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.

ObjectiveTo study the mechanism of Huanglian Jiedutang （HLJDT） in treating mice with atherosclerosis （AS） by improving ferroptosis. MethodsA total of 10 SPF C57BL/6J mice were selected as a normal group， and 50 ApoE^-/- mice were randomly divided into five groups： model group， low-dose group of HLJDT， medium-dose group of HLJDT， high-dose group of HLJDT， and atorvastatin （ATV） group. ApoE^-/- mice were fed a high-fat diet for eight weeks to establish the AS model， and at the 9th week， they were given normal saline， low， medium， and high doses of HLJDT （3.9， 7.8， 15.6 g·kg^-1·d^-1）， and atorvastatin calcium tablets （0.01 g·kg^-1·d^-1）， respectively， for a total of eight weeks. The formation of aortic plaque in mice was observed by gross oil red O staining and Masson staining. The levels of total cholesterol （TC）， low-density lipoprotein cholesterol （LDL-C）， triglyceride （TG）， and high-density lipoprotein cholesterol （HDL-C） in blood fat were measured by the automatic biochemical analyzer， and the mitochondrial structure of the aorta was observed by transmission electron microscopy. The content of serum superoxide dismutase （SOD） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. The content of reduced glutathione （GSH） in serum was detected by the microplate method， and that of malondialdehyde （MDA） in serum was detected by the TBA method. The protein expression of nuclear factor E₂-associated factor 2 （Nrf2）/glutathione peroxidase 4 （GPX4） signaling pathway was detected by Western blot. ResultsCompared with those of the normal group， the contents of TC， LDL-C， TG， HDL-C， and MDA in the serum and the aortic vascular plaque deposition of the model group were significantly increased （P<0.01）， while the expression levels of SOD and GSH in serum， as well as Nrf2， solute carrier family 7 member 11 （SLC7A11）， and GPX4 in aorta were significantly decreased （P<0.01）. Mice in the model group appeared mitochondrial fragmentation and vacuolation in the aorta， volume atrophy， mitochondrial crista reduction， or a loose and disorganized form. Compared with those in the model group， the aortic vascular plaque deposition was significantly decreased in the low-dose， medium-dose， and high-dose groups of HLJDT and ATV group， and the contents of serum TC， LDL-C， TG， and MDA in serum were significantly decreased （P<0.05， P<0.01）. The contents of serum SOD and GSH and the expression levels of Nrf2， SLC7A11， and GPX4 in the aorta were increased （P<0.05， P<0.01）， and the symptoms of aortic mitochondrial vacuolation were alleviated. The number of cristae was increased， and they were ordered neatly. ConclusionHLJDT can reduce aortic vascular plaque deposition， decrease blood lipid and MDA expression， increase SOD and GSH expression， and ameliorate the pathological changes of ferroptosis， the mechanism of which is related to the Nrf2/GPX4 signaling pathway.

Oral solid dosage forms require processes such as disintegration and dissolution to release the drug before it can be absorbed and utilized by the body. In this manuscript, imaging technology was used to continuously visualize and characterize the in vitro static drug release process of gliclazide modified release tablets from 15 manufacturers, combined with the traditional method of in vitro dissolution testing, to determine the release profile of gliclazide modified release tablets, to evaluate the similarity of the release profiles by using the similarity factor (f₂) method and based on the analysis of the release profiles fitted with a variety of mathematical models. The results indicate that the gliclazide modified release tablets produced by 14 companies are hydrophilic gel matrix tablets. Compared to the reference listed drug, the release profiles of formulations from 11 companies show high similarity (f₂ > 50) to the reference. Among these, formulations with visual characteristics similar to the reference exhibit similar release curves. This study provides an alternative method for the in vitro consistency evaluation of gliclazide modified release tablets, aiming to assess the in vitro release behaviour of generic formulations more accurately and comprehensively.

