Aim To explore the potential genes and mechanism of alisol B in the treatment of non-small cell lung cancer(NSCLC).Methods The proliferation and migration of NSCLC cells were detected by CCK-8 and Transwell.Genes of NSCLC and alisol B were col-lected through TCGA and compound gene prediction database,and their intersection genes were obtained.The network of protein-protein interaction(PPI)was constructed by using String database,and the top 20 key nodes were screened out,and the prognosis-related proteins related to the prognosis of NSCLC were screened out by using R language,and the intersection of them was obtained.The potential mechanism of ali-sol B on NSCLC was explored by KEGG and GO en-richment analysis and the relationship between related genes and immune cells,which was verified by cell-lev-el experiments.Results Alisol B inhibited the cell activity and migration ability of NSCLC cells.Five im-portant genes were identified by network pharmacologi-cal analysis:CCNE1,CDK1,COL1A1,COL1A2 and COL3A1.The results of cell experiment showed that al-isol B down-regulated the expression of Cyclin E1,CDK1 and COL1A2 in NSCLC cells.In addition,alisol B could inhibit the expression of COL1A2 and M2 macrophage marker CD206 in macrophages.Conclu-sions Alisol B may inhibit the proliferation of tumor cells by down-regulating CDK1 and Cyclin E1,and may affect the function of macrophages by inhibiting COL1A2,thus regulating the tumor immune microenvi-ronment and inhibiting NSCLC.

AIM To investigate the effects of extraction,concentration,centrifugation,sterilization processes on overall quality of Yanggan Jiangcao Mixture.METHODS The analysis was performed on a 20℃ thermostatic Phenomenex Luna C18column(250 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1% phosphoric acid flowing at 1 mL/min in a gradient elution manner.The HPLC fingerprints were established,after which the contents of salidroside,geniposide,forsythoside A,specnuezhenide,glycyrrhizic acid and atractylenolideⅢ,along with the calculation of their transfer rates.RESULTS There were twenty common peaks in the fingerprints for fifteen batches of samples under different processes with the similarities of more than 0.950.Six constituents showed good linear relationships within their own ranges(r≥0.999 0),whose average recoveries were 96.96%-106.69% with the RSDs of 0.97%-2.87%,and the transfer rates and total transfer rates were more than 80% and more than 90%,respectively.CONCLUSION Yanggan Jiangcao Mixture demonstrates good stability under different processes.

AIM To investigate the effects of Zuogui Jiangtang Tongmai Recipe on necroptosis pathway in a rat model of type 2 diabetes mellitus(T2DM)complicated with cerebral infarction(CI).METHODS The SD rats were randomly divided into the sham operation group,the model group,the metformin group(0.045 g/kg),and the low,medium and high dose Zuogui Jiangtang Tongmai Recipe groups(6.5,13,26 g/kg),with 9 rats in each group.In contrast to rats of the sham operation group,rats of the other groups were given 4 weeks feeding of high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin to establish a T2DM rat model with one week stable blood glucose,followed by gavage of corresponding drugs 3 days before the establishment of the middle cerebral artery occlusion(MCAO)model.After 7 days of administration,the rats had their CI injury assessed by mNSS method and TTC staining;their level of blood glucose detected by blood glucose meter;their levels of glycated serum protein,serum TNF-α and IL-1β detected by ELISA;their cerebral mRNA expressions of FADD,RIPK1,RIPK3 and MLKL detected by RT-qPCR;and their cerebral protein expressions of FADD,p-RIPK1,p-RIPK3 and p-MLKL detected by Western blot.RESULTS Compared with the sham operation group,the model group displayed increased levels of blood glucose value,glycosylated serum protein,neurological function score,cerebral infarction volume,cerebral FADD,RIPK1,RIPK3 and MLKL mRNA expressions,cerebral FADD,p-RIPK1,p-RIPK3 and p-MLKL protein expressions,serum TNF-α and IL-1β levels(P＜0.01);and more disordered and morphologically diverse neurons with smaller nucleus.Compared with the model group,the groups intervened with medium or high dose Zuogui Jiangtang Tongmai Recipe,or metformin shared improvement in terms of the aforementioned indices(P＜0.05,P＜0.01);and more neurons with regular morphology neat arrangement,and reduced cell gap.CONCLUSION Zuogui Jiangtang Tongmai Recipe can improve the neurological dysfunction of the rat model of T2DM complicated with CI,which may associate with the inhibited activation of necroptosis signaling pathway.

