BackgroundInsomnia， a common sleep disorder， is robustly associated with cardiovascular diseases， diabetes， and psychiatric disorders， substantially impairing quality of life. Although clinically commonly used medications are effective， long-term use may lead to drug resistance and dependence. While the efficacy of Ganmai Dazao Decoction in improving insomnia is definite， its underlying molecular mechanisms remain unclear. ObjectiveTo explore the active ingredients and core targets of Ganmai Dazao Decoction in the treatment of insomnia， systematically reveal its potential molecular pharmacological mechanism， and to provide references for clinical application. MethodsIn November 2024， the active ingredients and related targets of Ganmai Dazao Decoction were screened from the INPUT database. Insomnia-related datasets were acquired from the Gene Expression Omnibus （GEO） database， followed by differential expression analysis using GEO2R to identify differentially expressed genes （DEGs） associated with insomnia. The shared targets were obtained through Venn diagrams， and the protein-protein interaction （PPI） network was constructed using the STRING database and Cytoscape 3.9.1. Enrichment analyses were conducted on the shared targets using Gene Ontology （GO） and the Kyoto Encyclopedia of Genes and Genomes （KEGG）. The top 3 key active ingredients and the top 10 core targets in terms of node degree values were selected. Molecular docking and molecular dynamics simulation of receptors and ligands were performed using AutoDock 4.4.6， and the results were visualized using Pymol 3.0.3 to further verify the stability of the receptor-ligand complex system. ResultsA total of 337 active ingredients and 5 265 drug-related targets in Ganmai Dazao Decoction were retrieved， along with 1 061 insomnia-related DEGs. 287 shared targets were identified between Ganmai Dazao Decoction and insomnia. The traditional Chinese medicine-active ingredients-shared targets-disease network showed that quercetin， catechins and kaempferol were the key components of Ganmai Dazao Decoction in treating insomnia. These three components alleviate insomnia by acting on ten core targets， including nuclear factor kappa B inhibitor alpha （NFKBIA）， fibronectin 1 （FN1）， interleukin-6 （IL6）， protein c-Fos （FOS）， histone acetyltransferase p300 （EP300）， histone deacetylase 1 （HDAC1）， transcription factor Jun （JUN）， heat shock protein HSP 90-alpha 1 （HSP90AA1）， glyceraldehyde 3-phosphate dehydrogenase （GAPDH）， and interleukin-1 beta （IL1β）. GO and KEGG enrichment analyses indicated that Ganmai Dazao Decoction may alleviate insomnia through the IL17 signaling pathways， lipid and atherosclerosis signaling pathways， and other mechanisms. The results of molecular docking demonstrated strong binding affinity between the 3 key components and the 10 core targets of Ganmai Dazao Decoction. Molecular dynamics simulations further confirmed the stability of the quercetin-GAPDH， catechin-HDAC1 and kaempferol-EP300 complexes. ConclusionThe key components of Ganmai Dazao Decoction， namely quercetin， catechin， and kaempferol， exert therapeutic effects on insomnia by targeting 10 core proteins and modulating multiple pathways， including the IL17 signaling pathway， lipids and atherosclerotic-related pathways. ［Funded by Chengdu Medical College Level Scientific Research Project （number， CYZYB23-01）］

Acetamiprid and pyridaben is a compound pesticide, Due to its characteristics of high efficiency, and environmental safety, it has been widely used in agriculture in recent years.This article reports a case of poisoning from a mixture of pymetrozine and propargite pesticides. The patient experienced severe symptoms of poisoning such as vomiting, shortness of breath, abdominal discomfort, generalized cyanosis, bradycardia, and shock after self-administering the pesticide. After admission to the hospital, the patient was treated with atropine to increase heart rate, endotracheal intubation to assist breathing, and norepinephrine to maintain blood pressure. In addition, the patient received gastric lavage, catharsis, hemoperfusion, and continuous blood purification therapy to eliminate the toxins. The patient was successfully treated and discharged after recovery.

Acetamiprid and pyridaben is a compound pesticide, Due to its characteristics of high efficiency, and environmental safety, it has been widely used in agriculture in recent years.This article reports a case of poisoning from a mixture of pymetrozine and propargite pesticides. The patient experienced severe symptoms of poisoning such as vomiting, shortness of breath, abdominal discomfort, generalized cyanosis, bradycardia, and shock after self-administering the pesticide. After admission to the hospital, the patient was treated with atropine to increase heart rate, endotracheal intubation to assist breathing, and norepinephrine to maintain blood pressure. In addition, the patient received gastric lavage, catharsis, hemoperfusion, and continuous blood purification therapy to eliminate the toxins. The patient was successfully treated and discharged after recovery.

