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*

Objective:To identify the genetic causes of adverse pregnancy outcomes in five families and provide a basis for rational guidance on genetic and reproductive counseling.Methods:A retrospective analysis was conducted on five families with a history of adverse pregnancy outcomes, where one partner was found to have a cryptic balanced translocation. These cases were identified among pregnant women who underwent invasive prenatal diagnosis at the Department of Medical Genetics and Prenatal Diagnosis, Wuxi Maternity and Child Health Care Hospital, from January 2016 to June 2023. Conventional G-banding karyotyping and chromosomal microarray analysis (CMA) were performed on affected children/fetus samples. Chromosome G-banding and fluorescence in situ hybridization (FISH) were used for parental tracing. Genetic counseling was provided for all cases, and the pregnancy outcomes were followed up. Descriptive analysis was applied in this study. Results:Case 1 involved an eldest son with unexplained intellectual disability, and the CMA results showed an 8.6 Mb deletion in the 4p16.3p16.1 region and a 1.0 Mb duplication in the 7q36.3 region. Unbalanced translocations were detected in the current pregnancies of the other four cases, which were a 6.1 Mb duplication in the 14q32.2q32.33 region with a 1.6 Mb deletion in the 21q22.3 region (Case 2), a 10.8 Mb duplication in the 6q25.3q27 region with a 1.2 Mb deletion in the 15q26.3 region (Case 3), a 1.0 Mb duplication in the 5p15.33 region with a 2.9 Mb deletion in the 6q27 region (Case 4), and a 3.2 Mb deletion in the 1q44 region with a 4.8 Mb duplication in the 19p13.3 region (Case 5). FISH confirmed that either the husband or the wife in each of the five families was a carrier of a cryptic balanced translocation. Further CMA testing on amniotic fluid samples from previous terminated pregnancies of Cases 3 and 4 also indicated unbalanced translocations. Both the current pregnancy of Case 1 and the subsequent pregnancy of Case 2 gave birth to healthy babies after non-abnormal prenatal diagnosis and genetic counseling. Cases 3 to 5 are currently preparing for pregnancy.Conclusion:Advances and combined application of genetic technologies will be conducive to improving the diagnosis of cryptic balanced translocations in some families with adverse pregnancy outcomes, providing a sound basis for genetic counseling in future pregnancy.

Objective To develop a matrix metalloproteinase(MMP)-responsive hyaluronic acid(HA)-based controlled-release material for brain-derived neurotrophic factor(BDNF)to provide a novel therapeutic strategy for intervention and repair of traumatic brain injury(TBI).Methods HA was modified with amination,followed by condensation with Suflo-SMCC carboxyl group to form amide,and then linked with glutathione(GSH)to synthesize HA-GSH.The recombinant glutathione S-transferase(GST)-tissue inhibitor of metalloproteinase(TIMP)-BDNF(GST-TIMP-BDNF)expression plasmid was constructed using molecular cloning technique with double enzyme digestion by Bam H Ⅰ and Eco R Ⅰ.The recombinant GST-TIMP-BDNF protein was expressed in the Escherichia coli prokaryotic expression system,and purified by ion exchange chromatography,confirmed by Western blotting.MMP diluents were supplemented with PBS,MMP inhibitor marimastat,and varing concentrations(0.4,0.6,0.8 mg/ml)of GST-TIMP-BDNF or GST-BDNF.MMP-2 activity was analyzed using an MMP activity detection kit to evaluate the inhibitory effect of the recombinant protein on MMP.Primary rat neurons were extracted and cultured to establish an iron death model induced by RSL3.The effect of recombinant protein GST-TIMP-BDNF on neuronal injury was detected by immunofluorescence staining.Results MRI hydrogen spectrum identification confirmed the successful synthesis of HA-GSH.Western blotting results showed the successful expression of the recombinant protein GST-TIMP-BDNF containing the GST tag using the E.coli prokaryotic expression system.MMP activity detection results indicated that the recombinant protein GST-TIMP-BDNF had a superior inhibitory effect on MMP-2 activity compared to GST-BDNF(P<0.05).Immunofluorescence staining results showed a significant increase in fluorescence intensity in rat neurons treated with GST-TIMP-BDNF after RSL3 induction(P<0.05).Conclusion A MMP-responsive HA-based BDNF controlled-release material has been successfully developed,exhibiting a protective effect on neuron damage.

