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 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.

Objective To investigate whether the mechanism of action of electro-acupuncture in MPTP-induced Parkinson's disease(PD)model mice is related to neuroinflammation mediated by the forkhead box protein O1(FoXO1)/NOD-like receptor heat protein structural domain-related protein 3(NLRP3)signaling pathway.Methods Thirty mice were randomly divided into control(Ctrl),model(MPTP),and electro-acupuncture(EA)groups,with 10 mice in each group.PD mouse model was prepared by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)30 mg/(kg·d)for 7 d consecutively.Behavioral changes of mice were observed by open field and pole-climbing experiments;α-synuclein(α-syn)expression in the substantia nigra of the midbrain was observed by immunohistochemistry;FoXO1,NLRP3,ASC,and Caspase-1 were deter-mined by Western blot;and the expression of inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)was detected by ELISA in the substantia nigra of the midbrain.Results Mice in the MPTP group showed significant behavioral deficits,with a significant increase in midbrain substantia nigra α-syn(P＜0.01),elevated levels of Iba-1,FoXO1,NLRP3,ASC,and Caspase-1 proteins(P＜0.05 and P＜0.01,respectively),and a significant increase in the levels of TNF-α,and IL-1β(P＜0.01).The treatment of EA was effective in improve the dyskinesia of PD mice,improve motor balance and coordination(P＜0.01),reduce the abnormal aggregation of α-syn(P＜0.01),reduce the abnormal activation of microglia,and alleviate neuroinflammation(P＜0.05).Conclusion Electro-acupuncture may play a protective role in PD by modulating FoXO1/NLRP3 pathway-mediated neuroinflammation.

To construct a single-cell transcriptomic map of testicular tissue under hypobaric hypoxia exposure and perform diversity analysis of supportive cells, aiming to provide new insights into the mechanisms of reproductive toxicity for future research.

Objective To investigate the effects of electroacupuncture on the intestinal NIMA-related kinase 7(NEK7)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway in mice with Parkinson's disease.Methods According to the randomized number table method,36 C57BL/6 mice were randomly divided into the control group,the model group,and the electroacupuncture group,with 12 mice per group.The Parkinson's disease mouse model was established by gavage of rotenone solution(10 mg/kg)for 28 d.After molding,the electroacupuncture group was stimulated with"Fengfu"(DU17),"Taichong"(LR3),and"Zusanli"(ST36)for 14 d,while the control group and the model group were only treated with immobilization.The motor ability of mice was detected by pole climbing test and hindlimb rating score,the positive expressions of nigral tyrosine hydroxylase(TH)and colon occludin were detected by immunohistochemistry,the histological morphology of colon was observed by hematoxylin-eosin staining,and Western blotting was used to detect the protein expressions of NEK7,NLRP3,Caspase-1,and interleukin-1β(IL-1β).Results Compared with the control group,mice in the model group had lower score on the pole climbing test and a higher hindlimb rating score(P＜0.01);the average optical densities of nigral TH and colon occludin were decreased(P＜0.01);significant inflammatory infiltration was observed in the colonic tissue,and the muscularis propria was thinned;and the protein expressions of NEK7,NLRP3,Caspase-1,and IL-1β in the colonic tissue were elevated(P＜0.01).Compared with the model group,mice in the electroacupuncture group had higher score on the pole climbing test and a lower hindlimb rating score(P＜0.01);the average optical densities of nigral TH and colon occludin were increased(P＜0.01);the degree of inflammatory infiltration of colonic tissues decreased,and the muscularis propria was thickened;and the protein expressions of NEK7,NLRP3,Caspase-1,and IL-1β of colonic tissues were decreased(P＜0.01).Conclusion Electroacupuncture at"Fengfu"(DU17),"Taichong"(LR3),and"Zusanli"(ST36)can improve motor functional impairments in mice with Parkinson's disease,and the mechanism may be through the inhibition of intestinal NEK7/NLRP3 pathway,improving the intestinal barrier damage,relieving the intestinal inflammation,and improving the dopaminergic neuron injury.

