The lab module of exploratory experiment is newly designed in the practical course of bio-chemistry.Here we describe one of the experimental projects,and it originates from new scientific re-search results on the dynamic structure of ATP synthase.This exploratory experiment is organized in the form of real scientific research,which would fully mobilize the initiative and creativity of students in learning theoretical knowledge and experimental technology.Students work in groups and start with refer-ence reading.Through cooperation,they must develop certain experimental plan,handle samples with photocrosslinking technique and utilize the high-throughput electrophoresis method to analyze the dynamic structural change of ε subunit in ATP synthase under different physiological conditions.High quality re-sults from high-throughput electrophoresis can only be obtained through optimized operation and treat-ment,from which students would experience the process of technological innovation.The teaching process of this lab module embodies the student-centered teaching concept and is widely approved and supported by students.The project of ATP synthase closely combines the content of lab course with cut-ting-edge technology.Students can deeply experience the importance of experimental technology innova-tion in solving scientific problems.The practical ability of students would be comprehensively improved through this lab module.

Objective To explore the role of plasma exosome microRNA-622(miR-622)in cachexia of hep-atocellular carcinoma(HCC).Methods Select preoperative blood samples from 40 patients with stage Ⅲ-ⅣHCC and 33 patients with non HCC liver benign diseases collected at Huashan Hospital,Fudan University from 2021 to 2024 as the research subjects.Exosomes were isolated from the plasma of patients,human HCC cell lines(HepG2,Hep3B,PLC,Huh7),and the culture supernatants of primary human hepatocytes(PHH)by differential centrifugation.The relative expression level of miR-622 was detected by real-time fluorescence quantitative polymerase chain reaction.The level of lipolysis was assessed by determining the concentration of glycerol and fatty acids in adipocyte culture medium.Results Plasma exosome miR-622 levels in HCC pa-tients were positively correlated with subcutaneous adipose index and visceral adipose index(r=0.516,0.539,P＜0.05).HCC cell conditional medium was able to significantly increase the lipolysis level of adipo-cytes.The relative expression levels of miR-622 in exosomes from HCC cells(HepG2,Hep3B,PLC,Huh7)were significantly lower than those in exosomes from PHH,with values of 0.13±0.04,0.28±0.08,0.23±0.07,and 0.24±0.04,respectively,compared to 1.00±0.18 in PHH exosomes.Further studies revealed that plasma exosome of HCC patients treatment was able to lead to a decrease in the relative expression level of miR-622 in adipocytes,as well as an enhancement of lipolysis.Conclusion The expression of plasma exosomal miR-622 is downregulated in HCC patients.HCC cells can deliver miR-622 to adipocytes via exosomes to reg-ulate lipolysis.Plasma exosomal miR-622 may serve as a potential biomarker for predicting HCC cachexia and a therapeutic target.

