent prediction for this BCS class Ⅱ drug. Conclusion: BCS and PBPK are complementary methods that enable comprehensive research of BCS parameters, intestinal absorption rate, metabolism, prediction of human absorption fraction and bioavailability, simulation of PK, and drug absorption in various intestinal segments across species. This combined approach may facilitate a more comprehensive and accurate analysis of the absorption characteristics of active ingredients of Chinese medicine from in vitro and in vivo perspectives.

Objective:To investigate the expression of oxidative stress and related pathway factors in fibroblasts from human hypertrophic scar and normal skin tissue.Methods:Select and collect the scar tissue and normal full-thickness skin tissue of the donor area from 8 hypertrophic scar patients who were treated with surgical resection at the Department of Burns and Plastic Surgery, the Fourth Medical Center of Chinese PLA General Hospital from June 2019 to July 2020, extract and culture primary fibroblasts by weaving method, subculture them, and perform the following experiments when the cells were subcultured to the third generation: (1)Detect the proliferation ability of hypertrophic scar fibroblasts(HSF)and normal skin fibroblasts(NSF)with CCK-8 method.(2)Detect the number of reactive oxygen species in HSF and NSF by flow cytometry.(3)Colorimetric method for detecting relative activity of superoxide dismutase(SOD)in two kinds of cells.(4)Detect the NQO1 gene expression in two kinds of cells with RT-PCR.(5)Detect the Nrf2 gene expression in two kinds of cells with RT-PCR.(6)Detects Nrf2 and Bcl2 protein content in two kinds of cells with Western blotting.(7)Detect the expression and distribution of Nrf2 in cells with cellular immunohistochemical staining. The results were analyzed by SPSS 25.0 statistical software, independent sample t test was performed, and P<0.05 was considered as the difference was statistically significant. Results:(1) Compared with NSF, the number of the two kinds of cells is statistically different from the third day (The statistical results from the third day to the seventh day were as follows: t=2.631, P=0.039; t=7.025, P<0.001; t=5.031, P<0.001; t=4.241, P=0.002; t=6.525, P=0.003). (2) The reactive oxygen species content in HSF was 75.39%±3.06%, which was significantly higher than 45.36%±3.66% in the NSF group ( t=-17.804, P<0.001). (3) The relative activity of SOD of the HSF group was (50.76±0.52) U/ml, which is higher than that of the NSF group (42.76±1.35) U/ml ( t=15.674, P<0.001). (4) The relative expression of NQO1 mRNA in HSF was 0.859±0.076, which was lower than that in the NSF group at 1.595±0.181 ( t=3.763, P=0.020). (5) The relative expression of Nrf2 mRNA in HSF was 0.590±0.055, which was lower than that in the NSF group at 1.595±0.146 ( t=6.445, P=0.003). (6) The relative expression of Nrf2 protein of HSF was 0.314±0.035, which was significantly lower than 0.912±0.039 in the NSF group ( t=22.554, P<0.001). The relative expression of Bcl2 protein of HSF was 0.466±0.020, which was significantly lower than 0.734±0.066 in the NSF group ( t=7.780, P<0.001). (7) Nrf2 protein express in NSF and HSF cells, and protein expression was found in both nucleus and cytoplasm. Conclusions:HSF has a high degree of oxidative stress, which may be due to some reasons that reduce the content of Nrf2, resultsing in a decrease in the expression of various antioxidant enzymes, and ultimately leading to the accumulation of reactive oxygen species. Compared with NSF, HSF has stronger proliferation and apoptosis abilities, and reactive oxygen species can cause cell apoptosis, indicating that oxidative stress may be one of the pathogenesis of hypertrophic scars.