Objective To study the in vitro expression of three phenylalanine hydroxylase(PAH)mutants(p.R243Q,p.R241C,and p.Y356X)and determine their pathogenicity.Methods Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein.Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells.Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants,and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay.Results Bioinformatics analysis predicted that all three mutants were pathogenic.The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control(P>0.05),while the mRNA expression level of the p.Y356X mutant significantly decreased(P<0.05).The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control(P<0.05).The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control(P<0.05),while there was no significant difference between the p.R243Q mutant and the wild type control(P>0.05).Conclusions p.R243Q,p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells,with p.R241C and p.Y356X mutants also affecting the function of PAH protein.These three PAH mutants are to be pathogenic.[Chinese Journal of Contemporary Pediatrics,2024,26(2):188-193]

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Objective:To investigate the effect of methylene blue (MB) on motor dysfunction and its mechanism in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse models.Methods:Forty healthy male C57BL/6 mice were randomly divided into 4 groups: control group, model group, low-dose treatment group and medium-dose treatment group ( n=10); PD mouse models were established by intraperitoneal injection of 25 mg/kg/d MPTP for a consecutive 7 d; low-dose treatment group and medium-dose treatment group were pretreated intraperitoneally with MB 2 mg/kg/d or MB 10 mg/kg/d for a consecutive 3 d, respectively; and then, MPTP 25 mg/kg/d+MB 2 mg/kg/d or MPTP 25 mg/kg/d+MB 10 mg/kg/d were injected intraperitoneally into the low-dose treatment group or medium-dose treatment group for a consecutive 7 d (MPTP and MB were given at 12 h of interval). Eight d after modeling, open field experiment, pole climbing experiment and rod rotating experiment were carried out to evaluate the spontaneous movement, coordination, endurance and motor ability. And then, the mice were sacrificed; immunofluorescent staining was used to observe tyrosine hydroxylase (TH) expression in the substantia nigra; Western blotting was used to detect the expressions of TH, α-synuclein, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cleaved-Caspase-1 and Gasdermin D (GSDMD) in the striatum and substantia nigra of mice. Contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-18 in the substantia nigra and striatum of mice were detected by ELISA. Results:Compared with the control group, the model group had shortened residence time in rod rotating, prolonged descent time in rod climbing, reduced total movement distance in open field, decreased number of TH-positive cells in the substania nigra, decreased TH protein levels in the substania nigra and striatum, and increased NLRP3, ASC, cleaved-Caspase-1, GSDMD and GSDMD-N protein levels in the substania nigra and striatum, and increased TNF-α, IL-1β and IL-18 contents in the substania nigra and striatum, with significant differences ( P<0.05). Compared with the model group, low-dose treatment group and medium-dose treatment group had prolonged residence time in rod rotating, shortened descent time in rod climbing, increased total movement distance in open field, increased number of TH-positive cells in the substania nigra, and increased TH protein levels in the substania nigra and striatum, decreased NLRP3, ASC, and cleaved-Caspase-1 levels in the substania nigra and striatum, and decreased TNF-α, IL-1β and IL-18 contents in the substania nigra and striatum, with significant differences ( P<0.05). No statistical differences in the above indexes were noted between the low-dose treatment group and medium-dose treatment group ( P>0.05). Conclusion:Low-/medium-dose MB can ameliorate motor dysfunction in PD mouse models, whose mechanism may be related to downregulate NLRP3 inflammasome and inhibit neuroinflammatory response to reduce dopaminergic neuron pyroptosis.