OBJECTIVE: To investigate the anti-inflammatory effect of rutaecarpine (RUT) on monosodium urate crystal (MSU)-induced murine peritonitis in mice and further explored the underlying mechanism of RUT in lipopolysaccharide (LPS)/MSU-induced gout model in vitro. METHODS: In MSU-induced mice, 36 male C57BL/6 mice were randomly divided into 6 groups of 8 mice each group, including the control group, model group, RUT low-, medium-, and high-doses groups, and prednisone acetate group. The mice in each group were orally administered the corresponding drugs or vehicle once a day for 7 consecutive days. The gout inflammation model was established by intraperitoneal injection of MSU to evaluate the anti-gout inflammatory effects of RUT. Then the proinflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the proportions of infiltrating neutrophils cytokines were detected by flow cytometry. In LPS/MSU-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and proinflammatory cytokines were measured by ELISA. The percentage of pyroptotic cells were detected by flow cytometry. Respectively, the mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, the nuclear translocation of nuclear factor κB (NF-κB) p65 was observed by laser confocal imaging. Additionally, surface plasmon resonance (SPR) and molecular docking were applied to validate the binding ability of RUT components to tumor necrosis factor α (TNF-α) targets. RESULTS: RUT reduced the levels of infiltrating neutrophils and monocytes and decreased the levels of the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 6 (IL-6, all P<0.01). In vitro, RUT reduced the production of IL-1β, IL-6 and TNF-α. In addition, RT-PCR revealed the inhibitory effects of RUT on the mRNA levels of IL-1β, IL-6, cyclooxygenase-2 and TNF-α (P<0.05 or P<0.01). Mechanistically, RUT markedly reduced protein expressions of tumor necrosis factor receptor (TNFR), phospho-mitogen-activated protein kinase (p-MAPK), phospho-extracellular signal-regulated kinase, phospho-c-Jun N-terminal kinase, phospho-NF-κB, phospho-kinase α/β, NOD-like receptor thermal protein domain associated protein 3 (NLRPS), cleaved-cysteinyl aspartate specific proteinase-1 and cleaved-gasdermin D in macrophages (P<0.05 or P<0.01). Molecularly, SPR revealed that RUT bound to TNF-α with a calculated equilibrium dissociation constant of 31.7 µmol/L. Molecular docking further confirmed that RUT could interact directly with the TNF-α protein via hydrogen bonding, van der Waals interactions, and carbon-hydrogen bonding. CONCLUSION RUT alleviated MSU-induced peritonitis and inhibited the TNFR1-MAPK/NF-κB and NLRP3 inflammasome signaling pathway to attenuate gouty inflammation induced by LPS/MSU in THP-1 macrophages, suggesting that RUT could be a potential therapeutic candidate for gout.
Animals ; NF-kappa B/metabolism* ; Male ; Indole Alkaloids/therapeutic use* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Inflammation/complications* ; Uric Acid ; Quinazolines/therapeutic use* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Humans ; Gout/chemically induced* ; Inflammasomes/metabolism* ; Cytokines/metabolism* ; THP-1 Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Mice ; Molecular Docking Simulation ; Lipopolysaccharides ; Quinazolinones

Sjögren's syndrome (SS) is an autoimmune disease characterized primarily by oral and periocular dryness. Astragalus-Salvia (AS) and Ophiopogon-Dendrobium (OD) represent two frequently utilized herb pairs in SS treatment. While the combination of AS-OD herb pairs demonstrates clinical efficacy in alleviating SS symptoms, its underlying mechanism remains unclear. This investigation sought to assess the therapeutic effects and elucidate the potential mechanisms of AS-OD in non-obese diabetic (NOD)/Ltj mice with SS. The study utilized NOD/Ltj mice as SS models, administering AS-OD treatment for 10 weeks at doses of 113.1, 226.2, and 339.3 mg·d^-1·20 g^-1. Results demonstrated that AS-OD improved SS symptoms, evidenced by enhanced salivary flow rate, decreased anti-SSA/Ro and anti-SSB/La antibody levels, increased swimming duration, and reduced lactate (LA) and blood urea nitrogen (BUN) levels in NOD/Ltj mice. AS-OD reduced lymphocyte infiltration, enhanced Aquaporin-5 (AQP5) expression in the submandibular gland, decreased inflammatory cytokine levels in the submandibular gland, and reduced the T helper type 17/regulatory T lymphocyte (Th17/Treg) cell ratio in the spleen. Transcriptomic and proteomic analyses indicated AS-OD's involvement in regulating phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and Janus kinase 3/signal transducer and activator of transcription 3 (JAK1/STAT3) pathways, with inhibitory effects validated in both NOD/Ltj mice submandibular gland and A-253 cells. Furthermore, AS-OD enhanced cell viability and reduced A-253 cell apoptosis through the PI3K/AKT pathway. In A-253 cells, AS-OD reduced inflammatory cytokine levels, CXC chemokine ligand 9/10 (CXCL9/10), and T-cell chemotaxis by inhibiting the JAK1/STAT3 pathway. AS-OD mitigates SS by suppressing inflammation and immune responses through the PI3K/AKT and JAK1/STAT3 pathways.
