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 compare the safety and efficacy of direct thrombectomy versus bridging thrombectomy in the treatment of acute anterior circulation large vessel occlusion stroke under different collateral circulation statuses.Methods Totally 93 patients with acute anterior circulation ischemic stroke admitted to the First Affiliated Hospital of Xinxiang Medical University from September 2020 to March 2023 were selected as the research subjects.Patients were divided into direct throm-bectomy group(n=47)and bridging thrombectomy group(n=46)based on the type of thrombectomy.Patients in the direct thrombectomy group received direct intravascular thrombectomy,while patients in the bridging thrombectomy group received intravenous thrombolysis with alteplase combined with mechanical thrombectomy.According computed tomography angiography,the collateral circulation Tan classification was applied to divide the patients into good collateral circulation sub-group and poor collateral circulation sub-group.The modified thrombolysis in cerebral infarction grading(mTICI)was used to evaluate vessel recanalization.Head computed tomography plain scan was performed at 24-48 hours postoperatively to assess if there was hemorrhagic transformation,and modified Rankin Scale score was performed at 90 days postoperatively.Information such as imaging examination time,femoral artery puncture time,vessel recanalization time after thrombectomy,prognosis and spontaneous non-traumatic symptomatic intracerebral hemorrhage(SICH)were collected.Results The age,gender,baseline Alberta stroke program early computed tomography score,baseline national institutes of health stroke scale score,proportions of hypertension,diabetes and atrial fibrillation,baseline systolic pressure,creatinine,baseline blood glucose,platelet count,occlusion site,stroke etiologies and collateral circulation status of patients in the two groups were not statistically significantly different(P＞0.05).There were no significant differences in the post-admission imaging examination time,femoral artery puncture time,vessel recanalization time after thrombectomy,successful vascular reperfusion rate,good prognosis rate,mortality rate,and SICH incidence between the two groups(P＞0.05).The hemorrhagic transformation rate of patients in the direct thrombectomy group was significantly lower than that in the bridging thrombectomy group(P＜0.05).There were no significant differences in the post-admission imaging examination time,femoral artery puncture time,vessel recanalization time after thrombectomy,successful vascular reperfusion rate,good prognosis rate,mortality rate,and SICH incidence between patients with good collateral circulation and patients with poor collateral circulation in the two groups(P＞0.05).The hemorrhagic transformation rate of patients with good and poor collateral circulation in the direct thrombectomy group was significantly lower than that in the bridging thrombectomy group(P＜0.05).Conclusion Under different collateral circulation conditions,the safety and efficacy of direct thrombectomy and bridging thrombectomy in the treatment of acute anterior circulation large vessel occlusion stroke are similar,but bridging thrombectomy is more likely to result in cerebral hemorrhage transformation compared with direct thrombectomy.

ObjectiveTo investigate the effects of maltol aluminum exposure on miR-193a-3p, demethylase AlkB homolog 5 (ALKBH5), phosphatase and tensin homolog deleted on chromosome ten (PTEN) and protein kinase B (AKT), and whether miR-193a-3p is involved in aluminum-induced cognitive impairment by regulating ALKBH5/PTEN/AKT signaling pathway. Methods Specific pathogen-free male SD rats were randomly divided into control group and low-, medium- and high- dose groups according to their body weight, with eight rats in each group. Rats in the low-, medium-, and high- dose groups were intraperitoneally injected with maltol aluminum solution at concentrations of 10.00, 20.00, and 40.00 μmol/kg body weight, respectively, while the rats in control group were given an equal volume of 0.9% sodium chloride solution. Rats were injected for five days every week for three months. After injection, the novel object recognized test was used to assess the learning and memory ability of the rats. The relative expression of miR-193a-3p and B-cell lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine aspartate protease-3 (Caspase-3) mRNA in rat hippocampus was detected using the real-time quantitative polymerase chain reaction. The relative protein expression of ALKBH5, PTEN, and AKT2 in the rat hippocampus was detected using Western blot. Results The discrimination index and the preference index of the new object recognition test of the rats in high-dose group were lower than those in control group and low-dose group (all P<0.05). The relative expression of miR-193a-3p and Bcl-2 mRNA in the hippocampus of the rats in high-dose group was lower than those in control group and low-dose group (all P<0.05). The relative mRNA expression of Bax in the high-dose group was higher than those in the control group and low-dose group (both P<0.05). The relative mRNA expression of Caspase-3 of the rats in the high-dose group was higher than that in the other three groups (both P<0.05). The relative protein expression of ALKBH5 in the hippocampus of the rats in the high-dose group was lower than that in the control group (P<0.05). The relative expression of PTEN protein was higher than those in the control group and low-dose group (both P<0.05). The relative protein expression of AKT2 was lower than those in the control group and low-dose group (both P<0.05). Conclusion Sub-chronic aluminum exposure can inhibit the expression of miR-193a-3p in the hippocampus of rats, which may disrupt the ALKBH5/PTEN/AKT pathway and affect normal neuronal homeostasis and cellular function. This pathway may play an important role in aluminum-induced cognitive impairment.

