This study aims to investigate the in vitro mechanisms underlying the beneficial effects of puerarin on hepatic insulin resistance(IR) based on the carbohydrate response element-binding protein(ChREBP)/peroxisome proliferator-activated receptor(PPAR)α/PPARγ axis involved in glucose and lipid metabolism. An IR-HepG2 cell model was established by treating cells with dexamethasone for 48 h, and the cells were then treated with 10, 20, and 40 μmol·L~(-1) puerarin for 24 h. Glucose levels and output in the extracellular fluid were measured by the glucose oxidase method, while cell viability was assessed by the cell counting kit-8(CCK-8) assay. The adenosine triphosphate(ATP) content and glycogen synthesis were evaluated through chemiluminescence and periodic acid-Schiff staining, respectively. Western blot was employed to quantify the protein levels of forkhead box protein O1(FoxO1), phosphorylated forkhead box protein O1 [p-FoxO1(Ser256)], glucagon, phosphofructokinase, liver type(PFKL), pyruvate kinase L-R(PKLR), pyruvate dehydrogenase complex 1(PDHA1), insulin receptor substrate 2(IRS2), phosphatidylinositol 3-kinase p85(PI3KR1), phosphorylated protein kinase B [p-Akt(Thr308)], glycogen synthase(GYS), glycogen phosphorylase, liver type(PYGL), adiponectin(ADPN), ChREBP, PPARα, and PPARγ. Additionally, the protein levels of acetyl-CoA carboxylase 1(ACC1), phosphorylated ATP citrate lyase [p-ACLY(Ser455)], sterol regulatory element binding protein 1c(SREBP-1c), peroxisome proliferator-activated receptor gamma coactivator 1α(PGC1α), carnitine palmitoyltransferase 1α(CPT1α), and glucagon receptor(GCGR) were also determined. Immunofluorescence was employed to visualize the expression and nuclear location of ChREBP/PPARα/PPARγ. Furthermore, quantitative PCR with the antagonists GW6471 and GW9662 was employed to assess Pparα, Pparγ, and Chrebp. The findings indicated that puerarin effectively reduced both the glucose level and glucose output in the extracellular fluid of IR-HepG2 cells without obvious effect on the cell viability, and it increased intracellular glycogen and ATP levels. Puerarin down-regulated the protein levels of FoxO1 and glucagon while up-regulating the protein levels of p-FoxO1(Ser256), PFKL, PKLR, PDHA1, IRS2, PI3KR1, p-Akt(Thr308), GYS, PYGL, ADPN, ACC1, SREBP-1c, p-ACLY(Ser455), PGC1α, CPT1α, and GCGR in IR-HepG2 cells. Furthermore, puerarin up-regulated both the mRNA and protein levels of ChREBP, PPARα, and PPARγ and promoted the translocation into the nucleus. GW6471 was observed to down-regulate the expression of Pparα while up-regulating the expression of Chrebp and Pparγ. GW9662 down-regulated the expression of Pparγ while up-regulating the expression of Pparα, with no significant effect on Chrebp. In summary, puerarin activated the hepatic ChREBP/PPARα/PPARγ axis, thereby coordinating the glucose and lipid metabolism, promoting the conversion of glucose to lipids to exert the blood glucose-lowering effect.
Isoflavones/pharmacology* ; Humans ; PPAR gamma/genetics* ; Hep G2 Cells ; Glucose/metabolism* ; Lipid Metabolism/drug effects* ; PPAR alpha/genetics* ; Liver/drug effects* ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics* ; Insulin Resistance