Animals ; STAT3 Transcription Factor/genetics* ; Sjogren's Syndrome/immunology* ; Mice, Inbred NOD ; Proto-Oncogene Proteins c-akt/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Janus Kinase 1/genetics* ; Humans ; Female ; Astragalus Plant/chemistry* ; Male

This study aims to investigate the mechanism by which resveratrol(RES) alleviates cerebral vascular endothelial damage in sepsis-associated encephalopathy(SAE) through network pharmacology and animal experiments. By using network pharmacology, the study identified common targets and genes associated with RES and SAE and constructed a protein-protein interaction( PPI) network. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were performed to pinpoint key signaling pathways, followed by molecular docking validation. In the animal experiments, a cecum ligation and puncture(CLP) method was employed to induce SAE in mice. The mice were randomly assigned to the sham group, CLP group, and medium-dose and high-dose groups of RES. The sham group underwent open surgery without CLP, and the CLP group received an intraperitoneal injection of 0. 9% sodium chloride solution after surgery. The medium-dose and high-dose groups of RES were injected intraperitoneally with 40 mg·kg-1 and 60 mg·kg~(-1) of RES after modeling, respectively, and samples were collected 12 hours later. Neurological function scores were assessed, and the wet-dry weight ratio of brain tissue was detected. Serum superoxide dismutase(SOD), catalase( CAT) activity, and malondialdehyde( MDA) content were measured by oxidative stress kit. Histopathological changes in brain tissue were examined using hematoxylin-eosin(HE) staining. Transmission electron microscopy was employed to evaluate tight cell junctions and mitochondrial ultrastructure changes in cerebral vascular endothelium. Western blot analysis was performed to detect the expression of zonula occludens1( ZO-1), occludin, claudins-5, optic atrophy 1( OPA1), mitofusin 2(Mfn2), dynamin-related protein 1(Drp1), fission 1(Fis1), and hypoxia-inducible factor-1α(HIF-1α). Network pharmacology identified 76 intersecting targets for RES and SAE, with the top five core targets being EGFR, PTGS2, ESR1, HIF-1α, and APP. GO enrichment analysis showed that RES participated in the SAE mechanism through oxidative stress reaction. KEGG enrichment analysis indicated that RES participated in SAE therapy through HIF-1α, Rap1, and other signaling pathways. Molecular docking results showed favorable docking activity between RES and key targets such as HIF-1α. Animal experiment results demonstrated that compared to the sham group, the CLP group exhibited reduced nervous reflexes, decreased water content in brain tissue, as well as serum SOD and CAT activity, and increased MDA content. In addition, the CLP group exhibited disrupted tight junctions in cerebral vascular endothelium and abnormal mitochondrial morphology. The protein expression levels of Drp1, Fis1, and HIF-1α in brain tissue were increased, while those of ZO-1, occludin, claudin-5, Mfn2, and OPA1 were decreased. In contrast, the medium-dose and high-dose groups of RES showed improved neurological function, increased water content in brain tissue and SOD and CAT activity, and decreased MDA content. Cell morphology in brain tissue, tight junctions between endothelial cells, and mitochondrial structure were improved. The protein expressions of Drp1, Fis1, and HIF-1α were decreased, while those of ZO-1, occludin, claudin-5, Mfn2, and OPA1 were increased. This study suggested that RES could ameliorate cerebrovascular endothelial barrier function and maintain mitochondrial homeostasis by inhibiting oxidative stress after SAE damage, potentially through modulation of the HIF-1α signaling pathway.