Objective To investigate the therapeutic effect and mechanism of paeoniflorin(PAE)on thrombosis angiitis obliterans(TAO)in rats.Methods TAO rat model was established by sodium laurate injection.Rats were randomly divided into sham operation group(intraperitoneal injection of 0.9％NaCl),model group(intraperitoneal injection of 0.9％NaCl),experimental-L,-H groups(intraperitoneal injection of PAE 5,20 mg·kg-1·d-1),experimental-H+agonist group(intraperitoneal injection of 20 mg·kg-1·d-1 PAE+caudal vein injection of 10 ng·mL-1·kg 1·d-1 740 Y-P).Thrombin time(TT)was measured by magnetic bead coagulation;the levels of interleukin(IL)-1 β and endothelin 1(ET-1)were detected by enzyme-linked immunosorbent assay kit;the expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated-PI3K(p-PI3 K),protein kinase B(AKT),p-AKT,nuclear factor(NF)-κB p65,p-NF-κB p65 were detected by Western blotting.Results The TT of sham operation group,model group,experimental-L,-H groups and experimental-H+agonist group were(14.88±1.32),(10.02±0.95),(12.65±1.22),(14.70±1.36)and(10.64±1.21)s;IL-1β were(154.23±13.45),(356.69±31.17),(268.62±23.58),(199.64±20.87)and(337.48±31.46)pg·mL-1;ET-1 were(6.78±0.68),(14.43±1.14),(11.23±1.07),(8.20±0.81)and(13.33±1.27)pg·mL-1;p-PI3K/PI3K were 0.36±0.04,0.76±0.07,0.59±0.05,0.44±0.04 and 0.69±0.07;p-AKT/AKT were 0.52±0.05,0.90±0.09,0.74±0.08,0.61±0.06 and 0.86±0.08;p-NF-κB p65/NF-κB p65 were 0.28±0.03,0.95±0.04,0.69±0.07,0.35±0.05 and 0.87±0.08,respectively.There were statistically significant differences between model group and sham operation group(all P＜0.05);the above indexes in experimental-L group and experimental-H group were significantly different from those in medel group(all P＜0.05);the above indexes in experimental-H+agonist group were significantly different from those in experimental-H group(all P＜0.05).Conclusion PAE may improve disease progression in TAO rats by inhibiting the PI3K/AKT/NF-κB signaling pathway.

The success of the Human Genome Project has significantly deepened our understanding of genomics and catalyzed a growing focus on proteomics, as researchers aim to decipher the complex relationship between genes and proteins. Given the central role of proteins in regulating physiological processes—including DNA replication, metabolic reactions, signal transduction, pH balance, and cellular structure—developing advanced protein sequencing technologies is critical. Proteins are fundamental to nearly all biological activities, making their detailed study essential for understanding cellular functions and disease mechanisms. The Edman degradation method, developed in the 1950s, was a breakthrough in sequencing short peptides. However, its limitations in read length (fewer than 50 amino acids) and slow cycle time fall short of modern demands. Mass spectrometry has since emerged as the gold standard in protein sequencing due to its high accuracy, throughput, and reproducibility. The method is enhanced by a robust sample preparation workflow and advances in mass spectrometry technology. Despite these strengths, mass spectrometry faces limitations in dynamic range, sensitivity, read length, and sequence coverage, hindering complete de novo protein sequencing. These technological gaps underscore the need for innovative methods to provide more detailed and accurate protein sequence data. In the past decade, new protein sequencing methods, including tunneling current, fluorescence fingerprinting, and real-time dynamic fluorescence, have shown significant developmental potential. However, these methods are not yet ready for widespread application, as each still faces technical hurdles. Meanwhile, advances in nanopore DNA sequencing have sparked interest in applying nanopore technology to protein sequencing, particularly owing to its speed, convenience, and cost-effectiveness. Unlike DNA sequencing, protein sequencing presents greater challenges due to proteins’ complex three-dimensional structures, heterogeneous electrical charges, difficulties in directional movement, and diverse amino acid compositions, further complicated by post-translational modifications. Researchers have made significant strides in addressing these challenges, such as using unfolding enzymes, high temperatures, high voltage, and deformers to unravel protein structures, and employing charged sequences and electroosmotic flow to control peptide translocation. The latest strategies for nanopore protein sequencing can be broadly categorized into three approaches: strand sequencing, enzyme-assisted nanopore sequencing, and nanopore fingerprinting. In strand sequencing, dragging a protein-oligonucleotide conjugate through a nanopore with the aid of protein motors generates stepped current signals produced by the peptide strand. In enzyme-assisted nanopore sequencing, 20 proteinogenic amino acids and various post-translational modifications have been distinguished using nanopores, and sequencing of short peptides has also been demonstrated. In nanopore fingerprinting, polypeptide fragments resulting from protease digestion of a protein can be identified through nanopore sensing. Despite these advances, further improvements in protein engineering, data processing, identification accuracy, and read length are needed to make these strategies practically useful. This review provides an overview of the current major approaches to nanopore protein sequencing, emphasizing the strategies, recent advances, breakthroughs and challenges in nanopore protein sequencing. As nanopore technology continues to evolve, it is expected to offer more efficient and accurate sequencing solutions in proteomics, potentially leading to new technological breakthroughs in biochemistry and biomedicine.

