ObjectiveTo investigate the effect of Qingrun prescription（QRP）-containing serum on improving insulin resistance in HepG2 cells and its potential mechanisms. MethodsAn insulin resistance model was established in HepG2 cells with 1×10^-6 mol·L^-1 insulin. Branched-chain α-keto acid dehydrogenase （BCKDH） gene silencing was achieved using siRNA， and the cells were divided into 8 groups： normal group， model group （1×10^-6 mol·L^-1 insulin）， metformin group （1 mmol·L^-1 metformin）， high-， medium-， and low-dose QRP groups （20%， 10%， and 5% QRP-containing serum， respectively）， QRP + siRNA-silenced BCKDH （si-BCKDH） group （10% QRP-containing serum + si-BCKDH）， and QRP + si-NC group （10% QRP-containing serum + si-NC）. Glucose levels in the supernatant were measured with a glucose assay kit， while glycogen content was assessed using a glycogen assay kit. Levels of branched-chain amino acids （BCAAs） and branched-chain keto acids （BCKAs） were determined using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. mRNA transcription and protein expression levels of BCKDH， dishevelled， Egl-10， and pleckstrin （DEP） domain-containing mammalian target of rapamycin （mTOR）-interacting protein （DEPTOR）， mTOR， and ribosomal protein S6 kinase 1 （S6K1） were detected using real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultsCompared to the normal group， the model group exhibited significantly decreased glucose consumption and glycogen content， increased levels of BCAAs and BCKAs， downregulated expression of BCKDH and DEPTOR， and upregulated mTOR and S6K1 expression （P<0.01）. In comparison to the model group， QRP treatment at all doses significantly enhanced glucose consumption and glycogen content while reducing BCAAs and BCKAs levels （P<0.01）. The high- and medium-dose QRP groups demonstrated significant upregulation of BCKDH mRNA transcription and protein expression， as well as DEPTOR mRNA transcription. Moreover， the DEPTOR protein expression level was significantly increased in high-， medium-， and low-dose QRP groups， while mTOR and S6K1 mRNA and protein expression levels were markedly downregulated （P<0.05， P<0.01）. Compared to the QRP + si-NC group， the QRP + si-BCKDH group exhibited increased BCAAs and BCKAs levels， significantly decreased BCKDH mRNA transcription and protein expression， downregulated DEPTOR mRNA and protein expression， and upregulated mTOR and S6K1 mRNA and protein expression （P<0.05， P<0.01）. ConclusionQRP may improve insulin resistance by reprogramming BCAAs metabolism. This effect involves upregulating BCKDH， reducing BCAAs and BCKAs levels， and suppressing the mTOR pathway activation.