OBJECTIVE： To study the long-term toxicity of Liqi sanjie extractum in rats after intragastric administration， and to provide reference for safety evaluation before clinical practice. METHODS： A total of 160 rats were randomly divided into control group （normal saline） and Liqi sanjie extractum low-dose， medium-dose and high-dose groups （7.828 0， 15.656 0， 31.312 0 g/kg， calculated by crude drug）， with 40 rats in each group. They were given relevant medicine intragastrically once a day from Monday to Saturday. The experimental period was 120 days， and the recovery period was 30 days after the end of the experiment. General information of rats was observed， and body weight and feed consumption of rats were measured once a week. At the 61st day of administration， the end of administration and the end of recovery period， 10， 20 and 10 rats were collected from each group to observe their hematology， blood biochemistry， organ coefficient and histopathology changes. RESULTS： From 61st day to 120th day of administration， the rats of Liqi sanjie extractum high-dose group had hair loss and erection， and recovered after withdrawal of medicine. During medication， the body weight of mice in Liqi sanjie extractum low-dose and medium-dose groups increased faster than control group， while the body weight of rats in Liqi sanjie extractum high-dose group increased slower than control group. Compared with control group， the feed consumption of Liqi sanjie extractum low-dose group increased， while those of Liqi sanjie extractum medium-dose and high-dose groups decreased； the rats were recovered after drug withdrawal. On the 61st day of administration and after the end of administration， some hematological indexes， blood biochemical indexes and organ coefficients of rats in administration group were significantly different from those of control group （P＜0.05 or P＜0.01）. The hematology， blood biochemistry and organ coefficients of rats were basically recovered after the end of the recovery period. The number of erythrocyte， hematocrit， standard deviation of erythrocyte width， albumin， globulin ratio and potassium K+ levels in Liqi sanjie extractum low-dose group were significantly lower than those in control group （P＜0.05 or P＜0.01）. The absolute value of intermediate cells in blood of rats in Liqi sanjie extractum medium-dose group was significantly higher than that of control group （P＜0.05）， and the mean hemoglobin concentration， K+ and uterine coefficient in blood were significantly lower than those of control group （P＜0.05）. The number of white blood cells， absolute value of lymphocyte， absolute value of intermediate cells， the percentage of intermediate cells， prothrombin time and spleen coefficient in Liqi sanjie extractum high-dose group were significantly higher than those in the control group （P＜0.05 or P＜0.01）. Mean hemoglobin concentration， granulocyte percentage， albumin， alkaline phosphatase and K+ were significantly lower than those in the control group （P＜0.05 or P＜0.01）. No abnormalities in systemic autopsy and histopathology were noticed in rats. CONCLUSIONS： Long-term intragastric administration of Liqi sanjie extractum can cause certain toxic reactions in rats， and low dose of Liqi sanjie extractum causes less and lighter toxic reactions which can be automatically recovered after drug withdrawal. It can provide reference for the determination of clinical safe dose.

OBJECTIVE： To investigate the improvement effects of Liqi sanjie granule on liver-qi stagnation model rats. METHODS： According to the weight， totally 80 rats were randomly divided into blank control group （normal saline）， model control group （normal saline）， Xiaoyao pill control group （positive control a， 750 mg/kg ，calculated by crude drug）， Xiaojin pill control group （positive control b， 200 mg/kg， calculated by pill weight）， Liqi sanjie pill control group （prototype control， 1 957 mg， calculated by crude drug） and Liqi sanjie granule low-dose， medium-dose and high-dose groups （978.5， 1 957， 3 914 mg/kg， calculated by crude drug）， with 10 rats in each group. Each group was given medicine 20 mL/kg intragastrically once a day， for consecutive 21 d. 1 h after per medication， liver-qi stagnation model was established in those groups by binding method except for blank control group. The syrup preference of rats was determined by designing syrup preference test. Rattail suspension test was adopted to determine the hanging immobility time and struggling times of mice. Open-field behavior test was used to determine total behavior score so as to judge the extent of liver-qi stagnation and effect of the drug in rats. RESULTS： Compared with blank control group， hanging immobility time of model control group was significantly prolonged， the syrup preference and the total behavior score of open field test were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Compared with model control group， the struggling times of rats were increased significantly in Xiaojin pill control group， Liqi sanjie pill control group and Liqi sanjie granule medium-dose group （P＜0.05 or P＜0.01）； the hanging immobility time of Xiaoyao pill control group， Xiaojin pill control group， Liqi sanjie pill control group， Liqi sanjie granule low-dose and medium-dose groups were shortened significantly； syrup preference and total behavior score of open-field behavior test were increased significantly （P＜0.05 or P＜0.01）. Compared with Liqi sanjie pill control group， the struggling times of rats were decreased significantly and hanging immobility time were prolonged significantly only in Liqi sanjie granule high-dose group （P＜0.05 or P＜0.01）； there was no statistical significance in above indexes of rats in Liqi sanjie granule low-dose and medium-dose groups （P＞0.05）. CONCLUSIONS： Liqi sanjie granule can significantly improve liver-qi stagnation caused by binding method， and the effects of low-dose and medium-dose Liqi sanjie granule are similar to those of Liqi sanjie pill.