ObjectiveNeuroinflammation plays a crucial role in both the onset and progression of ischemic stroke, exerting a significant impact on the recovery of the central nervous system. Excessive neuroinflammation can lead to secondary neuronal damage, further exacerbating brain injury and impairing functional recovery. As a result, effectively modulating and reducing neuroinflammation in the brain has become a key therapeutic strategy for improving outcomes in ischemic stroke patients. Among various approaches, targeting immune regulation to control inflammation has gained increasing attention. This study aims to investigate the role of in vitro induced regulatory T cells (Treg cells) in suppressing neuroinflammation after ischemic stroke, as well as their potential therapeutic effects. By exploring the mechanisms through which Tregs exert their immunomodulatory functions, this research is expected to provide new insights into stroke treatment strategies. MethodsNaive CD4⁺ T cells were isolated from mouse spleens using a negative selection method to ensure high purity, and then they were induced in vitro to differentiate into Treg cells by adding specific cytokines. The anti-inflammatory effects and therapeutic potential of Treg cells transplantation in a mouse model of ischemic stroke was evaluated. In the middle cerebral artery occlusion (MCAO) model, after Treg cells transplantation, their ability to successfully migrate to the infarcted brain region and their impact on neuroinflammation levels were examined. To further investigate the role of Treg cells in stroke recovery, the changes in cytokine expression and their effects on immune cell interactions was analyzed. Additionally, infarct size and behavioral scores were measured to assess the neuroprotective effects of Treg cells. By integrating multiple indicators, the comprehensive evaluation of potential benefits of Treg cells in the treatment of ischemic stroke was performed. ResultsTreg cells significantly regulated the expression levels of both pro-inflammatory and anti-inflammatory cytokines in vitro and in vivo, effectively balancing the immune response and suppressing excessive inflammation. Additionally, Treg cells inhibited the activation and activity of inflammatory cells, thereby reducing neuroinflammation. In the MCAO mouse model, Treg cells were observed to accumulate in the infarcted brain region, where they significantly reduced the infarct size, demonstrating their neuroprotective effects. Furthermore, Treg cell therapy notably improved behavioral scores, suggesting its role in promoting functional recovery, and increased the survival rate of ischemic stroke mice, highlighting its potential as a promising therapeutic strategy for stroke treatment. ConclusionIn vitro induced Treg cells can effectively suppress neuroinflammation caused by ischemic stroke, demonstrating promising clinical application potential. By regulating the balance between pro-inflammatory and anti-inflammatory cytokines, Treg cells can inhibit immune responses in the nervous system, thereby reducing neuronal damage. Additionally, they can modulate the immune microenvironment, suppress the activation of inflammatory cells, and promote tissue repair. The therapeutic effects of Treg cells also include enhancing post-stroke recovery, improving behavioral outcomes, and increasing the survival rate of ischemic stroke mice. With their ability to suppress neuroinflammation, Treg cell therapy provides a novel and effective strategy for the treatment of ischemic stroke, offering broad application prospects in clinical immunotherapy and regenerative medicine.