This study aims to investigate the mechanism of Qingrun Decoction in alleviating hepatic insulin resistance in type 2 diabetes mellitus(T2DM) rats through the reprogramming of amino acid metabolism. A T2DM rat model was established by inducing insulin resistance through a high-fat diet combined with intraperitoneal injection of streptozotocin. The model rats were randomly divided into five groups: model group, high-, medium-, and low-dose Qingrun Decoction groups, and metformin group. A normal control group was also established. The rats in the normal and model groups received 10 mL·kg~(-1) distilled water daily by gavage. The metformin group received 150 mg·kg~(-1) metformin suspension by gavage, and the Qingrun Decoction groups received 11.2, 5.6, and 2.8 g·kg~(-1) Qingrun Decoction by gavage for 8 weeks. Blood lipid levels were measured in different groups of rats. Pathological damage in rat liver tissue was assessed by hematoxylin-eosin(HE) staining and oil red O staining. Transcriptome sequencing and untargeted metabolomics were performed on rat liver and serum samples, integrated with bioinformatics analyses. Key metabolites(branched-chain amino acids, BCAAs), amino acid transporters, amino acid metabolites, critical enzymes for amino acid metabolism, resistin, adiponectin(ADPN), and mammalian target of rapamycin(mTOR) pathway-related molecules were quantified using quantitative real-time polymerase chain reaction(qRT-PCR), Western blot, and enzyme-linked immunosorbent assay(ELISA). The results showed that compared with the normal group, the model group had significantly increased serum levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), and resistin and significantly decreased ADPN levels. Hepatocytes in the model group exhibited loose arrangement, significant lipid accumulation, fatty degeneration, and pronounced inflammatory cell infiltration. In liver tissue, the mRNA transcriptional levels of solute carrier family 7 member 2(Slc7a2), solute carrier family 38 member 2(Slc38a2), solute carrier family 38 member 4(Slc38a4), and arginase(ARG) were significantly downregulated, while the mRNA transcriptional levels of solute carrier family 1 member 4(Slc1a4), solute carrier family 16 member 1(Slc16a1), and methionine adenosyltransferase(MAT) were upregulated. Furthermore, the mRNA transcription and protein expression levels of branched-chain α-keto acid dehydrogenase E1α(BCKDHA) and DEP domain-containing mTOR-interacting protein(DEPTOR) were downregulated, while mRNA transcription and protein expression levels of mTOR, as well as ribosomal protein S6 kinase 1(S6K1), were upregulated. The levels of BCAAs and S-adenosyl-L-methionine(SAM) were elevated. The serum level of 6-hydroxymelatonin was significantly reduced, while imidazole-4-one-5-propionic acid and N-(5-phospho-D-ribosyl)anthranilic acid levels were significantly increased. Compared with the model group, Qingrun Decoction significantly reduced blood lipid and resistin levels while increasing ADPN levels. Hepatocytes had improved morphology with reduced inflammatory cells, and fatty degeneration and lipid deposition were alleviated. Differentially expressed genes and differential metabolites were mainly enriched in amino acid metabolic pathways. The expression levels of Slc7a2, Slc38a2, Slc38a4, and ARG in the liver tissue were significantly upregulated, while Slc1a4, Slc16a1, and MAT expression levels were significantly downregulated. BCKDHA and DEPTOR expression levels were upregulated, while mTOR and S6K1 expression levels were downregulated. Additionally, the levels of BCAAs and SAM were significantly decreased. The serum level of 6-hydroxymelatonin was increased, while those of imidazole-4-one-5-propionic acid and N-(5-phospho-D-ribosyl)anthranilic acid were decreased. In summary, Qingrun Decoction may improve amino acid metabolism reprogramming, inhibit mTOR pathway activation, alleviate insulin resistance in the liver, and mitigate pathological damage of liver tissue in T2DM rats by downregulating hepatic BCAAs and SAM and regulating key enzymes involved in amino acid metabolism, such as BCKDHA, ARG, and MAT, as well as amino acid metabolites and transporters.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Insulin Resistance ; Diabetes Mellitus, Type 2/genetics* ; Male ; Liver/drug effects* ; Amino Acids/metabolism* ; Rats, Sprague-Dawley ; Humans ; Metabolic Reprogramming

The accumulation of deoxyribonucleic acid (DNA) oxidative damage mediated by reactive oxygen species (ROS) is closely associated with liver diseases. 8-Oxoguanine (8-OxoG), a prevalent DNA oxidation product, plays a significant role in liver disease progression. The base excision repair (BER) pathway, comprising over 30 proteins including 8-OxoG DNA glycosylase1 (OGG1), MutY homolog (MUTYH), and MutT homolog protein 1 (MTH1), is responsible for the clearance and mismatch repair of 8-OxoG. Abnormally high levels of 8-OxoG and dysregulated expression and function of 8-OxoG repair enzymes contribute to the onset and development of liver diseases. Consequently, targeting the 8-OxoG production and repair system with agonists or inhibitors may offer a promising approach to liver disease treatment. This review summarizes the impact of 8-OxoG accumulation and dysregulated repair enzymes on various liver diseases, including viral liver disease, alcoholic liver disease (ALD), metabolic dysfunction-associated steatotic liver disease (MASLD), cholestatic liver disease (CLD), liver fibrosis, cirrhosis, and liver cancer. Additionally, we review natural constituents as potential therapeutic agents that regulate 8-OxoG production, repair enzymes, and repair system-related signal pathways in oxidative damage-induced liver diseases.
Humans ; Liver Diseases/genetics* ; Biological Products/pharmacology* ; DNA Repair/drug effects* ; Guanine/metabolism* ; Animals ; Disease Progression ; DNA Damage ; Oxidative Stress