N-type voltage-gated calcium (Ca²⁺) channels (N-type VGCC, Ca_V2.2) mediate Ca²⁺ influx in response to action potential at the presynaptic terminal, and play an important role in synaptogenesis, neurotransmitter release and nociceptive signal transduction. It is a new target for the development of drugs for the treatment of neuralgia (chronic pain) and other major diseases. Due to the difficulty of calcium channel expression in vitro and the detection of channel current, there is a great lack of new drug screening models. In this study, we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of Ca_V2.2 in vitro (this study were reviewed and approved by the Ethics Committee of Guangxi University, approval number: GXU-2023-0249). Firstly, the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat Ca_V2.2 were used as templates for in vitro transcription to generate their related mRNA (cRNA), after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression. The two-electrode voltage clamp (TEVC) technique was used to detect the inward current produced by Ca_V2.2. At the same time, the expression conditions of Ca_V2.2 were optimized, and its gating function was characterized from the aspects of channel activation and inactivation. The results showed that 3-5 days after cRNA microinjection, stable Ca_V2.2-mediated barium ion (Ba²⁺) currents were successfully detected. The interference of endogenous potassium channels and Ca²⁺-activated chloride channels can be eliminated by tetraethylammonium hydroxide (TEAOH) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM) treatment. The maximum potential for Ca_V2.2 activation is 0 mV, and the current reverses to be outward when the membrane potential is greater than +50 mV. By fitting the steady-state activation and inactivation curves, the half-maximal activation potential and half-maximal inactivation potential of Ca_V2.2 are identified as -15.9 and -60.2 mV. In this study, a stable Ca_V2.2 expression system was established based on Xenopus laevis oocytes. The in vitro expression system can provide a new way for the screening of Ca_V2.2 active compounds or lead drugs.

Conducting local tolerance testing on parentaral drugs is of great significance for evaluating the clinical medication risks of drugs.Although relevant domestic and international guidelines provide detailed instructions on how to conduct local tolerance testing,it was found that some products still provide non-standard application materials,which affects the efficiency of drug development.This article summarizes the information on domestic and international guidance related to the local tolerance testing and elaborates on common problems based on specific application cases,with the aim of of providing reference for related work.

This article summarizes the domestic and international research progress of recombinant human follicle stimulating hormone(rFSH).According to relevant guidelines and application cases,the general requirements and common problems for non-clinical evaluation of rFSH are summarized.The clinical development prospects of long-acting rFSH products which is a hot research topic in recent years are analyzed and corresponding suggestions are given in order to provide reference for related work.

Objective To investigate the attributable risk(AR)of Acinetobacter baumannii(AB)infection in criti-cally ill patients.Methods A multicenter retrospective cohort study was conducted among adult patients in inten-sive care unit(ICU).Patients with AB isolated from sterile body fluid and confirmed with AB infection in each cen-ter were selected as the infected group.According to the matching criteria that patients should be from the same pe-riod,in the same ICU,as well as with similar APACHE Ⅱ score(±5 points)and primary diagnosis,patients who did not infect with AB were selected as the non-infected group in a 1:2 ratio.The AR was calculated.Results The in-hospital mortality of patients with AB infection in sterile body fluid was 33.3％,and that of non-infected group was 23.1％,with no statistically significant difference between the two groups(P=0.069).The AR was 10.2％(95％CI:-2.3％-22.8％).There is no statistically significant difference in mortality between non-infected pa-tients and infected patients from whose blood,cerebrospinal fluid and other specimen sources AB were isolated(P＞0.05).After infected with AB,critically ill patients with the major diagnosis of pulmonary infection had the high-est AR.There was no statistically significant difference in mortality between patients in the infected and non-infec-ted groups(P＞0.05),or between other diagnostic classifications.Conclusion The prognosis of AB infection in critically ill patients is highly overestimated,but active healthcare-associated infection control for AB in the ICU should still be carried out.

Objective:To investigate the association of serum levels of tumor necrosis factor receptor-related factor 6(TRAF6)and histone deacetylase 3(HDAC3)with severity of coronary artery disease and prognosis in patients with acute myocardial infarction(AMI).Methods:A total of 122 AMI patients admitted Macheng Traditional Chinese Medicine Hospital between July 2019 and December 2020 were selected,divided into low score group(n=73,＜20 points)and high score group(n=49,≥20 points)according to Gensini score.Association of serum levels of TRAF6 and HDAC3 with Gensini score was analyzed by Pearson method.According to survival condition during hospitalization within 28d,they were divided into survival group(n=85)and death group(n=37).Cox regression model was used to analyze the influencing factors for death within 28d in AMI patients;receiver operating charac-teristic(ROC)curve was used to analyze the predictive efficacy of TRAF6 and HDAC3 for death within 28d in AMI patients.Results:Patients in high score group had significant higher serum levels of TRAF6[(6.01±2.39)μg/ml vs.(5.06±1.74)μg/ml,P=0.012]and HDAC3[(4.14±1.94)ng/ml vs.(2.87±1.37)ng/ml,P＜0.001]compared with low score group.Pearson correlation analysis indicated that serum levels of TRAF6 and HDAC3 were positively correlated with Gensini score(r=0.879,0.837,P＜0.001 all).Patients in death group had significant higher serum levels of TRAF6[(6.89±1.67)μg/ml vs.(4.81±1.24)μg/ml,P＜0.001]and HDAC3[(5.37±1.77)ng/ml vs.(2.52±0.76)ng/ml,P＜0.001]compared with those in survival group.Cox regression analysis indicated that Gensini score[HR=1.857,95％CI 1.259～2.737,P=0.001],TRAF6[HR=1.659,95％CI 1.083～2.543,P=0.022],HDAC3[HR=1.779,95％CI 1.192～2.653,P=0.004]were independent risk factors for death within 28d in AMI patients.ROC curve analysis indicated that AUC of TRAF6,HDAC3 and their combina-tion predicting death within 28d in AMI patients was 0.862,0.859 and 0.971 respectively,and AUC of combination was significantly higher than those of TRAF6 and HDAC3 alone(Z=2.535,2.032,P=0.011,0.042).Conclu-sion:Serum levels of TRAF6 and HDAC3 are closely related to severity of coronary artery disease and prognosis in AMI patients,and dual combination possesses good predictive value for prognosis in AMI patients.