OBJECTIVE To explore whether pachymic acid （Pac） regulates mitochondrial autophagy mediated by the PTEN- induced kinase 1 （PINK1）/Parkin RBR E3 ubiquitin-protein ligase （Parkin） signaling pathway to alleviate myocardial injury in coronary heart disease （CHD） rats. METHODS SD rats were divided into control （Con） group， CHD group， Pac low-dose group （Pac-L group）， Pac high-dose group （Pac-H group）， Pac-H+PINK1/Parkin signaling pathway inhibitor group （Pac-H+3-MA group）， with 10 rats in each group. Except for the Con group， CHD models were established in the remaining groups of rats. After successful modeling， the rats in each group were intraperitoneally injected with the corresponding drugs or normal saline. After continuous intervention for 4 weeks， the left ventricular ejection fraction （LVEF）， left ventricular end-diastolic volume （LVEDV）， left ventricular end-systolic volume （LVESV）， and mean arterial pressure （MAP） of the rats were detected. The levels of creatine kinase isoenzyme （CK-MB）， lactate dehydrogenase （LDH）， cardiac troponin I （cTnI）， and cardiac troponin T （cTnT） in the serum， as well as the levels of tumor necrosis factor-α （TNF-α）， interleukin-10 （IL-10）， IL-1β， reactive oxygen species （ROS）， malondialdehyde （MDA） in the myocardial tissue， and the activities of catalase （CAT） and superoxide dismutase （SOD）， as well as the expression levels of p62， cleaved caspase-3， Parkin， PINK1 proteins and the ratio of microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）/LC3Ⅰ ratio were measured. The morphology of myocardial tissue and mitochondrial autophagic vesicles were observed， and the number of mitochondrial autophagic vesicles per unit area and the rate of cardiomyocyte apoptosis were counted. RESULTS Compared with CHD group， LVEF， MAP， IL-10 levels， CAT and SOD activities， p62， Parkin， PINK1 protein expressions， LC3Ⅱ/LC3Ⅰ ratio， the numbers of mitochondrial autophagic vesicles per unit area in the Pac-L and Pac-H E-mail：hzdpft@163.com groups were increased significantly （P＜0.05）； the levels of LVEDV， LVESV， CK-MB， LDH， cTnI， cTnT， TNF-α， IL-1β， ROS and MDA， cell apoptosis rates， and protein expression of cleaved caspase-3 were all decreased significantly （P＜0.05）； and the changes in various indicators were more pronounced in the Pac-H group （P＜0.05）； both groups showed varying degree of improvement in myocardial histopathological morphology. Compared with the Pac-H group， the aforementioned indicators in rats from the Pac-H+3-MA group were all significantly reversed （P＜0.05）. CONCLUSIONS Pac may promote mitochondrial autophagy in cardiomyocytes of CHD rats by activating the PINK1/ Parkin signaling pathway， thereby reducing inflammatory responses and oxidative stress and improving myocardial injury.

ObjectiveTo study the plasma pharmacokinetics and tissue distribution of five representative components in Wujiwan， and to illustrate the difference of metabolism and tissue distribution before and after compatibility. MethodHealthy male SD rats were divided into four groups， including Wujiwan group（A group， 62.96 g·L^-1）， Coptidis Rhizoma group（B group， 38.4 g·L^-1）， processed Euodiae Fructus group（C group， 5.88 g·L^-1） and fried Paeoniae Radix Alba group（D group， 18.68 g·L^-1）， with 65 rats in each group， and were administered the drugs according to the clinical dose of decoction pieces converted into the dose of the extracts. Then plasma， liver， small intestine and brain were taken at pharmacokinetic set time in each group after administration. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry was developed for the quantitative analysis of five representative components［berberine（Ber）， palmatine（Pal）， evodiamine（Evo）， rutecarpine（Rut） and paeoniflorin（Pae）］ in Wujiwan， their concentrations in plasma， liver， small intestine and brain were detected at different time， plasma samples were processed by protein precipitation， and tissue samples were pretreated by protein precipitation plus liquid-liquid extraction. Non-atrioventricular model was used to calculate the pharmacokinetic parameters of each component， and the parameters of each group were compared. ResultPharmacokinetic results of A group showed that area under the curve（AUC_0-t） of the five representative components were ranked as follows：Ber and Pal were small intestine>liver>blood， Evo and Rut were liver>small intestine>plasma， Pae was small intestine>plasma， which was not detected in the liver， no other components were detected in brain except for Ber. In comparison with plasma and other tissues， peak concentration（C_max） of Ber， Pal， Evo， and Rut were the highest and time to peak（t_max） were the lowest in the liver of A group. In plasma， the AUC_0-t and C_max of Evo and Rut were increased in A group compared with C group， t_max of Pea was elevated and its C_max was decreased in A group compared with D group. In the liver， compared with B-D groups， C_max values of 5 representative components except Pae were elevated， AUC_0-t of Pae was decreased and AUC_0-t of Evo and Rut were increased in the A group. In the small intestine， half-life（t_1/2） of each representative components in A group was elevated and t_max was decreased， and C_max of each representative ingredient except Pal was decreased， AUC_0-t values of Ber and Pal were increased， whereas the AUC_0-t values of Evo and Rut were decreased. ConclusionThe small intestine， as the effector organ， is the most distributed， followed by the liver. The pharmacokinetic parameters of the representative components in Wujiwan are changed before and after compatibility， which is more favorable to the exertion of its pharmacodynamic effects.