This study was aimed at establishing a sensitive and specific sandwich ELISA detection method for Brucella.We screened monoclonal capture antibodies and detection antibodies for Brucella detection,and optimized and determined the opti-mal antibody coating time and concentration,as well as the optimal blocking solution,blocking time,and yin-yang critical val-ue.The specificity of this method was verified by examination of other bacteria prone to cross-reacting with Brucella.The sen-sitivity of the method was verified by detection of a gradient dilution of inactivated Brucella.Moreover,the sandwich ELISA detection results were compared with test tube agglutination and qPCR results.The selected capture antibody was 4A12,and the selected detection antibody was 6C12.Experimental analysis indicated that the optimal coating concentration for the 4A12 capture antibody was 5 μg/mL,and the optimal dilution ratio for the 6C12 detection antibody was 1∶2000.The optimal coating conditions were overnight at 4℃,and blocking with 5％skim milk powder for 2 hours.The established double antibody sand-wich ELISA method reacted with only Brucella but not other bacteria,thus demonstrating the method's good specificity.Inac-tivated Brucella solution was still detectable after dilution to 1 × 105 CFU/mL,thus demonstrating the method's good sensitiv-ity.The intra-and inter batch coefficients of variation were both below 10％,thus indicating the method's good repeatability.Thus,this study successfully established a dual antibody sandwich ELISA method for Brucella detection,which has good spe-cificity and sensitivity,and might provide an effective approach for the precise diagnosis and effective prevention and control of brucellosis.

Objective To explore the behavioral patterns and hippocampal neurogenesis of CHD8+mice,and to provide behavioral and morphological basis for improving autism like behavior and neurogenesis.Methods Genotype of wild type(WT)and CHD8+/-mice was identified.Weight measurement was conducted on both male and female mice of the WT and CHD8+/-strains.Subsequently,a battery of behavioral tests was administered,which included three-chamber test,self-grooming test,nesting test,Y-maze spontaneous alternation test,food burial test,open-field test and light-dark transition test.Afterwards,the mice were administered 2％pentobarbital sodium(2 ml/kg)to induce anesthesia.Their brains were frozen with 4％paraformaldehyde,removed for photography and analysis to identify any alterations in brain size.Western blotting and immunofluorescent labeling were used to detect changes in the process of hippocampus neurogenesis.Results Western blotting analysis demonstrated a decrease in the amounts of chromodomain helicase DNA binding protein 8(CHD8)protein in both male and female mice with CHD8+genotype,as compared to WT mice.There were no notable disparities in body weight between male and female WT and CHD8+mice,as well as in brain size.The three-chamber social behavior test revealed that both male and female CHD8+/-mice had social deficiencies(P＜0.05).During the open field test,there was no significant difference in the total distance moved by male and female WT and CHD8+/-mice.However,the amount of time spent in the central region was considerably lower in CHD8+/-mice compared to the WT mice(P＜0.01).Furthermore,the light-dark transition test revealed that both male and female CHD8+/-mice spent considerably less time investigating the white box compared to the WT mice(P＜0.05).Nevertheless,there were no notable alterations found in self-grooming,nesting,spontaneous alternation of Y-maze,and food burial experiments.In addition,Western blotting result demonstrated a significant drop in doublecortin(DCX)expression(P＜0.001),and immunofluorescent staining revealed a notable reduction in the number of DCX+cells(P＜0.01)in the hippocampus of CHD8+/-mice.Conclusion CHD8+/-mice exhibit social disorders and anxiety-like behaviors,with a decrease in the number of newly generated neurons in the hippocampus and neurogenesis disorders.

