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.

Objective To understand the surveillance situation of poliovirus in Guangzhou from 2011 to 2024, and to further strengthen polio surveillance and ensure the continued maintenance of a polio-free status. Methods An analysis was conducted on the discovery and investigation results of six cases of vaccine-derived poliovirus (VDPV) detected in Guangzhou. Results A total of 6 VDPV incidents were reported in Guangzhou from 2011 to June 2024, among which 5 incidents were from sewage sample testing in the Liede Sewage Treatment Plant in Guangzhou, all of which were confirmed as VDPV, with 1 for type I, 1 for type II, and 3 for type III. In addition, one confirmed HFMD case was identified as a type VDPV II carrier. No presence of any wild poliovirus (WPV), VDPV cases, or circulating VDPV (cVDPV) was reported. Conclusion Guangzhou City has maintained a high level of vigilance and effectiveness in the monitoring and prevention of polio. Continuously strengthening the construction of the polio monitoring network, optimizing vaccination strategies, and comprehensively improving public health awareness are still the focus of the prevention and control work in the future.

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by various complications, and the exact etiology remains unclear. Treatments for SLE encompass hormone therapy, plasma exchange and immunoadsorption, and targeted biological therapies. Berbamine (BBM), a cellular immunopotentiator with diverse biological functions, has not been reported to have immunomodulatory and therapeutic effects on SLE. The mice were divided into control group, model group, positive control group, low, medium and high BBM groups. In control group, C57BL/6J wild mice received intraperitoneal injection of saline. In model group, MRL/lpr lupus mice were treated with intraperitoneal injection of saline. In positive control group, MRL/lpr lupus mice received intragastric administration of hydroxychloroquine sulfate tablets [Plaquenil, 150 mg/(kg·d)]. In BBM groups, MRL/lpr lupus mice received intragastric administration of different concentration of BBM respectively [20 mg/(kg·d), 50 mg/(kg·d), 100 mg/(kg·d)]. After 8 weeks of treatment, blood was collected from the retro-orbital venous plexus, and ELISA was used to detect the levels of anti-double-stranded DNA (dsDNA) antibodies, antinuclear antibodies (ANA), and anti-small nuclear ribonucleoprotein/Sm (snRNP/Sm) antibodies. Spleen tissues were collected for analysis of Th1/Th2 ratio by flow cytometry. The RNA and protein of spleen were extracted, and the levels of T-box transcription factor T-bet and GATA3 (GATA binding protein 3) mRNA and protein were detected by qRT-PCR and Western blot. The proliferation of white blood cells in the blood was tested by blood routine test. The histopathological changes of kidneys of each group were detected by HE staining. Compared with the model group, the levels of ANA, anti-dsDNA, and anti-snRNP/Sm antibodies were significantly reduced in the BBM-treated groups. The Th1/Th2 ratio was significantly decreased in the model group, but reversed by BBM. Compared with the control group, T-bet expression was significantly downregulated, while GATA3 expression was significantly upregulated in the model group. After BBM intervention, T-bet expression significantly increased, while GATA3 expression decreased compared with the model group. The number of white blood cells significantly decreased in the model group, and increased in the BBM-treated groups. In the model group, the glomerular mesangial and endothelial cells showed significant hyperplasia, clear thrombus was observed in the dilated capillaries, and inflammatory cells infiltrated in the renal interstitium. In medium and high BBM groups, the infiltration of inflammatory cells and capillary thrombosis were significantly decreased. In conclusion, BBM exhibits certain immunomodulatory effects on SLE and promotes the proliferation of white blood cells.
Animals ; Lupus Erythematosus, Systemic/immunology* ; Mice ; Mice, Inbred C57BL ; Mice, Inbred MRL lpr ; Female ; Benzylisoquinolines/pharmacology*

Based on the pharmacokinetics theory, this study investigated the pharmacokinetic characteristics of albiflorin, paeoniflorin, wogonoside, and wogonin in normal and febrile rats and summarized absorption and elimination rules of Dayuanyin in them to provide reference for further development and clinical application of Dayuanyin. Blood samples were taken from the fundus venous plexus of normal and model rats after intragastric administration of Dayuanyin at different time points. The concentration of each substance in blood was determined by ultra performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS) technique at different time points. DAS 2.0, a piece of pharmacokinetics software, was used to calculate the pharmacokinetic parameters of each component. The results show that the 4 components had good linear relationship in their respective ranges, and the results of methodological investigation met the requirements. The pharmacokinetic parameters of C_(max), T_(max), t_(1/2), AUC_(0-t), AUC_(0-∞), and MRT_(0-t) were calculated by the DAS 2.0 non-compartmental model. Compared with those in the normal group, C_(max) and AUC_(0-t) of the 4 components in the model group were significantly increased. There were significant differences in the pharmacokinetic characteristics between the normal and model groups, suggesting that the absorption and elimination of Dayuanyin may be affected by the changes of internal environment of the body in different physiological states.
Animals ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Rats, Sprague-Dawley ; Fever/metabolism* ; Tandem Mass Spectrometry ; Chromatography, High Pressure Liquid ; Glucosides/pharmacokinetics* ; Monoterpenes

