Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Objective To explore the effect of Polysaccharides of Panax notoginseng(PPN)on anti-exercise fatigue in mice.Methods One hundred male KM mice were randomly divided into negative control group,positive control group and experimental-L,-M,-H groups,with 20 cases per group.Experimental-L,-M,-H groups was given 100,200,400 mg·kg-1 PPN,respectively;positive control group was given 200 mg·kg-1 vitamin C;negative control group was given 0.1 mL·10 g-1 0.9％NaCl.Five groups were gavaged once a day for 28 days.After the last administration,the loaded swimming time was measured;after 90 minutes of the unloaded swimming test,the mice were allowed to rest for 30 minutes,the levels of lactic acid(LD),blood urea nitrogen(BUN),glycogen,and malondialdehyde(MDA)were measured,the safety of PPN with organ indices and histopathology.Results LD levels in negative control group,positive control group and experimental-L,-M,-Hgroupswere(4.76±0.84),(2.86±0.34),(3.00±0.69),(2.35±0.65)and(1.39±0.48)mg·kg-1;BUN contents were(13.65±1.25),(12.55±0.91),(12.12±1.24),(11.06±1.30)and(9.85±1.05)mmol·L-1;liver glycogen contents were(3.24±0.56),(11.11±2.16),(5.61±1.41),(6.60±1.49)and(12.05±2.25)mg·g-1;MDA levels were(2.36±0.21),(1.23±0.41),(1.93±0.23),(1.73±0.21)and(1.04±0.18)mg prot·mL-1.Compared with negative control group,the differences of above indexes in the positive control group and experimental-L,-M,-H groups were statistically significant(P＜0.05,P＜0.01,P＜0.001).Conclusion PPN can increase exercise endurance in mice and has an anti-fatigue effect.This study provides a theoretical basis for the application of PPN in the field of anti-fatigue research.

Objective To study the antioxidant activity and organ protection of different components of Panax notoginseng polysaccharide(PNPS)in D-galactose-induced oxidative damage aging model mice.Methods KM mice were randomly divided into normal group,model group,vitamin C(VC)group(given 200 mg·kg-1 VC),crude polysaccharide from Panax notoginseng(CPPN)group,neutral polysaccharide from Panax notoginseng(NPPN)group and acidic polysaccharide from Panax notoginseng(APPN-Ⅰ,APPN-Ⅱ,APPN-Ⅲ)group(given 400 mg·kg-1 CPPN,NPPN,APPN-Ⅰ,APPN-Ⅱ,APPN-Ⅲ,respectively).Except for the normal group,oxidative injury aging mouse models were established by intraperitoneal injection of 1 g·kg-1 D-galactose.The mice were sacrificed after continuous administration for 42 days,and serum and liver homogenate were prepared.Malondialdehyde(MDA)was determined by thiobarbituric acid method;superoxide dismutase(SOD)was determined by tetrazole salt method;glutathione peroxidase(GSH-Px)was determined by double antibody sandwich method.Results Serum SOD in the normal group,model group,VC group,CPPN group,NPPN group and APPN-Ⅰ,APPN-Ⅱ,APPN-Ⅲ groups were(15.07±0.69),(12.79±1.51),(15.56±1.01),(13.69±0.96),(14.27±0.64),(14.31±0.99),(14.18±0.79)and(15.85±0.89)U·mL-1;serum GSH-Px were(105.35±4.97),(90.36±4.31),(111.51±7.00),(113.03±8.06),(118.77±5.19),(123.60±8.08),(131.65±3.60)and(149.22±13.32)ng·L-1;serum MDA were(1.72±0.26),(4.16±0.92),(2.26±0.59),(2.82±0.47),(2.46±0.50),(1.98±0.41),(2.39±0.39)and(2.07±0.24)nmol·mL-1;the liver SOD were(234.22±3.84),(205.04±7.28),(234.63±6.37),(214.99±17.66),(234.13±3.63),(234.63±3.44),(233.87±5.63)and(235.42±2.33)U·mgprot-1;liver GSH-Px were(274.27±23.72),(207.00±15.22),(257.68±16.39),(249.79±18.78),(252.62±10.92),(256.25±21.83),(261.20±17.52)and(263.16±17.98)ng·L-1;liver MDA were(35.70±3.52),(49.65±6.32),(36.15±2.48),(39.17±4.29),(37.40±6.19),(35.34±4.06)and(35.90±5.36),(33.31±7.64)nmol·mgprot-1.Compared with the normal group,SOD,GSH-Px in serum and liver of mice in the model group were significantly reduced,and the content of MDA was significantly increased(all P＜0.01).After treatment with different components of Panax notoginseng polysaccharide,the oxidative indicators in mice were significantly improved,among which APPN-Ⅲ have the best antioxidant activity,which could significantly increase the activities of SOD,GSH-Px in serum and liver,and reduce the content of MDA(all P＜0.01).Conclusion Different components of Panax notoginseng polysaccharide have antioxidant activity and organ protection in vivo,among which APPN-Ⅲ has the best antioxidant activity and has a good organ protection effect.