The influence of material properties on fingerprint development effects were systematically studied.Firstly,carbon dots/NaYF4 and Cu nanoclusters/starch nanocomposites respectively possessing different fluorescent intensities and dual fluorescent colors,as well as NaYF4 micro-/nano-materials exhibiting different morphologies,sizes,and surface properties were chemically synthesized and further used for latent fingerprint development.Then,the developing effects were comprehensively evaluated by visual analysis combining with spectral characterization and Python-based calculation.Finally,the influences of material properties on latent fingerprint development were quantitatively evaluated from three dimensions including contrast,sensitivity,and selectivity.The fluorescence properties of developing materials and substrates could significantly affect the developing contrast,namely,the stronger the developing signal,the higher the contrast;the weaker the background noise,the higher the contrast.The sizes and morphologies of the developing materials could respectively influence the quantity and quality of developed minutiae,and significantly affect the developing sensitivity,namely,the smaller the particle size of developing materials,the more the quantity of developed minutiae and the higher the sensitivity;the smaller the surface area of developing materials,the higher the quality of developed minutiae and the higher the sensitivity.The surface properties together with the sizes and morphologies of developing materials could influence their adsorption performances,and significantly affect the developing selectivity,namely,the stronger the specific adsorption of developing materials with fingerprint substance,and the weaker the non-specific adsorption of developing materials with substrate,the higher the selectivity.Moreover,the fingerprint development using the materials with suitable surface area and appropriate mass would have a high selectivity.

Objective To analyze the characteristics of methadone-related poisoning cases and provide a reference for forensic identification.Methods A total of 71 cases of methadone-related poisoning re-ported from 1998 to 2023 in China and 26 cases of methadone-related deaths reported from 2013 to 2018 in Italy were retrieved from databases including PubMed,Wanfang and CNKI.The general infor-mation,forensic pathological and toxicological characteristics were analyzed.Results Among the 71 methadone-related poisoning cases in China,there were 54 cases(76.06%)of poisoning without death and 17 cases(23.94%)of death from poisoning.There were 54 male cases(76.06%),and 51 cases(71.83%)aged 19 to 39 years old.There were 35 cases(49.30%)of poisoning caused by methadone alone,and 32 cases(45.07%)were poisoning caused by methadone combined with other substances or drugs including heroin and benzodiazepines.Most of the poisoned showed coma,respiratory depres-sion and miosis.Signs of asphyxia were often found by autopsy.The mass concentration of methadone detected in the blood of 6 deceased ranged from 0.112 to 3.000 mg/L.Among the 26 methadone-related deaths in Italy,22 cases were male(84.62%).There were 6 cases(23.08%)caused by methadone alone,and 20 cases(76.92%)died from methadone combined with other substances or drugs.The mass concentration of methadone in blood ranged from 0.181 to 4.059 mg/L.Conclusion The propor-tions of poisoning cases caused by methadone alone and methadone combined with other substances or drugs are comparable in China.The majority of deceased caused by methadone poisoning shows typi-cal triad of coma,respiratory depression and miosis,which helps forensic experts determine the cause of death related to methadone.Additionally,it is necessary to increase the routine testing of the con-centration of methadone and its combined substances or drugs in deceased,and collect data for the in-terpretation of the results of related cases.