Animals ; Mice ; Network Pharmacology ; Resveratrol/administration & dosage* ; Male ; Sepsis-Associated Encephalopathy/genetics* ; Signal Transduction/drug effects* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Endothelium, Vascular/metabolism* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Humans ; Sepsis/complications* ; Oxidative Stress/drug effects*

Objective To observe the impact of brain-computer interface(BCI)robot combined with task-oriented training(TOT)on hand function and activities of daily living in stroke patients.Methods A total of 40 patients with subacute stroke who were hospitalized in the Department of Rehabilitation Medicine of Anhui No.2 Province People's Hospital from December 2022 to December 2024 were prospectively and consecutively included in this study.The stroke patients were randomly assigned to the experimental group and the control group using the random number table method,with 20 cases in each group.Baseline data were collected from all patients,including gender,age,personal history(smoking,drinking),past medical history(hypertension,diabetes),stroke type(hemorrhagic stroke,ischemic stroke),hemiplegia side(left,right),disease course,stroke location(basal ganglia,internal capsule),and admission assessment indicators(including kinesthetic and visual imagery questionnaire[KVIQ-20]score,mini-mental state examination[MMSE]score and National Institutes of Health stroke scale[NIHSS]score).Both groups of patients received conventional rehabilitation training and TOT.The experimental group then underwent BCI robot training combined with TOT on this basis.Both groups received treatments for 4 weeks,and the upper limb and hand functions of all patients were evaluated using the Fugl-Meyer upper extremity function assessment scale(FMA-UE),the wrist-hand part of the FMA-UE scale(FMA-WH),the Wolf motor function test(WMFT),and the modified Ashworth scale(MAS)before and after the treatment;the hand function related part(feeding[10 score],bathing[5 score],dressing[10 score],grooming[5 score],and toilet using[10 score])of modified Barthel index(MBI)was used to assess the patients'hand dexterity to perform daily activities.Results(1)No statistically significant differences were found in the baseline data between the two groups of patients(all P＞0.05).(2)Before treatment,the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI in the experimental group were(18.75±7.38),(2.95±1.54),(26.90±8.69),(1.10±0.66),and(15.45±1.82)respectively,while those in the control group were(15.90±5.39),(2.25±1.12),(24.15±6.78),(1.25±0.60),and(15.65±3.12)respectively.There were no statistically significant differences in the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI between the two groups before treatment(all P＞0.05).After 4 weeks of treatment,the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI in the experimental group were(27.10±7.76),(5.75±2.97),(40.85±10.19),(0.73±0.57),and(21.15±2.66)respectively,while those in the control group were(21.25±5.29),(4.00±1.49),(31.85±7.60),(0.73±0.64),and(17.40±3.14)respectively.The time main effects(Ftime values were 925.061,138.138,624.635 and 405.986 respectively,all P＜0.01),group main effects(Fgroup values were 4.460,4.562,5.011 and 4.411 respectively,all P＜0.05),and the interaction effects of time and group(Ftime×group values were 44.358,7.356,52.506 and 114.128 respectively,all P＜0.05)of FMA-UE,FMA-WH,WMFT and hand function related score of MBI scores were all significant.The time main effect of MAS scores was significant(Ftime value was 59.478,P＜0.01),while the group main effect(Fgroup value was 0.162,P＞0.05),the interaction effects of time and group(Ftime×group value was 1.652,P＞0.05)were not significant.Conclusion The combined task-oriented training with BCI robots can improve the upper limb and hand functions of stroke patients,enhance their ability to perform daily activities,and the effect is superior to that of single task-oriented training.