ObjectiveSodium hyaluronate (HA) was used as the research object to modify it with phenolic acid in order to obtain the molecular structure with better antioxidant activity or even new activity. MethodsIn this study, 5 kinds of phenolic acid-sodium hyaluronate was prepared by free radical-mediated grafting method, and the grafts with the highest grafting degree were selected to optimize the synthesis conditions. Then, grafts structure and physicochemical properties were analyzed. The grafts were characterized by IR, UV, ¹H NMR, FESEM and TGA spectra. The in vitro antioxidant capacity of grafts was determined by the scavenging ability of DPPH·, ABTS⁺· and O^2-·. ResultsAmong 5 kinds of phenolic acid-sodium hyaluronate, the grafting rate of ferulic acid-sodium hyaluronate copolymer (FA-HA) was highest , which was chosen as experimental sample in the following tests. Firstly, the reaction conditions were investigated and the highest grafting rate was (16.59±0.31) mg/g at the optimal preparation conditions. Then, FA-HA structure and physicochemical properties were analyzed. Data from UV, IR, ¹H NMR analyses, TGA showed that FA were successfully grafted to HA. Compared with HA, the results of gel permeation chrematography (GPC) showed that the molecular mass distribution ofFA-HA copolymer decreased from 34.4 to 31.5 ku, but the uniformity of molecular distribution was improved. FESEM results showed that the structure of copolymer exhibited a closely connected lamellar structure with a relatively smooth surface. TGA results showed that thermal stability of FA-HA had a little decline. The antioxidant performance in vitro results showed that, during 0.25-10 g/L, FA-HA can eliminate (83.76±4.86)% DPPH·, (76.95±5.06)% ABTS⁺· and (83.08±2.51)% O^2-· respectively at 10 g/L. which were higher than that of native HA and FA. ConclusionFA and HA were successfully grafted together by free radical grafting, and the grafted FA-HA had better antioxidant activity in vitro, which provided a theoretical basis for further research and development of phenolic acid-HA grafts.

In order to study the compounds from Glechoma longituba (Nakai) Kupr. and explore the substance bases of its dissipating blood stasis, MCI, silica gel, Sephadex LH-20, ODS column chromatography and preparative thin layer chromatography were used to isolate and purify the compounds. The structures were identified by MS, 1D NMR, and 2D NMR spectra analysis, etc. Eight triterpenoids were isolated from dichloromethane fraction of ethanol extract from G. longituba and identified as 2α,3α,16β-trihydroxy-13α,27-cyclours-11-en-28-oic acid (1), 28-norurs-12-ene-3β,17β-diol (2), 28-norolean-12-ene-3β,17β-diol (3), oleanonic acid (4), 3β,13β-dihydroxyurs-11-en-28-oic acid (5), uvaol (6), oleanolic acid (7), ursolic acid (8). Compound 1 is a new hexacyclic triterpene with 13α,27-cyclopropane structure, named euscaphic acid H; compounds 2 and 3 were isolated from Lamiaceae for the first time while compounds 4 and 5 were isolated from this genus. The in vitro anticoagulant activity of triterpenoids was evaluated by four coagulation indexes (activated partial thromboplastin time, APTT; thrombin time, TT; prothrombin time, PT; fibrinogen, FIB). Among them, compounds 1 and 6 showed obvious anticoagulant effects.