Objective To evaluate the bioequivalence and safety of two voriconazole for injection in healthy Chinese subjects,and to explore the safety of the excipient sulfobutyl-β-cyclodextrin.Methods A single-center,single-dose,randomized,open-label,two-preparation,two-period,double-crossover trial design.A total of 18 healthy subjects were enrolled and administrated with a single intravenous infusion of voriconazole test drug and reference drug at 4 mg·kg-1 under fasting conditions,with a sequence determined by randomization.The concentrations of voriconazole in plasma and sulfobutyl-β-cyclodextrin in urine were determined by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Phoenix WinNonlin 8.2 software was used to calculate pharmacokinetic parameters of voriconazole in plasma,and SAS(version 9.4)software was used to calculate pharmacokinetic parameters of sulfobutyl-β-cyclodextrin in urine and bioequivalence analysis.Results Major pharmacokinetic parameters of voriconazole in plasma after a single intravenous infusion of voriconazole test drug and reference drug in 18 healthy subjects in a fasted state were as follows:Cmax were(2 177.00±399.10)and(2 265.00±378.70)ng·mL-1;AUC0-t were(14 612.07±8 182.95)and(15 144.69±7 814.02)ng·h·mL-1;AUC0-∞ were(16 217.48±10 862.78)and(16 863.18±10695.75)ng·h·mL-1;tmax were 2.00 and 1.98 h,respectively.The 90％confidence intervals of the geometric mean ratios of Cmax,AUC0-t and AUC0-∞ for the two drugs were within the equivalence range of 80.00％to 125.00％.Conclusion The two voriconazole for injection preparations were bioequivalent and safe when administered by intravenous infusion under fasting conditions in healthy Chinese subjects.

Objective To evaluate the pharmacokinetics(PK)of tenofovir alafenamide Fumarate tablets(25 mg)in healthy Chinese subjects after single oral administration to provide a basis for bioequivalence evaluation.Methods Using a single-dose,randomized,open-lable,two-period,two-way crossover design under fasting condition,while three-way crossover design under fed condition,42 healthy subjects respectively for fasting and fed study were enrolled,and randomized into two groups to receive a single dose of test product(T)or reference product(R)25 mg.Plasma concentration of tenofovir alafenamide and tenofovir were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS)method.The pharmacokinetic parameters were calculated by WinNonlin software(8.1 version)using non-compartmental model,and bioequivalence evaluation was performed for the two preparations.Relevant safety evaluations were performed during the trial.Results The test product and the reference product under fasting study,the main PK parameters of tenofovir alafenamide were as follows:Cmax were(215.17±94.24)and(199.30±71.11)ng·mL-1;AUC0-t were(135.44±71.60)and(123.91±53.82)h·ng·mL-1;the main PK parameters of tenofovir were as follows:Cmax were(7.30±2.27)and(7.12±1.74)ng·mL-1,AUC0-t of tenofovir were(237.16±47.09)and(230.06±43.41)h·ng·mL-1,respectively.The test product and the reference product under fed study,the main PK parameters of tenofovir were as follows:Cmax were(197.69±82.19)and(197.10±110.54)ng·mL-1;AUC0-t were(197.69±82.19)and(197.10±110.54)h·ng·mL-1;the main PK parameters of tenofovir were as follows:CMax were(2.57±1.37)and(2.58±1.31)ng·mL-1;AUC0-t were(227.08±74.33)and(238.51±128.30)h·ng·mL-1,respectively.The 90％confidence interval for geometric mean ratio of Cmax,AUC0-tof T and R under fed condition were between 80.00％-125.00％,respectively.The incidence of adverse events in fasting and fed tests was 21.43％and 30.95％,respectively,and no serious adverse event was reported.Conclusion The test formulation and reference formulation of tenofovir alafenamide fumarate tablets were equivalent and was safe.