This paper aims to investigate the hypoglycemic effect and mechanism of berberine in improving energy metabolism based on the multi-pathway regulation of brain and muscle aromatic hydrocarbon receptor nuclear translocal protein 1(BMAL1): cyclin kaput complex of day-night spontaneous output cyclin kaput(CLOCK). The dexamethasone-induced hepatic insulin resistance(IR) HepG2 cell model was used; 0.5, 1, 5, 10, 20 μmol·L~(-1) berberine were administered at 15, 18, 21, 24, 30, 36 h. The time-dose effect of glucose content in extracellular fluid was detected by glucose oxidase method. The optimal dosage and time of berberine were determined for the follow-up study. Glucose oxidase method and chemiluminescence method were respectively performed to detect hepatic glucose output and relative content of ATP in cells; Ca~(2+), reactive oxygen species(ROS), mitochondrial structure and membrane potential were detected by fluorescent probes. Moreover, ultraviolet colorimetry method was used to detect the liver type of pyruvate kinase(L-PK) and phosphoenol pyruvate carboxykinase(PEPCK). In addition, pyruvate dehydrogenase E1 subunit α1(PDHA1), phosphate fructocrine-liver type(PFKL), forkhead box protein O1(FoxO1), peroxisome proliferator-activated receptor gamma co-activator 1α(PGC1α), glucose-6-phosphatase(G6Pase), glucagon, phosphorylated nuclear factor-red blood cell 2-related factor 2(p-Nrf2)(Ser40), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), fibroblast growth factor 21(FGF21), uncoupled protein(UCP) 1 and UCP2 were detected by Western blot. BMAL1:CLOCK complex was detected by immunofluorescence double-staining method, combined with small molecule inhibitor CLK8. Western blot was used to detect PDHA1, PFKL, FoxO1, PGC1α, G6Pase, glucagon, Nrf2, HO-1, NQO1, FGF21, UCP1 and UCP2 in the CLK8 group. The results showed that berberine downregulated the glucose content in extracellular fluid in IR-HepG2 cells in a time-and dose-dependent manner. Moreover, berberine inhibited hepatic glucose output and reduced intracellular Ca~(2+) and ROS whereas elevated JC-1 membrane potential and improved mitochondrial structure to enhance ATP production. In addition, berberine upregulated the rate-limiting enzymes such as PFKL, L-PK and PDHA1 to promote glycolysis and aerobic oxidation but also downregulated PGC1α, FoxO1, G6Pase, PEPCK and glucagon to inhibit hepatic gluconeogenesis. Berberine not only upregulated p-Nrf2(Ser40), HO-1 and NQO1 to enhance antioxidant capacity but also upregulated FGF21, UCP1 and UCP2 to promote energy metabolism. Moreover, berberine increased BMAL1, CLOCK and nuclear BMAL1:CLOCK complex whereas CLK8 reduced the nuclear BMAL1:CLOCK complex. Finally, CLK8 decreased PDHA1, PFKL, Nrf2, HO-1, NQO1, FGF21, UCP1, UCP2 and increased FoxO1, PGC1α, G6Pase and glucagon compared with the 20 μmol·L~(-1) berberine group. BMAL1:CLOCK complex inhibited gluconeogenesis, promoted glycolysis and glucose aerobic oxidation pathways, improved the reduction status within mitochondria, protected mitochondrial structure and function, increased ATP energy storage and promoted energy consumption in IR-HepG2 cells. These results suggested that berberine mediated BMAL1:CLOCK complex to coordinate the regulation of hepatic IR cells to improve energy metabolism in vitro.
Humans ; Berberine/pharmacology* ; Gluconeogenesis/drug effects* ; Hep G2 Cells ; Glucose/metabolism* ; Liver/drug effects* ; Energy Metabolism/drug effects* ; Hypoglycemic Agents/pharmacology* ; ARNTL Transcription Factors/genetics* ; Glycolysis/drug effects* ; Oxidation-Reduction/drug effects*

Objective To explore the effects of dihydromyricetin on the proliferation,apoptosis,and invasion of endometrial cancer(EC)cells and its possible mechanisms.Methods Ishikawa cells in the logarithmic growth phase were taken and divided into the control group,20 μmol/L dihydromyricetin intervention group,40 μmol/L dihydromyricetin intervention group,and 80 μmol/L dihydromyricetin intervention group,which were treated with different final concentrations of dihydromyricetin(0 μmol/L,20 μmol/L,40 μmol/L,80 μmol/L).Then,CCK-8 assay and flow cytometry were used to detect the effects of dihydromyricetin on the cell proliferation and apoptosis.Transwell experiment was used to detect the effect of dihydromyricetin on the cell invasion.qRT-PCR and Western blot were used to detect the effects of dihydromyricetin on the cell expression of miR-21 and PTEN.Results Compared with the control group,the cell proliferation inhibition rate and apoptosis rate in the dihydromyricetin intervention group were significantly increased(P<0.05),and gradually increased with the increase of dihydromyricetin concentration(P<0.05).Compared with the control group,the numbers of migration and invasion cells in the dihydromyricetin intervention group were significantly decreased(P<0.05),and gradually decreased with the increase of dihydromyricetin concentration(P<0.05).Compared with the control group,the cell expression of miR-21 in the dihydromyricetin intervention group was significantly decreased(P<0.05),and gradually decreased with the increase of dihydromyricetin concentration(P<0.05),the expression levels of PTEN mRNA and protein were significantly increased(P<0.05),and gradually increased with the increase of dihydromyricetin concentration(P<0.05).Conclusion Dihydromyricetin can inhibit the growth and metastasis of EC cells,and the inhibitory effect is positively correlated with its concentration.The mechanism may be related to the effect of dihydromyricetin on the miR-21/PTEN signaling pathway of EC cells.