In this study, fluvoxamine maleate sustained-release pellet system tablets were prepared and were used to evaluate their release behaviors in vitro. Fluvoxamine maleate pellets were prepared using centrifugal-spherization method and coated by fluidized bed as bottom-spray. The multi-unit sustained-release pellets and appropriate excipients for prescription volumes were mixed uniformly and then compressed to tablets. Screening and determining the optimal formulation of drug loaded pellets through L8 (2⁴) Taguchi experiment. Using Minitab software to design a DOE experiment with 2⁴ partial factors, including material temperature, fan speed, atomization pressure, and spray rate to optimize the bottom spray coating process. Taking monostearate glycerol ester with a particle size of 24-40 mesh as the main diluent for tableting to relieve the delamination phenomenon between pellets and excipients during tablet pressing and reduce mechanical damage to the coating film. By examining the powder fluidity indexes such as angle of repose, bulk density, tapped density, and Hausner ratio of mixed particles, it was found that the flowability and compressibility are good and suitable for direct compression. Evaluate the basic properties of the sustained-release tablets, investigate the in vitro release behavior and study the release mechanism. The results of in vitro release test showed that the self-made sustained-release tablets could disintegrate into independent pellet units in phosphate buffer at pH 6.8 and release slowly within 24 h, which conformed to the first-order drug release model. The fluvoxamine maleate sustained-release pellet system tablets meet the requirements of preparation design and has a great commercial prospect.

Objective:To investigate the regulatory effect of transient receptor potential cation channel subfamily C member 3 (TRPC3) on the retina in oxygen-induced retinopathy (OIR) mice and biological behavior of human retinal vascular endothelial cells (HREC).Methods:A total of 32 healthy SPF grade 7-day-old C57BL/6 mice were selected and randomly divided into a control group and an OIR group by the random number table method, with 16 mice in each group.The control group received no special treatment, and the OIR model was established in the OIR group.On postnatal day 17 (PN17), the success of the model establishment was verified by immunofluorescence staining of the retinal patch.The in vitro cultured HREC were divided into a normal control group, a transfection reagent group, and a si-TRPC3 group.The normal control group received no special treatment, while the transfection reagent group and the si-TRPC3 group were transfected with transfection reagent or transfection reagent + si-TRPC3.The relative expression of TRPC3 mRNA was detected by real-time quantitative fluorescence PCR.The relative expressions of TRPC3, transcription factor NF-E2 related factor (Nrf2), and superoxide dismutase (SOD) proteins were determined by Western blot.HREC were further divided into a normal control group, a vascular endothelial growth factor (VEGF) group, a si-TRPC3 group, and a Pyr3 (TRPC3 channel inhibitor) group, which were cultured in complete medium, medium containing 20 ng/ml VEGF recombinant protein, medium containing 20 ng/ml VEGF recombinant protein (si-TRPC3 transfection for 72 hours), and medium containing 20 ng/ml VEGF recombinant protein+ 1 μmol/L Pyr3 for 48 hours, respectively.The proliferation ability of HREC was detected using cell counting kit 8 (CCK-8). The horizontal and vertical migration ability of cells were detected by cell scratch assay and transwell assay, respectively.This study followed the 3R principles of animal welfare and was approved by the Ethics Committee of Hebei Eye Hospital (No.2023LW04). Results:Pathological neovascular clusters with strong fluorescent staining appeared in the retina of OIR mice on PN17.The relative expressions of TRPC3 mRNA and protein in the retina of OIR mice were 2.057±0.244 and 1.517±0.290, respectively, significantly higher than 0.983±0.033 and 0.874±0.052 of control group ( t=6.165, 3.094; both at P<0.05). The relative expression levels of TRPC3 mRNA and protein were significantly lower, and the relative expression levels of Nrf2 and SOD proteins were higher in the si-TRPC3 group than in the normal control and transfection reagent groups, and the differences were statistically significant (all at P<0.05). The CCK-8 experiment results showed that the cell absorbance value was higher in the VEGF group than in the normal control group, and lower in the si-TRPC3 and Pyr3 groups than in the VEGF group, with statistically significant differences (all at P<0.05). The results of the cell scratch experiment showed that the lateral migration rate of VEGF group cells was higher than that of normal control group, while the lateral migration rate of si-TRPC3 group and Pyr3 group cells was lower than that of VEGF group, and the differences were statistically significant (all at P<0.05). The transwell experiment results showed that the number of stained cells in the VEGF group was higher than that in the normal control group, and the number of stained cells in the si-TRPC3 group and Pyr3 group was lower than that in the VEGF group, with statistically significant differences (all at P<0.05). Conclusions:Hypoxia induces increased TRPC3 expression in OIR mouse retina, and downregulation of TRPC3 inhibits HREC proliferation and migration.The mechanism is related to the activation of the Nrf2-related oxidative stress pathway.