In this study, we designed and synthesized 12 novel aloperine derivatives with different core structures. Among them, compound 3 with a ten-membered ring core was obtained through a special ring expansion reaction after γ-H Huffman elimination of quaternary ammonium salt, and the structure was verified by X-single crystal diffraction. Furthermore, their antiviral activity against human β-coronavirus HCoV-OC43 was evaluated by CCK-8 assay. Quaternary ammonium salt 2a and 3 had a good inhibitory effect against HCoV-OC43, and 2a had the highest anti-HCoV-OC43 activity with an EC₅₀ values of 3.77 μmol·L^-1 and a SI value of over 53.1. Schrӧdinger molecular docking results showed that both 2a and 3 might display their anti-HCoV-OC43 activity by directly acting on host TMPRSS2 and SR-B1. The results expanded the structural types of endocyclic aloperine and the function against coronavirus, and provided useful scientific data for the development of pharmaceutical applications of these compounds.

Objective:To discuss the protective effect of velvet antler peptide(VAP)in the osteoporosis(OP)model rats,and to clarify the possible mechanism.Methods:Sixty 12-week-old SD rats were randomly divided into control group,model group,positive drug group(treated with 1 mg·kg-1·d-1 of alendronate sodium by gavage),low dose of VAP group(treated with 100 mg·kg-1·d-1 VAP),medium dose of VAP group(treated with 200 mg·kg-1·d-1 VAP),and high dose of VAP group(treated with 300 mg·kg-1·d-1 VAP),and there were ten rats in each group.Except for control group,the rats in the other groups were injected with dexamethasone(2 mg·kg-1)to replicate the OP rat model,while the rats in control group were injected with the equivalent volume of saline twice a week for 11 consecutive weeks.Dual-energy X-ray absorptiometry was used to detect the bone mineral density(BMD)of femur tissue of the rats in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the levels of serum calcium(Ca2+),phosphate(P),osteoprotegerin(OPG),alkaline phosphatase(ALP),and osteocalcin(OCN)in serum of the rats in various groups;biochemical method was used to detect the malondialdehyde(MDA)level and superoxide dismutase(SOD)activity in serum of the rats in various groups;HE staining was used to observe the pathomorphology of bone tissue of the rats in various groups;Western blotting method was used to detect the expression levels of silent information regulator 1(SIRT1),catalase(CAT),Runt-related transcription factor 2(RUNX2),and forkhead box protein O1(FOXO1)proteins in bone tissue of the rats in various groups.Results:Compared with control group,the BMD of femoral tissue of the rats in model group was decreased(P<0.05);compared with model group,the BMD of femur tissue of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were increased(P<0.05 or P<0.01).Compared with control group,the levels of Ca2+,P,OPG,and SOD activities in serum of the rats in model group were decreased(P<0.05),and the levels of ALP,OCN,and MDA were increased(P<0.05);compared with model group,the level of OPG in serum of the rats in low dose of VAP group was significantly increased(P<0.05),the levels of Ca2+,P,OPG,and activities of SOD in serum of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were significantly increased(P<0.05 or P<0.01),and the levels of ALP,OCN,and MDA in serum of the rats in positive drug group and different doses of VAP groups were decreased(P<0.05 or P<0.01).The HE staining results showed that compared with control group,the rats in model group had fewer bone cells and disordered arrangements in the bone tissue,thinner bone trabeculae with large fractures,and an expanded marrow cavity;compared with model group,the rats in positive drug group,medium dose of VAP group,and high dose of VAP group had thicker bone trabeculae arranged more tightly.The Western blotting results showed that compared with control group,the expression levels of SIRT1,CAT,RUNX2,and FOXO1 proteins in bone tissue of the rats in model group were decreased(P<0.05);compared with model group,the expression levels of SIRT1,CAT,RUNX2,and FOXO1 proteins in bone tissue of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were significantly increased(P<0.05 or P<0.01).Conclusion:VAP has the protective effect against OP in the rats,and its mechanism may be related to mediating the antioxidant stress action through the SIRT1/FOXO1 signaling pathway.