This study aimed to explore the therapeutic mechanisms of Colquhounia Root Tablets(CRT) in treating diabetic kidney disease(DKD) by integrating biomolecular network mining with animal model verification. By analyzing clinical transcriptomics data, an interaction network was constructed between candidate targets of CRT and DKD-related genes. Based on the topological eigenvalues of network nodes, 101 core network targets of CRT against DKD were identified. These targets were found to be closely related to multiple pathways associated with type 2 diabetes, immune response, and metabolic reprogramming. Given that immune-inflammatory imbalance driven by metabolic reprogramming is one of the key pathogenic mechanisms of DKD, and that many core network targets of CRT are involved in this pathological process, receptor for advanced glycation end products(RAGE)-reactive oxygen species(ROS)-phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)-nuclear factor-κB(NF-κB)-NOD-like receptor family pyrin domain containing 3(NLRP3) signaling axis was selected as a candidate target for in-depth research. Further, a rat model of DKD induced by a high-sugar, high-fat diet and streptozotocin was established to evaluate the pharmacological effects of CRT and verify the expression of related targets. The experimental results showed that CRT could effectively correct metabolic disturbances in DKD, restore immune-inflammatory balance, and improve renal function and its pathological changes by inhibiting the activation of the RAGE-ROS-PI3K-AKT-NF-κB-NLRP3 signaling axis. In conclusion, this study reveals that CRT alleviates the progression of DKD through dual regulation of metabolic reprogramming and immune-inflammatory responses, providing strong experimental evidence for its clinical application in DKD.
Animals ; Diabetic Nephropathies/metabolism* ; Receptor for Advanced Glycation End Products/genetics* ; NF-kappa B/genetics* ; Signal Transduction/drug effects* ; Rats ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Phosphatidylinositol 3-Kinases/genetics* ; Reactive Oxygen Species/metabolism* ; Humans ; Plant Roots/chemistry* ; Rats, Sprague-Dawley ; Tablets/administration & dosage*

This study aims to investigate the molecular mechanism by which naringin alleviates cerebral ischemia/reperfusion(CI/R) injury through DRP1/LRRK2/MCU signaling axis. A total of 60 SD rats were randomly divided into the sham group, the model group, the sodium Danshensu group, and low-, medium-, and high-dose(50, 100, and 200 mg·kg~(-1)) naringin groups, with 10 rats in each group. Except for the sham group, a transient middle cerebral artery occlusion/reperfusion(tMCAO/R) model was established in SD rats using the suture method. Longa 5-point scale was used to assess neurological deficits. 2,3,5-Triphenyl tetrazolium chloride(TTC) staining was used to detect the volume percentage of cerebral infarction in rats. Hematoxylin-eosin(HE) staining and Nissl staining were employed to assess neuronal structural alterations and the number of Nissl bodies in cortex, respectively. Western blot was used to determine the protein expression levels of B-cell lymphoma-2 gene(Bcl-2), Bcl-2-associated X protein(Bax), cleaved cysteine-aspartate protease-3(cleaved caspase-3), mitochondrial calcium uniporter(MCU), microtubule-associated protein 1 light chain 3(LC3), and P62. Mitochondrial structure and autophagy in cortical neurons were observed by transmission electron microscopy. Immunofluorescence assay was used to quantify the fluorescence intensities of MCU and mitochondrial calcium ion, as well as the co-localization of dynamin-related protein 1(DRP1) with leucine-rich repeat kinase 2(LRRK2) and translocase of outer mitochondrial membrane 20(TOMM20) with LC3 in cortical mitochondria. The results showed that compared with the model group, naringin significantly decreased the volume percentage of cerebral infarction and neurological deficit score in tMCAO/R rats, alleviated the structural damage and Nissl body loss of cortical neurons in tMCAO/R rats, inhibited autophagosomes in cortical neurons, and increased the average diameter of cortical mitochondria. The Western blot results showed that compared to the sham group, the model group exhibited increased levels of cleaved caspase-3, Bax, MCU, and the LC3Ⅱ/LC3Ⅰ ratio in the cortex and reduced protein levels of Bcl-2 and P62. However, naringin down-regulated the protein expression of cleaved caspase-3, Bax, MCU and the ratio of LC3Ⅱ/LC3Ⅰ ratio and up-regulated the expression of Bcl-2 and P62 proteins in cortical area. In addition, immunofluorescence analysis showed that compared with the model group, naringin and positive drug treatments significantly decreased the fluorescence intensities of MCU and mitochondrial calcium ion. Meanwhile, the co-localization of DRP1 with LRRK2 and TOMM20 with LC3 in cortical mitochondria was also decreased significantly after the intervention. These findings suggest that naringin can alleviate cortical neuronal damage in tMCAO/R rats by inhibiting DRP1/LRRK2/MCU-mediated mitochondrial fragmentation and the resultant excessive mitophagy.
Animals ; Rats, Sprague-Dawley ; Reperfusion Injury/genetics* ; Flavanones/administration & dosage* ; Rats ; Dynamins/genetics* ; Male ; Brain Ischemia/genetics* ; Protein Serine-Threonine Kinases/genetics* ; Signal Transduction/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage*