This study aims to investigate the mechanism of berberine in regulating the metabolism network via clock-controlled genes represented by brain and muscle arnt-like 1(BMAL1) to ameliorate insulin resistance(IR) of hepatocytes in vitro. The HepG2 cell model of dexamethasone-induced IR(IR-HepG2) was established and treated with 5, 10, and 20 μmol·L~(-1) berberine, respectively, for 24 h. The glucose oxidase method and cell counting kit-8(CCK-8) assay were employed to measure extracellular glucose concentration and cell viability, respectively. Periodic acid-Schiff(PAS) staining and lipid fluorescence method were used to detect glycogen and lipids. The immunofluorescence(IF) assay was employed to detect the nuclear localization of BMAL1 and circadian locomotor output cycles kaput(CLOCK) in IR-HepG2 cells. Western blot was employed to determine the protein levels of BMAL1, CLOCK, period circadian clock 2(PER2), cryptochrome circadian regulator 1(CRY1), Rev-Erbα, carbohydrate response element-binding protein(ChREBP), peroxisome proliferator-activated receptors alpha and gamma(PPARα/γ), sterol regulatory element-binding protein 1C(SREBP-1C), mammalian target of rapamycin(mTOR), protein kinase B(Akt), glycogen synthase kinase-3β(GSK3β), acetyl coenzyme A carboxylase 1(ACC1), fatty acid synthase(FASN), carnitine palmitoyltransferase 1α(CPT1α), nicotinamide phosphoribosyltransferase(NAMPT), silent information regulator 1(SIRT1), adiponectin(ADPN), insulin receptor substrate 2(IRS2), and phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85). In addition, the levels of phosphorylated adenosine monophosphate-activated protein kinase alpha(AMPKα), Akt, GSK3β, BMAL1, and mTOR were determined. Furthermore, 20 μmol·L~(-1) CLK8 was added to measure the glucose consumption as well as the protein levels of ChREBP, PPARα, and mTOR in IR-HepG2 cells. The results showed that berberine increased the glucose consumption, lowered the lipid levels, increased the expression and nuclear localization of BMAL1 and CLOCK, and up-regulated the level of BMAL1 in IR-HepG2 cells. Furthermore, berberine up-regulated the levels of ADPN, IRS2, PI3Kp85, p-Akt(Ser473)/Akt, p-mTOR(Ser2448)/mTOR, PPARα, and CPT1α, and down-regulated the levels of p-GSK3β(Ser9)/GSK3β, ChREBP, SREBP-1C, ACC1, and FASN. The addition of CLK8 reduced glucose consumption in IR-HepG2 cells, up-regulated the ChREBP level, and down-regulated PPARα and mTOR levels by inhibiting the BMAL1 and CLOCK interaction. In summary, berberine regulated glucose and lipid metabolism via clock-controlled genes with BMAL1 at the core to ameliorate IR of hepatocytes.
Humans ; Hepatocytes/drug effects* ; Lipid Metabolism/drug effects* ; Glucose/metabolism* ; Berberine/pharmacology* ; Insulin Resistance ; Hep G2 Cells ; CLOCK Proteins/genetics* ; ARNTL Transcription Factors/genetics*

Abstract Allergic diseases can occur in all systems of the body, covering the whole life cycle, from children to adults and to old age, can be lifelong onset and even fatal in severe cases. Children account for the largest proportion of the victims of allergic disease, Children s allergies start from scratch, ranging from mild to severe, from less to more, from single to multiple systems and systemic performance, so the prevention and treatment of allergic diseases in children is of great importance, which can not only prevent high risk allergic conditions from developing into allergic diseases, but also further block the process of allergy. At present, there is no consensus on the management system of allergic children in kindergartens and primary schools. The "Consensus on Allergy Management and Prevention in Kindergartens and Primary Schools", which includes the organizational structure, system construction and management of allergic children, provides evidence informed recommendations for the long term comprehensive management of allergic children in kindergartens and primary schools, and provides a basis for the establishment of the prevention system for allergic children.

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.
Rats ; Animals ; Proto-Oncogene Proteins c-akt ; Endoplasmic Reticulum Chaperone BiP ; Caspase 3 ; Caspase 9 ; Diabetes Mellitus, Experimental ; Caspase 12 ; Calcium/pharmacology* ; Molecular Docking Simulation ; Endoplasmic Reticulum Stress ; Protein Serine-Threonine Kinases/genetics* ; Liver ; Apoptosis ; Insulin ; Glucose ; Glycogen/pharmacology* ; RNA, Messenger

Human neutrophil elastase (hNE) is a serine proteolytic enzyme mainly distributed in neutrophils. When the balance between anti-hNE protein and hNE is broken, excessive release of hNE can cause the occurrence of various diseases. Therefore, inhibition of hNE is a promising therapeutic strategy. In this paper, the structure, action mechanism, physiological function of hNE and the development of hNE inhibitors were briefly summarized, in order to provide information for the related research.