Objective To investigate the risk factors for bronchopulmonary dysplasia(BPD)in twin preterm infants with a gestational age of<34 weeks,and to provide a basis for early identification of BPD in twin preterm infants in clinical practice.Methods A retrospective analysis was performed for the twin preterm infants with a gestational age of<34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020.According to their conditions,they were divided into group A(both twins had BPD),group B(only one twin had BPD),and group C(neither twin had BPD).The risk factors for BPD in twin preterm infants were analyzed.Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins.Results A total of 904 pairs of twins with a gestational age of<34 weeks were included in this study.The multivariate logistic regression analysis showed that compared with group C,birth weight discordance of>25%between the twins was an independent risk factor for BPD in one of the twins(OR=3.370,95%CI:1.500-7.568,P<0.05),and high gestational age at birth was a protective factor against BPD(P<0.05).The conditional logistic regression analysis of group B showed that small-for-gestational-age(SGA)birth was an independent risk factor for BPD in individual twins(OR=5.017,95%CI:1.040-24.190,P<0.05).Conclusions The development of BPD in twin preterm infants is associated with gestational age,birth weight discordance between the twins,and SGA birth.

Aim To explore the ameliorative effects of berberine(BBR)on diabetic nephropathy(DN)in mice and investigate its potential mechanisms through transcriptomic analysis.Methods 8-week-old db/db mice were randomly assigned into four groups:model group(DN group),BBR 50 mg·kg-1 group(BBR-L group),BBR 100 mg·kg-1 group(BBR-H group),and empagliflozin 10 mg·kg-1 group(EMPA group).Age-matched db/m mice were used as the control group(NC group),with eight mice in each group.Each group received intragastric administration once daily for eight weeks.After the treatment,serum,u-rine,and kidney samples were collected to evaluate re-nal function indicators and observe renal pathological changes.Differentially expressed genes(DEGs)in kidney tissue were identified through transcriptomic a-nalysis,followed by KEGG and GO enrichment analy-sis.Potential targets were further validated using mo-lecular docking,molecular dynamics simulations,West-ern blot,and immunohistochemistry.Results Both BBR and EMPA significantly reduced fasting blood glu-cose levels in DN mice,improved renal function,and alleviated renal injury and fibrosis.Compared to the NC group,855 DEGs were identified in the DN group,while 194 DEGs were identified in the BBR-H group compared to the DN group.KEGG enrichment analysis indicated that the mechanisms underlying BBR's effects on DN were primarily related to type 1 diabetes and bile secretion pathways.Molecular docking results demonstrated a strong binding affinity between BBR and FXR and a moderate binding affinity with SHP.Molecular dynamics simulations corroborated the doc-king results.FXR and SHP protein expression signifi-cantly decreased in the DN group compared to the NC group.At the same time,BBR treatment significantly increased the expression of these proteins compared to the DN group.Conclusion BBR may mitigate DN-in-duced renal injury by modulating bile acid and lipid homeostasis through the FXR-SHP pathway.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.