Objective:To investigate the association of serum levels of tumor necrosis factor receptor-related factor 6(TRAF6)and histone deacetylase 3(HDAC3)with severity of coronary artery disease and prognosis in patients with acute myocardial infarction(AMI).Methods:A total of 122 AMI patients admitted Macheng Traditional Chinese Medicine Hospital between July 2019 and December 2020 were selected,divided into low score group(n=73,＜20 points)and high score group(n=49,≥20 points)according to Gensini score.Association of serum levels of TRAF6 and HDAC3 with Gensini score was analyzed by Pearson method.According to survival condition during hospitalization within 28d,they were divided into survival group(n=85)and death group(n=37).Cox regression model was used to analyze the influencing factors for death within 28d in AMI patients;receiver operating charac-teristic(ROC)curve was used to analyze the predictive efficacy of TRAF6 and HDAC3 for death within 28d in AMI patients.Results:Patients in high score group had significant higher serum levels of TRAF6[(6.01±2.39)μg/ml vs.(5.06±1.74)μg/ml,P=0.012]and HDAC3[(4.14±1.94)ng/ml vs.(2.87±1.37)ng/ml,P＜0.001]compared with low score group.Pearson correlation analysis indicated that serum levels of TRAF6 and HDAC3 were positively correlated with Gensini score(r=0.879,0.837,P＜0.001 all).Patients in death group had significant higher serum levels of TRAF6[(6.89±1.67)μg/ml vs.(4.81±1.24)μg/ml,P＜0.001]and HDAC3[(5.37±1.77)ng/ml vs.(2.52±0.76)ng/ml,P＜0.001]compared with those in survival group.Cox regression analysis indicated that Gensini score[HR=1.857,95％CI 1.259～2.737,P=0.001],TRAF6[HR=1.659,95％CI 1.083～2.543,P=0.022],HDAC3[HR=1.779,95％CI 1.192～2.653,P=0.004]were independent risk factors for death within 28d in AMI patients.ROC curve analysis indicated that AUC of TRAF6,HDAC3 and their combina-tion predicting death within 28d in AMI patients was 0.862,0.859 and 0.971 respectively,and AUC of combination was significantly higher than those of TRAF6 and HDAC3 alone(Z=2.535,2.032,P=0.011,0.042).Conclu-sion:Serum levels of TRAF6 and HDAC3 are closely related to severity of coronary artery disease and prognosis in AMI patients,and dual combination possesses good predictive value for prognosis in AMI patients.

OBJECTIVES: To evaluate the report quality, methodological quality and evidence quality of the systematic reviews and meta-analyses (SRs/MAs) of acupuncture for in vitro fertilization-embryo transfer (IVF-ET). METHODS: The SRs/MAs of acupuncture for IVF-ET were searched electronically from databases of CNKI, Wanfang, VIP, SinoMed, PubMed, Embase, Cochrane Library, from inception of each database to September 27th, 2022. Two reviewers independently screened the literature and extracted the data. Using PRISMA statement, the AMSTAR 2 scale and the GRADE system, the report quality, methodological quality and evidence quality of the included SRs/MAs were assessed. RESULTS: A total of 28 SRs/MAs were included, with PRISMA scores ranging from 8.5 points to 27 points. The problems of report quality focused on protocol and registration, retrieval, risk of bias in studies, additional analysis, limitations and funding. The methodological quality of included studies was generally low, reflecting on items 2, 3, 7, 10, 12 and 16. A total of 85 outcome indexes were included in the GRADE system for evidence grade evaluation. Most of the evidences were low or very low in quality. The reasons for the downgrade were related to study limitations, inconsistency, imprecision and publication bias. CONCLUSIONS Acupuncture therapy improves the outcomes of IVF-ET, but the methodological quality and evidence quality of related SRs/MAs are low. It is recommended to conduct more high-quality studies in the future to provide more reliable evidences.
Acupuncture Therapy/methods* ; Databases, Factual ; Embryo Transfer ; Fertilization in Vitro ; Publication Bias ; Systematic Reviews as Topic