Objective: This cross-sectional investigation aimed to determine the incidence, clinical characteristics, prognosis, and related risk factors of olfactory and gustatory dysfunctions related to infection with the SARS-CoV-2 Omicron strain in mainland China. Methods: Data of patients with SARS-CoV-2 from December 28, 2022, to February 21, 2023, were collected through online and offline questionnaires from 45 tertiary hospitals and one center for disease control and prevention in mainland China. The questionnaire included demographic information, previous health history, smoking and alcohol drinking, SARS-CoV-2 vaccination, olfactory and gustatory function before and after infection, other symptoms after infection, as well as the duration and improvement of olfactory and gustatory dysfunction. The self-reported olfactory and gustatory functions of patients were evaluated using the Olfactory VAS scale and Gustatory VAS scale. Results: A total of 35 566 valid questionnaires were obtained, revealing a high incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain (67.75%). Females(χ2=367.013, P<0.001) and young people(χ2=120.210, P<0.001) were more likely to develop these dysfunctions. Gender(OR=1.564, 95%CI: 1.487-1.645), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), oral health status (OR=0.881, 95%CI: 0.839-0.926), smoking history (OR=1.152, 95%CI=1.080-1.229), and drinking history (OR=0.854, 95%CI: 0.785-0.928) were correlated with the occurrence of olfactory and taste dysfunctions related to SARS-CoV-2(above P<0.001). 44.62% (4 391/9 840) of the patients who had not recovered their sense of smell and taste also suffered from nasal congestion, runny nose, and 32.62% (3 210/9 840) suffered from dry mouth and sore throat. The improvement of olfactory and taste functions was correlated with the persistence of accompanying symptoms(χ2=10.873, P=0.001). The average score of olfactory and taste VAS scale was 8.41 and 8.51 respectively before SARS-CoV-2 infection, but decreased to3.69 and 4.29 respectively after SARS-CoV-2 infection, and recovered to 5.83and 6.55 respectively at the time of the survey. The median duration of olfactory and gustatory dysfunctions was 15 days and 12 days, respectively, with 0.5% (121/24 096) of patients experiencing these dysfunctions for more than 28 days. The overall self-reported improvement rate of smell and taste dysfunctions was 59.16% (14 256/24 096). Gender(OR=0.893, 95%CI: 0.839-0.951), SARS-CoV-2 vaccination status (OR=1.334, 95%CI: 1.164-1.530), history of head and facial trauma(OR=1.180, 95%CI: 1.036-1.344, P=0.013), nose (OR=1.104, 95%CI: 1.042-1.171, P=0.001) and oral (OR=1.162, 95%CI: 1.096-1.233) health status, smoking history(OR=0.765, 95%CI: 0.709-0.825), and the persistence of accompanying symptoms (OR=0.359, 95%CI: 0.332-0.388) were correlated with the recovery of olfactory and taste dysfunctions related to SARS-CoV-2 (above P<0.001 except for the indicated values). Conclusion: The incidence of olfactory and taste dysfunctions related to infection with the SARS-CoV-2 Omicron strain is high in mainland China, with females and young people more likely to develop these dysfunctions. Active and effective intervention measures may be required for cases that persist for a long time. The recovery of olfactory and taste functions is influenced by several factors, including gender, SARS-CoV-2 vaccination status, history of head and facial trauma, nasal and oral health status, smoking history, and persistence of accompanying symptoms.
Female ; Humans ; Adolescent ; SARS-CoV-2 ; Smell ; COVID-19/complications* ; Cross-Sectional Studies ; COVID-19 Vaccines ; Incidence ; Olfaction Disorders/etiology* ; Taste Disorders/etiology* ; Prognosis

OBJECTIVES: To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (V_acetonitrile∶V_methanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring. RESULTS: The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively. CONCLUSIONS This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.
Chromatography, Liquid/methods* ; Tandem Mass Spectrometry ; Methanol ; Carbamazepine/analysis* ; Benzodiazepines/analysis* ; Solvents ; Chromatography, High Pressure Liquid ; Solid Phase Extraction

Objective To design a remote psychological service platform to solve the problems of elementary troops in lack of psychologists,psychological intervention means and effects.Methods A remote psychological service platform was developed with B/S architecture,ASP.NET and ADO.NET technologies,Visual Studio.NET 2008 development platform and C# language,which was composed of four functional modules of psychological classroom,video teaching and psychological counseling,psy-chological assessment and file management,and system management.Results The platform developed provided elementary troops with remote psychological service for psychological assessment,psychological counseling,psychological guidance,mental health education,professional training for health personnel and emergency intervention.Conclusion The platform developed gains advantages in deployment,professional service,timely response and confidentiality,and facilitates the same psychological service effect as face-to-face communication.[Chinese Medical Equipment Journal,2023,44(9):49-52]