Constipation is a prevalent ailment which might significantly impact the quality of people's life and rise some associated deseases risks. In this study, a chronic constipation mouse model was established using loperamide hydrochloride. Mice were gavaged an Angelica sinensis Cistanche Fiber Compound, comprised of Dang Gui [Angelica sinensis (Oliv.) Diels], Rou Cong Rong (Cistanche deserticola Y.C.Ma), wheat fiber, and low-molecular-weight xylan at high dosage (3.6 g·kg^-1, 30 times the recommended human dose) and low dosage (0.6 g·kg^-1, 5 times the recommended human dose) for 14 days. The study assessed the therapeutic efficacy of the compound by observing fecal morphology, measuring water content, and conducting small intestine motility experiments. Furthermore, enzyme-linked immunosorbent assays (ELISA) were conducted to evaluate the serum levels of gastrointestinal hormones including motilin (MTL), gastrin (GAS), the inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Histopathological examination with H&E staining was employed to evaluate colonic tissue damage. Additionally, Western blot and immunohistochemistry experiments were conducted to examine the expression levels of aquaporin-3 (AQP3) and c-Kit in the colon. The results indicated that the Angelica sinensis Cistanche Fiber Compound could improve fecal morphology, increase fecal water content, enhance small intestinal transit rates in mice. Additionally, there was a significant elevation in the serum levels of gastrointestinal hormones and a notable reduction in the levels of inflammatory factors. The improvement in colonic histopathological damage was accompanied by a marked decrease in the expression of the colonic water channel protein AQP3 and a significant increase in c-Kit expression. These results collectively suggested the presence of a dose-response relationship. These findings indicate that the Angelica sinensis Cistanche Fiber Compound effectively alleviates constipation in mice. Its action is associated with the regulation of colonic water channel protein AQP3 and c-Kit expression, along with the modulation of serum inflammatory factors and gastrointestinal hormones. This experiment was approved by the Animal Experiment Ethics Committee of Jinan University.

Combined radiation and burn injury (CRBI) is a severe syndrome, which is induced by the simultaneous or successive radiation and burn; but no appropriate clinical therapies are available. Loong oil (LO) is a traditional Chinese medicine oil composed of the oil extracts of cuttlebone, safflower, walnut oil, and rapeseed oil, which has been demonstrated to own anti-radiation and tissue healing functions. In this study, glyceryl monostearate (GMO) was used for the preparation of lyotropic liquid crystals that loaded LO to obtain Loong oil-lyotropic liquid crystals (LOL) for the treatment of skin CRBI. The hexagonal phase structure of LOL was proved by small X-ray scattering (SAXS) analysis with an approximate 7:9:11 ratio of peaks and under the polarizing microscope with the birefringence phenomenon. LOL had a high LO loading capacity (4%) and good bio-adhesive force, favoring skin administration. Animal experiments were approved by the Ethics Committee of the Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-834). Compared to triethanolamine creams, LOL had higher skin permeability with total skin penetration within 1 h, benefiting the treatment of deep CRBI. A deep II degree burn CRBI mouse model was established after whole-body irradiation with 5 Gy ⁶⁰Co γ-ray and the next 6-second 100 ℃ burn injury. LOL promoted LO skin penetration, improved epithermal cell proliferation and the formation of hair follicles, accelerated wound healing, and inhibited scarring, which benefitted the therapy of CRBI. In this study, LO application fields are expanded and LO is a promising medication for the treatment of CRBI.