Objective To observe the impact of brain-computer interface(BCI)robot combined with task-oriented training(TOT)on hand function and activities of daily living in stroke patients.Methods A total of 40 patients with subacute stroke who were hospitalized in the Department of Rehabilitation Medicine of Anhui No.2 Province People's Hospital from December 2022 to December 2024 were prospectively and consecutively included in this study.The stroke patients were randomly assigned to the experimental group and the control group using the random number table method,with 20 cases in each group.Baseline data were collected from all patients,including gender,age,personal history(smoking,drinking),past medical history(hypertension,diabetes),stroke type(hemorrhagic stroke,ischemic stroke),hemiplegia side(left,right),disease course,stroke location(basal ganglia,internal capsule),and admission assessment indicators(including kinesthetic and visual imagery questionnaire[KVIQ-20]score,mini-mental state examination[MMSE]score and National Institutes of Health stroke scale[NIHSS]score).Both groups of patients received conventional rehabilitation training and TOT.The experimental group then underwent BCI robot training combined with TOT on this basis.Both groups received treatments for 4 weeks,and the upper limb and hand functions of all patients were evaluated using the Fugl-Meyer upper extremity function assessment scale(FMA-UE),the wrist-hand part of the FMA-UE scale(FMA-WH),the Wolf motor function test(WMFT),and the modified Ashworth scale(MAS)before and after the treatment;the hand function related part(feeding[10 score],bathing[5 score],dressing[10 score],grooming[5 score],and toilet using[10 score])of modified Barthel index(MBI)was used to assess the patients'hand dexterity to perform daily activities.Results(1)No statistically significant differences were found in the baseline data between the two groups of patients(all P＞0.05).(2)Before treatment,the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI in the experimental group were(18.75±7.38),(2.95±1.54),(26.90±8.69),(1.10±0.66),and(15.45±1.82)respectively,while those in the control group were(15.90±5.39),(2.25±1.12),(24.15±6.78),(1.25±0.60),and(15.65±3.12)respectively.There were no statistically significant differences in the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI between the two groups before treatment(all P＞0.05).After 4 weeks of treatment,the scores of FMA-UE,FMA-WH,WMFT,MAS and hand function related score of MBI in the experimental group were(27.10±7.76),(5.75±2.97),(40.85±10.19),(0.73±0.57),and(21.15±2.66)respectively,while those in the control group were(21.25±5.29),(4.00±1.49),(31.85±7.60),(0.73±0.64),and(17.40±3.14)respectively.The time main effects(Ftime values were 925.061,138.138,624.635 and 405.986 respectively,all P＜0.01),group main effects(Fgroup values were 4.460,4.562,5.011 and 4.411 respectively,all P＜0.05),and the interaction effects of time and group(Ftime×group values were 44.358,7.356,52.506 and 114.128 respectively,all P＜0.05)of FMA-UE,FMA-WH,WMFT and hand function related score of MBI scores were all significant.The time main effect of MAS scores was significant(Ftime value was 59.478,P＜0.01),while the group main effect(Fgroup value was 0.162,P＞0.05),the interaction effects of time and group(Ftime×group value was 1.652,P＞0.05)were not significant.Conclusion The combined task-oriented training with BCI robots can improve the upper limb and hand functions of stroke patients,enhance their ability to perform daily activities,and the effect is superior to that of single task-oriented training.

The lab module of exploratory experiment is newly designed in the practical course of bio-chemistry.Here we describe one of the experimental projects,and it originates from new scientific re-search results on the dynamic structure of ATP synthase.This exploratory experiment is organized in the form of real scientific research,which would fully mobilize the initiative and creativity of students in learning theoretical knowledge and experimental technology.Students work in groups and start with refer-ence reading.Through cooperation,they must develop certain experimental plan,handle samples with photocrosslinking technique and utilize the high-throughput electrophoresis method to analyze the dynamic structural change of ε subunit in ATP synthase under different physiological conditions.High quality re-sults from high-throughput electrophoresis can only be obtained through optimized operation and treat-ment,from which students would experience the process of technological innovation.The teaching process of this lab module embodies the student-centered teaching concept and is widely approved and supported by students.The project of ATP synthase closely combines the content of lab course with cut-ting-edge technology.Students can deeply experience the importance of experimental technology innova-tion in solving scientific problems.The practical ability of students would be comprehensively improved through this lab module.