Objective:To investigate the expression of oxidative stress and related pathway factors in fibroblasts from human hypertrophic scar and normal skin tissue.Methods:Select and collect the scar tissue and normal full-thickness skin tissue of the donor area from 8 hypertrophic scar patients who were treated with surgical resection at the Department of Burns and Plastic Surgery, the Fourth Medical Center of Chinese PLA General Hospital from June 2019 to July 2020, extract and culture primary fibroblasts by weaving method, subculture them, and perform the following experiments when the cells were subcultured to the third generation: (1)Detect the proliferation ability of hypertrophic scar fibroblasts(HSF)and normal skin fibroblasts(NSF)with CCK-8 method.(2)Detect the number of reactive oxygen species in HSF and NSF by flow cytometry.(3)Colorimetric method for detecting relative activity of superoxide dismutase(SOD)in two kinds of cells.(4)Detect the NQO1 gene expression in two kinds of cells with RT-PCR.(5)Detect the Nrf2 gene expression in two kinds of cells with RT-PCR.(6)Detects Nrf2 and Bcl2 protein content in two kinds of cells with Western blotting.(7)Detect the expression and distribution of Nrf2 in cells with cellular immunohistochemical staining. The results were analyzed by SPSS 25.0 statistical software, independent sample t test was performed, and P<0.05 was considered as the difference was statistically significant. Results:(1) Compared with NSF, the number of the two kinds of cells is statistically different from the third day (The statistical results from the third day to the seventh day were as follows: t=2.631, P=0.039; t=7.025, P<0.001; t=5.031, P<0.001; t=4.241, P=0.002; t=6.525, P=0.003). (2) The reactive oxygen species content in HSF was 75.39%±3.06%, which was significantly higher than 45.36%±3.66% in the NSF group ( t=-17.804, P<0.001). (3) The relative activity of SOD of the HSF group was (50.76±0.52) U/ml, which is higher than that of the NSF group (42.76±1.35) U/ml ( t=15.674, P<0.001). (4) The relative expression of NQO1 mRNA in HSF was 0.859±0.076, which was lower than that in the NSF group at 1.595±0.181 ( t=3.763, P=0.020). (5) The relative expression of Nrf2 mRNA in HSF was 0.590±0.055, which was lower than that in the NSF group at 1.595±0.146 ( t=6.445, P=0.003). (6) The relative expression of Nrf2 protein of HSF was 0.314±0.035, which was significantly lower than 0.912±0.039 in the NSF group ( t=22.554, P<0.001). The relative expression of Bcl2 protein of HSF was 0.466±0.020, which was significantly lower than 0.734±0.066 in the NSF group ( t=7.780, P<0.001). (7) Nrf2 protein express in NSF and HSF cells, and protein expression was found in both nucleus and cytoplasm. Conclusions:HSF has a high degree of oxidative stress, which may be due to some reasons that reduce the content of Nrf2, resultsing in a decrease in the expression of various antioxidant enzymes, and ultimately leading to the accumulation of reactive oxygen species. Compared with NSF, HSF has stronger proliferation and apoptosis abilities, and reactive oxygen species can cause cell apoptosis, indicating that oxidative stress may be one of the pathogenesis of hypertrophic scars.

Dry eye is a common ocular disease with a high morbidity.Many factors contribute to dry eye with cause pathophysiological changes of the ocular surface.In recent years,with the increasing attention of dry eye and the research progress,people have more in-depth understanding of the dry eye.This article reviews the researches in recent years and new progress in the treatment of dry eye.

Objective To discuss the alterations of functional brain properties in patients with idiopathic trigeminal neuralgia (ITN) based on graph theoretical approaches.Methods A total of 20 patients with pain in the distribution of left trigeminal nerve and 22 matched healthy controls were recruited in our hospital from May 2013 to January 2015.Resting-state functional MRI data in both groups were collected and analyzed with GRETNA software to obtain the matrices of brain network.From these connectivity matrices,a number of key characteristics were computed,including network efficiency,weighted shortest path length,weighted clustering coefficient and small-wordless.A two-tailed t test was used to appraise the significance of any inter-group differences in complex network measures investigated.Network-based statistic (NBS) approach was used to localize specific pairs of brain regions in which functional connectivity strength were altered in patients.Pearson's correlation analyses were performed to identify the correlations between clinical data and altered network properties in patients.Results ITN patients had significantly increased weighted clustering coefficient in the right calcarine (CAL) and bilateral thalamus,increased total efficiency in the left superior frontal gyrus (SFG),and increased regional efficiency in the right SFG,right CAL,bilateral cuneus and left pallidum,and significantly reduced weighted shortest path length for regional nodal characteristics in the right superior parietalis lobules (P＜0.05).The total efficiency in the left SFG and regional efficiency in the right SFG in ITN patients were negatively correlated with course of disease (r=-0.500,P=0.035;r=-0.501,P=0.034).As compared with the healthy controls,the ITN patients had enhanced functional loop connection between left SFG and right supplementary motor area,between left SFG and right SFG,between left SFG and right middle frontal gyrus,between left SFG and right orbital middle frontal gyrus,between left SFG and partes opercularis of right gyrus frontalis inferior (IFGoperc),between left SFG and bilateral putamen (PUT),between left SFG and right PAL,between right PUT and left interior superior frontal gyrus,and between left PUT and right caudate nucleus.The connectivity strength between left SFG and right IFGoperc was negatively correlated with disease duration (r=-0.487,P=-0.041).Conclusion Alterations in regional nodal characteristics may suggest central sensitization and modulation in ITN patients.