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

This study aims to explore the mechanism through which Yishen Jiangtang Decoction(YSJTD) regulates endoplasmic reticulum stress(ERS)-mediated NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome to improve diabetic nephropathy(DN) in db/db mice. Thirty db/db mice were randomly divided into the model group, YSJTD group, ERS inhibitor 4-phenylbutyric acid(4-PBA) group, with 10 mice in each group. Additionally, 10 db/m mice were selected as the control group. The YSJTD group was orally administered YSJTD at a dose of 0.01 mL·g~(-1), the 4-PBA group was orally administered 4-PBA at a dose of 0.5 mg·g~(-1), and the control and model groups were given an equal volume of carboxylmethyl cellulose sodium. The treatments were administered once daily for 8 weeks. Food intake, water consumption, and body weight were recorded every 2 weeks. After the intervention, fasting blood glucose(FBG), glycosylated hemoglobin(HbA1c), urine microalbumin(U-mALB), 24-hour urine volume, serum creatinine(Scr), and blood urea nitrogen(BUN) were measured. Inflammatory markers interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected using the enzyme-linked immunosorbent assay(ELISA). Renal pathology was assessed through hematoxylin-eosin(HE), periodic acid-Schiff(PAS), and Masson staining, and transmission electron microscopy(TEM). Western blot was used to detect the expression levels of glucose-regulated protein 78(GRP78), C/EBP homologous protein(CHOP), NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), cysteinyl aspartate-specific proteinase(caspase-1), and gasdermin D(GSDMD) in kidney tissues. The results showed that compared to the control group, the model group exhibited poor general condition, increased weight and food and water intake, and significantly higher levels of FBG, HbA1c, U-mALB, kidney index, 24-hour urine volume, IL-1β, and IL-18. Compared to the model group, the YSJTD and 4-PBA groups showed improved general condition, increased body weight, decreased food intake, and lower levels of FBG, U-mALB, kidney index, 24-hour urine volume, and IL-1β. Specifically, the YSJTD group showed a significant reduction in IL-18 levels compared to the model group, while the 4-PBA group exhibited decreased water intake and HbA1c levels compared to the model group. Although there was a decreasing trend in water intake and HbA1c in the YSJTD group, the differences were not statistically significant. No significant differences were observed in BUN, Scr, and kidney weight among the groups. Renal pathology revealed that the model group exhibited more severe renal damage compared to the control group. Kidney sections from the model group showed diffuse mesangial proliferation in the glomeruli, tubular edema, tubular dilation, significant inflammatory cell infiltration in the interstitium, and increased glycogen staining and blue collagen deposition in the basement membrane. In contrast, the YSJTD and 4-PBA groups showed varying degrees of improvement in renal damage, glycogen staining, and collagen deposition, with the YSJTD group showing more significant improvements. TEM analysis indicated that the model group had extensive cytoplasmic edema, homogeneous thickening of the basement membrane, fewer foot processes, and widening of fused foot processes. In the YSJTD and 4-PBA groups, cytoplasmic swelling of renal tissues was reduced, the basement membrane remained intact and uniform, and foot process fusion improved.Western blot results indicated that compared to the control group, the model group showed upregulation of GRP78, CHOP, GSDMD, NLRP3, ASC, and caspase-1 expression. In contrast, both the YSJTD and 4-PBA groups showed downregulation of these markers compared to the model group. These findings suggest that YSJTD exerts a protective effect against DN by alleviating NLRP3 inflammasome activation through the inhibition of ERS, thereby improving the inflammatory response in db/db DN mice.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Diabetic Nephropathies/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Inflammasomes/drug effects* ; Male ; Kidney/pathology* ; Endoplasmic Reticulum Chaperone BiP ; Humans ; Interleukin-18/genetics* ; Mice, Inbred C57BL