Objective To explore the eradication rate of human papillomavirus(HPV)and gestational outcome of patients with high-grade squamous intraepithelial disease of the cervix(HSIL)after loop electrosurgical excision procedure(LEEP)by transvaginal dissection of the vesicorectal form the cervix.Methods A total of 53 patients treated with LEEP by transvaginal dissection of the vesicorectal form the cervix in Obstetrics and Gynecology Hospital,Fudan University from Jan to Dec,2019 were investigated.Clinical information of cervical cytological examination,HPV test and cervical biopsy under colposcopy were followed up for 6,12 and 24 months post-LEEP were collected.HPV infection in these 53 patients were compared before and after LEEP surgery.The rate of successful fertility of the cohort,the HPV conversion rate of patients with hysterectomy and LEEP done were compared.The association between the pathological type and positive surgical margin and the association between HPV infection type and positive surgical margin were analyzed.Results HPV infection rate of was 94.3%(50/53)and the proportion of HPV16 and/or 18 infection was 75.5%(40/53).Mono-HPV infection rate(69.8%,37/53)was significantly higher than mixed HPV infection rate(22.7%,13/53).Thirty-eight patients(71.7%)were found with positive surgical margin in previous LEEP operation.Fifteen patients had recurrence(28.3%)and 40 patients(75.5%)successfully delivered baby after surgery.Postoperative pathology was mainly HSIL,accounting for 66%(30/53),and 28.3%patients(15/53)had no pathological change.Forty cases had satisfying fertility-conservative operation outcome with negative surgical margin,and 38 patients eradicated HPV infection after LEEP,which took up 95%of patients with satisfying fertility-conservative operation.There was no significant difference of positive resection margin rate in between groups of HPV16/18 infection and other types.Five cases had successful delivery(12.5%,5/40)with 1 case of vaginal delivery and 4 cases of cesarean section.Among these 5 cases,3 cases undertook preventive cervical cerclage,with 1 case of vaginal delivery and 2 cases of cesarean sections.Conclusion HPV eradication rate and surgical outcome could be significantly improved by LEEP with transvaginal dissection of the vesicorectal from the cervix,which satisfied the fertility preservation of females at reproductive age.

Surgical operation is the main treatment of oral and maxillofacial tumors.Dysphagia is a common postoperative complication.Swal-lowing disorder can not only lead to mis-aspiration,malnutrition,aspiration pneumonia and other serious consequences,but also may cause psychological problems and social communication barriers,affecting the quality of life of the patients.At present,there is no systematic evalua-tion and rehabilitation management plan for the problem of swallowing disorder after oral and maxillofacial tumor surgery in China.Combining the characteristics of postoperative swallowing disorder in patients with oral and maxillofacial tumors,summarizing the clinical experience of ex-perts in the field of tumor and rehabilitation,reviewing and summarizing relevant literature at home and abroad,and through joint discussion and modification,a group of national experts reached this consensus including the core contents of the screening of swallowing disorders,the phased assessment of prognosis and complications,and the implementation plan of comprehensive management such as nutrition management,respiratory management,swallowing function recovery,psychology and nursing during rehabilitation treatment,in order to improve the evalua-tion and rehabilitation of swallowing disorder after oral and maxillofacial tumor surgery in clinic.