ObjectiveTo investigate the effect of Tangzhi pills on the improvement of insulin resistance （IR） in the liver with type 2 diabetes （T2DM） by regulating phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway based on differential genes and its possible molecular mechanism. MethodT2DM rat models were prepared by high fat （HFD） diet combined with streptozotocin （STZ） intraperitoneal injection. The experiment was divided into blank group， model group， metformin hydrochloride group （0.18 g·kg^-1）， Tangzhi pills high （1.08 g·kg^-1）， medium （0.54 g·kg^-1） and low （0.27 g·kg^-1） dose groups. Rat serum， liver， and pancreatic tissue were collected， and the pathological tissue of the liver and pancreas was observed using hematoxylin-eosin （HE） staining. The fasting blood glucose level （FBG） was detected， and oral glucose tolerance （OGTT） tests were conducted. Enzyme-linked immunosorbent assay （ELISA） was used to detect fasting serum insulin （FINS） and glycated hemoglobin （GHb） levels in rats. IR homeostasis model index （HOMA-IR）， β cellular homeostasis index （HOMA-β）， and insulin sensitivity index （ISI） were calculated. Biochemical methods were used to determine the levels of triglyceride （TG）， total cholesterol （TC）， low-density lipoprotein （LDL-C）， and high-density lipoprotein （HDL-C） in rat serum. Transcriptomics obtained differentially expressed mRNA from liver tissue and enriched differentially expressed pathways. Real-time reverse transcriptase polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of cyclic adenylate responsive element binding protein 3-like protein 2 antibody （CREB3l2）， B-lymphocyte tumor 2 （Bcl-2）， Toll-like receptor 2 （TLR2）， cyclin-dependent kinase inhibitor 1A （CDNK1A）， and DNA damage induced transcription factor 4-like protein （DDIT4） in liver tissue. Western blot was used to detect the protein expression of phosphorylated phosphatidylinositol 3-kinase （p-PI3K）， phosphorylated protein kinase B （p-Akt）， glucose transporter 4 （GLUT4）， insulin receptor （INSR）， and insulin receptor substrate 2 （IRS2）. ResultThe pharmacodynamic experiment results showed that compared with model group， Tangzhi pills groups repaired liver and pancreatic tissue to varying degrees， reduced blood sugar （P<0.01）， and promoted a decrease in serum FINS， GHb， and HOMA-IR （P<0.05， P<0.01）. In addition， HOMA-β and ISI increased （P<0.05， P<0.01）. The levels of TC， TG， and LDL-C decreased （P<0.05， P<0.01）， while the levels of HDL-C increased （P<0.05， P<0.01）. The transcriptomics experimental results confirmed that the PI3K/Akt signaling pathway was significantly expressed in both the blank group and model group， as well as in the high-dose Tangzhi pills group and model group. CDNK1A， DDIT4， CREB3l2， Bcl-2， and TLR2 were significantly differentially expressed mRNA during TG intervention in T2DM. Compared with the model group， the protein expression of p-PI3K， p-Akt， GLUT4， INSR， and IRS2 increased in all Tangzhi pills groups （P<0.01）. The mRNA expression of CREB3l2， Bcl-2， and TLR2 increased （P<0.01）， while that of CDNK1A and DDIT4 decreased （P<0.01）. ConclusionTangzhi pills may regulate the PI3K/Akt signaling pathway based on the differential mRNA expression of CREB3l2， Bcl-2， TLR2， CDNK1A， and DDIT4， thereby improving IR in the liver with T2DM.

Diabetic nephropathy(DN)is one of the most serious microangiopathies in diabetes mellitus and the leading cause of death in patients with end-stage renal disease.Ferroptosis,as a mode of programmed cell death,is mainly manifested by excessive accumulation of intracellular lipid peroxides and iron.Ferroptosis is involved in a series of pathological processes such as damage to DN renal podocytes,mesangial cells,and renal tubular epithelial cells.Chinese materia medica has the characteristics of significant therapeutic effects and minimal adverse reactions in the treatment of diseases,and has been widely used in the prevention and treatment of DN.This article summarized the key factors regulating ferroptosis in DN,as well as the active components and TCM formulas targeting the inhibition of ferroptosis in the prevention and treatment of DN,providing reference for the development of DN targeted drugs.