This study was aimed at establishing a sensitive and specific sandwich ELISA detection method for Brucella.We screened monoclonal capture antibodies and detection antibodies for Brucella detection,and optimized and determined the opti-mal antibody coating time and concentration,as well as the optimal blocking solution,blocking time,and yin-yang critical val-ue.The specificity of this method was verified by examination of other bacteria prone to cross-reacting with Brucella.The sen-sitivity of the method was verified by detection of a gradient dilution of inactivated Brucella.Moreover,the sandwich ELISA detection results were compared with test tube agglutination and qPCR results.The selected capture antibody was 4A12,and the selected detection antibody was 6C12.Experimental analysis indicated that the optimal coating concentration for the 4A12 capture antibody was 5 μg/mL,and the optimal dilution ratio for the 6C12 detection antibody was 1∶2000.The optimal coating conditions were overnight at 4℃,and blocking with 5％skim milk powder for 2 hours.The established double antibody sand-wich ELISA method reacted with only Brucella but not other bacteria,thus demonstrating the method's good specificity.Inac-tivated Brucella solution was still detectable after dilution to 1 × 105 CFU/mL,thus demonstrating the method's good sensitiv-ity.The intra-and inter batch coefficients of variation were both below 10％,thus indicating the method's good repeatability.Thus,this study successfully established a dual antibody sandwich ELISA method for Brucella detection,which has good spe-cificity and sensitivity,and might provide an effective approach for the precise diagnosis and effective prevention and control of brucellosis.

ObjectiveThe scale of microalgae farming industry is huge. During farming, it is easy for microalgae to be affected by miscellaneous bacteria and other contaminants. Because of that, periodic test is necessary to ensure the growth of microalgae. Present microscopy imaging and spectral analysis methods have higher requirements for experiment personnel, equipment and sites, for which it is unable to achieve real-time portable detection. For the purpose of real-time portable microalgae detection, a real-time microalgae detection system of low detection requirement and fast detection speed is needed. MethodsThis study has developed a microalgae detection system based on deep learning. A microscopy imaging device based on bright field was constructed. With imaged captured from the device, a neural network based on YOLOv3 was trained and deployed on microcomputer, thus realizing real-time portable microalgae detection. This study has also improved the feature extraction network by introducing cross-region residual connection and attention mechanism and replacing optimizer with Adam optimizer using multistage and multimethod strategy. ResultsWith cross-region residual connection, the mAP value reached 0.92. Compared with manual result, the detection error was 2.47%. ConclusionThe system could achieve real-time portable microalgae detection and provide relatively accurate detection result, so it can be applied to periodic test in microalgae farming.

Objective:To explore the effect of different concentrations of Chinese herbal formula,Er-miao-san(EMS)immune regulatory gene on Gnαq expression in rats with collagen-induced arthritis(CIA).Methods:In the study,we established a CIA model,and randomly divided into model groups,EMS group with 1.5,3.0 and 4.5 g/kg(L-EMS,M-EMS,H-EMS)were adminis-tered to CIA rats by gavage,methotrexate was used as a positive control group(MTX).The rats in each group were given correspond-ing drugs for treatment,and the normal control group and model group were given the same amount of normal saline gavage.ELISA detects the expression of IL-1β,IL-6 and TNF-α in rats'serum.The mRNA and protein expression of Gnαq were analyzed using qRT-PCR and Western blot.The localization of Gnαq was performed by IHC.Results:The joint of rats in CIA group showed obvious damage,tissue hyperplasia and inflammatory infiltration;the joint tissue destruction and proliferation of MTX and EMS groups were reduced;the expressions of IL-1β,IL-6 and TNF-α in CIA group were extremely increased(P<0.01).The expressions of IL-1β(except H-EMS group),IL-6 and TNF-α in MTX and EMS groups were significantly lower than those in CIA group(P<0.05).The expressions of IL-1β,IL-6 and TNF-α in L-EMS group were lower than those in the M-EMS and H-EMS groups(P<0.05,P<0.01).The expression of Gnαq mRNA and protein in spleen and joints of CIA group were higher than those of Control group(P<0.01).The expression of Gnαq mRNA and protein in spleen and joints of EMS and MTX groups were lower than those of CIA group(P<0.05).The expression of Gnαq mRNA and protein in the dose of 1.5 g/kg of EMS group was lower than those of the other groups(P<0.01,P<0.05).Different degree of posi-tive signal was detected in spleen and joints in different groups.Conclusion:Gnαq is expressed in spleen and joints of CIA rats,may participate in the formation of inflammatory response in CIA rats,and then mediate the formation and development of RA by down-reg-ulating the expression of Gnαq.