Based on the ultra performance liquid chromatography-quadrupole Exactive Orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) technology, combined with related literature, databases, and reference material information, this study qualitatively analyzed the components of Dayuanyin in the tissue of rats after gavage and employed molecular docking technology to predict the rationality of the mechanism behind the antipyretic effect of the in vivo components in Dayuanyin. A total of 21, 26, 20, 21, 14, and 31 prototype components and 3, 16, 3, 7, 5, and 24 metabolites were identified from the heart, liver, spleen, lung, kidney, and hypothalamus of the rats, respectively, and the binding ability of key components and targets was further verified by molecular docking. The results showed that all components had good binding ability with targets. The established UPLC-Q-Exactive Orbitrap-MS could effectively and quickly identify the Dayuanyin components distributed in tissue and preliminarily identify their metabolites. Many components were identified in the hypothalamus, which suggested that the components delivered to the brain should be focused on in the study on Dayuanyin in the treatment of febrile diseases. The molecular docking technology was used to predict the rationality of the mechanism behind its antipyretic effect, which lays the foundation for the clarification of the material basis and action mechanism of Dayuanyin, the development of new preparations, and the prediction of quality markers.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Molecular Docking Simulation ; Male ; Antipyretics/metabolism* ; Rats, Sprague-Dawley ; Tissue Distribution ; Mass Spectrometry ; Chromatography, High Pressure Liquid ; Hypothalamus/metabolism*

OBJECTIVE: To investigate the short-to-medium-term clinical efficacy of total hip arthroplasty(THA) with Pinnacle ES constrained liner in the treatment of femoral neck fractures(FNF) associated with lower limb neuromuscular disorders. METHODS: A retrospective analysis was conducted on 16 patients who underwent primary THA using Pinnacle ES constrained liner for FNF with lower limb neuromuscular disorders and had complete follow-up data, treated between January 2020 and January 2023. There were 7 males and 9 females, with a mean age of (68.42±3.58) years old ranging from 61 to 75 years old. Among them, 10 cases had left-sided fractures and 6 had right-sided fractures;based on the Garden classification, 11 cases were type Ⅲ and 5 cases were type Ⅳ. The affected limbs were complicated with sequelae of poliomyelitis (2 cases), Parkinson's disease (2 cases), and sequelae of cerebral infarction (12 cases). All operations were performed via a posterolateral approach. Prosthesis position was evaluated using imaging data. Hip function was assessed by the Harris hip score(HHS) and Merle D'Aubigne hip score. During the follow-up period, the occurrence of complications such as prosthetic dislocation, loosening, and infection was recorded. RESULTS: One patient died of advanced tumor 2 years after surgery, and the remaining 15 patients were followed up for 24 to 64 months with a mean of (34.8±5.5) months. The operation time ranged from 50 to 90 minutes with a mean of (75.56±8.15) minutes, and the blood loss ranged from 150 to 200 ml with a mean of (170.32±12.56) ml. All patients achieved primary wound healing after surgery. Intraoperatively, femoral calcar splitting occurred in 2 cases, which were treated with titanium cable binding;no neurovascular injuries were observed in any case. The mean HHS increased from (18.95±2.25) preoperatively to (88.02±2.42) at the last follow-up, and the mean Merle D'Aubigne Score increased from (3.05±0.06) preoperatively to (16.65±0.93) at the last follow-up. Postoperative follow-up X-rays showed good prosthetic position, and no complications such as dislocation, prosthetic loosening, periprosthetic fracture, or deep infection occurred during the follow-up period. CONCLUSION Total hip arthroplasty with Pinnacle ES constrained liner is effective in the treatment of femoral neck fractures associated with lower limb neuromuscular disorders. It can significantly improve hip function, reduce the postoperative prosthetic dislocation rate, provide good initial stability, and achieve satisfactory short-to-medium-term clinical efficacy.
Humans ; Male ; Female ; Middle Aged ; Aged ; Arthroplasty, Replacement, Hip/methods* ; Femoral Neck Fractures/complications* ; Retrospective Studies ; Neuromuscular Diseases/surgery* ; Lower Extremity ; Treatment Outcome