ING1 family is a candidate for tumor suppressor,which has three splicing isoforms named p47ING1a,p33ING1b,and p24ING1c. Study of the effect of different isoforms of ING1 on HeLa cells proliferation and its molecular mechanism would help further identifying the functional relationship of ING1 isoforms,and finding important genes regulated by ING1. Cell growth curve and cell cycle analysis were used to observe the effect of ING1a,ING1b,and ING1c on HeLa cells growth,and the result indicated that they could all inhibit HeLa cells growth by arresting cell cycle at G0/G1 phase. PCR method was used to construct the PHD domain deletions of ING1a and ING1b. ING1a,ING1b,ING1c and the PHD domain deletions 1a?C and 1b?C were then overexpressed in HeLa cells. p16INK4a,PTEN/p27Kip1 and p53/p21Waf1 protein levels were detected by Western blot. The result showed that ING1a,ING1b,ING1c,and 1a?C except for 1b?C induced p16INK4a protein expression,in which ING1c had the most powerful effect. Luciferase assay identified that overexpression of pcDNA3.1(+)-1a?C facilitated p16INK4a transcription through enhancing p16INK4a promoter activity,while pcDNA3.1(+)-1b?C repressed the p16INK4a promoter activity . In a word,it was found for the first time that except for the p53/p21Waf1 pathway,three splicing isoforms of ING1 family could also inhibit HeLa cells proliferation though upregulation of p16INK4a and PTEN,and the PHD domain deletion of ING1a enhanced p16INK4a transcription. These findings provide new clews to further study on the mechanisms of ING1 family suppressing cancer cells growth.

OBJECTIVETo reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.

METHODSUsing a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.

RESULTSBoth 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.

CONCLUSIONSPI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.

Cells, Cultured ; Cellular Senescence ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; drug effects ; physiology ; G1 Phase ; drug effects ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors

Objective To investigate the effects of ?-2-macroglobulin on the aging process of human diploid fibroblasts. Methods pIRES-A2M sense and antisense vectors were constructed and transferred into 2BS cells mediated by lipofectamine.2BS/A2Ms and 2BS/A2Ma cell lines were verified by Southern and Northern blot analysis respectively.Cell growth curve,the population doublings,cell cycle analysis,staining of senescent-associated-?-galactosidase and expression of p16 and p21 in transfected cells were measured. Results Southern and Northern blot analysis verified that the exogenous cDNAs were integrated into genomic DNA in the transfected cells.The ultmost population doublings of 2BS/A2Ms cells were slightly higher than normal 2BS cells.Cell growth curve,cell cycle analysis,staining of senescent-associated-?-galactosidase and the population doublings all revealed that 2BS/A2Ms cells demonstrated obvious difference compared with 2BS/A2Ma cells((P0.05). Conclusions The aging process of 2BS cells is influenced slightly by expression of A2M.

Objective　To investigate the involvement of cell cycle negative regulator p16 in the replicative senescence of normal human diploid fibroblasts. 　Methods　 p16 cDNA and retroviral vector was introduced into normal human fibroblast 2BS cells by transfection technique. Then the effects of p16 on replicative cellular senescence of 2BS cells were examined. 　Results　 Compared with the control cells, 2BS cells transfected with p16 cDNA showed significant suppression of growth rate (decrease 50%) with cell cycle arrested at G1 phase. This phenomenon similar to that of the senescent cells, with the cellular response to stimulation by growth factor decreased 79.4%, showing the characteristics in morphology of senescent fibroblasts. 　Conclusions　 The over expression of p16 gene contributes to the process of cellular senescence of normal human diploid fibroblasts.

Objective　The relationship between DNA methylation and the overexpression of cell cycle negative regulator p16MTS1/INK4a in senescent cells was studied. 　Methods　PCR amplification of p16 exon I following digestion with Sma I , a methylation sensitive DNA endonuclease, was adapted to determine the methylation status at specific site. 　Results　 T-he increased expression of p16 in the aging process of human fetal lung diploid fibroblasts (2BS) was observed. In middle-aged and old cells, the p16 level was about 3 folds and 10 folds respectively as that in young cells. The methylation level of the Sma I site in p16 exon I tended to decline with aging, being about 64% and 41% in young and middle-aged cells respectively, but still maintain relatively as high as about 24% in senescent cells. 　Conclusions　 The overexpression of p16 in senescent human fibroblasts might be related to the alteration of methylation level of exon I, its mechanisms need to be defined further.