ObjectiveThe absorption of substances into blood is mainly dependent on the mesenteric lymphatic pathway and the portal venous pathway. The substances transported via the portal venous pathway can be metabolized by the biotransformation in the liver. On the contrary, the substances in the mesenteric lymph fluid enter the blood circulation without biotransformation and can affect the body directly. Alcohol consumption is strongly linked to global health risk. Previous reports have analyzed the changes of metabolites in plasma, serum, urine, liver and feces after alcohol consumption. Whether alcohol consumption affects the metabolites in lymph fluid is still unknown. Therefore, it is particularly important to explore the changes of substances transported via the mesenteric lymphatic pathway and analyze their harmfulness after alcohol drinking. MethodsIn this study, male Wistar rats were divided into high, medium, and low-dosage alcohol groups (receiving Chinese Baijiu at 56%, 28% and 5.6% ABV, respectively) and water groups. The experiment was conducted by alcohol gavage lasting 10 d, 10 ml·kg^-1·d^-1. Then mesenteric lymph fluid was collected for non-targeted metabolomic analysis by using liquid chromatography-mass spectrometry (LC-MS) and bioinformatic analysis. Principal component analysis and hierarchical clustering were performed by using Biodeep. Meanwhile, KEGG enrichment analysis of the differential metabolites was also performed by Biodeep. MetaboAnalyst was used to analyze the relationship between the differential metabolites and diseases. ResultsThe metabolites in the mesenteric lymph fluid of the high-dosage alcohol group change the most. Based on the KEGG enrichment analysis, the pathways of differential metabolites between the high-dosage alcohol group and the control group are mainly enriched in the central carbon metabolism in cancer, bile secretion, linoleic acid metabolism, biosynthesis of unsaturated fatty acids, etc. Interestingly, in the biosynthesis of unsaturated fatty acids category, the content of arachidonic acid is increased by 7.25 times, whereas the contents of palmitic acid, oleic acid, stearic acid, arachidic acid and erucic acid all decrease, indicating lipid substances in lymph fluid are absorbed selectively after alcohol intake. It’s worth noting that arachidonic acid is closely related to inflammatory response. Furthermore, the differential metabolites are mainly related with schizophrenia, Alzheimer’s disease and lung cancer. The differential metabolites between the medium-dosage alcohol and the control group were mainly enriched in phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, linoleic acid metabolism and cholesterol metabolism. The differential metabolites are mainly related to schizophrenia, Alzheimer’s disease, lung cancer and Parkinson’s disease. As the dose of alcohol increases, the contents of some metabolites in lymph fluid increase, including cholesterol, L-leucine, fumaric acid and mannitol, and the number of metabolites related to schizophrenia also tends to increase, indicatingthat some metabolites absorbed by the intestine-lymphatic pathway are dose-dependent on alcohol intake. ConclusionAfter alcohol intake, the metabolites transported via the intestinal-lymphatic pathway are significantly changed, especially in the high-dosage group. Some metabolites absorbed via the intestinal-lymphatic pathway are dose-dependent on alcohol intake. Most importantly, alcohol intake may cause inflammatory response and the occurrence of neurological diseases, psychiatric diseases and cancer diseases. High-dosage drinking may aggravate or accelerate the occurrence of related diseases. These results provide new insights into the pathogenesis of alcohol-related diseases based on the intestinal-lymphatic pathway.

OBJECTIVE To study the fingerprint of Denghong buyang huanwu granules （DBHG）， screen the quality markers， and establish the method for content determination of active ingredients. METHODS HPLC method was adopted. The fingerprints of 10 batches of DBHG （S1-S10） were established by using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）， and similarity evaluation was also performed. Traditional Chinese medicine pieces attribution analysis， cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were conducted for common peaks， and quality biomarkers were screened based on variable importance projection （VIP） values＞1. The contents of 10 batches of samples were determined by the same HPLC method， such as salidroside， tyrosol， calycosin-7-O-beta-D-glucoside， scutellarin and calycosin. RESULTS A total of 25 common peaks were obtained in the fingerprints for 10 batches of samples and 6 common peaks were identified， i.e. salidroside， tyrosol， paeoniflorin， calycosin-7-O-beta-D-glucoside， scutellarin， calycosin. Their similarities were greater than 0.9， and 10 common peaks of them were unique components of Erigeron breviscapus. DBHG could be clustered into 2 categories by using CA and PCA； S4-S5， and S7 could be clustered into one category and other samples could be clustered into one category. The corresponding components of peaks 16 （scutellarin）， 12， 15 （calycosin-7-O-beta-D-glucoside）， 13 （paeoniflorin）， and 14 were quality markers. The average contents of salidroside， tyrosol， calycosin-7-O-beta-D-glucoside， scutellarin and calycosin were 1.64， 0.45， 0.31， 0.73， 0.15 mg/g in 10 batches of samples. CONCLUSIONS HPLC fingerprint for DBHG and a method for determining the contents of five active ingredients including salidroside are successfully established. Five quality markers have been screened. It can be used for the quality control of the preparation.

ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.

Objective To explore the effects of electroacupuncture on motor function in Parkinson's disease(PD)model mice and NLRP3 inflammasome-related proteins in the midbrain substantia nigra(SN).Methods C57BL/6 mice were assigned to three groups according to the random number table method:control group,model group,and electroacupuncture(EA)group,12 mice per group.The PD model was reproduced by intragastric administration of rotenone solution 10 mg/(kg·d).EA group was administered at the three selected points,"Fengfu"(GV16),"Taichong"(LR3),and"Zusanli"(ST36),with a treatment cycle of 2 weeks.The control and model groups took the same time synchronous fixation operation for the control variable.Behavioral scores and open field tests were used to detect the exercise ability of mice in each group.Tyrosine hydroxylase(TH)and α-synuclein(α-syn)in the midbrain SN of mice in all groups were measured with an immunohistochemistry test.NLRP3 and cysteinyl aspartate specific proteinase-1(Caspase-1)protein expression levels in the midbrain SN of mice in the three groups were measured using Western blotting,and interleukin-1β(IL-1β)content was determined with an enzyme-linked immunosorbent assay.Results Compared to the control group,the behavioral scores of the mice in the model group were higher(P<0.01).Compared to the model group,the behavioral scores of the mice in the EA group were lower(P<0.01).Compared to the control group,the time ratio of the relative rest state of the mice in the model group(<100 mm/s)increased significantly(P<0.01),while the time ratio of the slow motion(100～200 mm/s)and time ratio of the fast motion(>200 mm/s)state decreased significantly(P<0.01).Compared to the model group,the time ratio spent in the relative rest state of mice in the EA group decreased significantly(P<0.01),while the time ratio of the slow motion state and time ratio of the fast motion state and movement rate increased significantly(P<0.01).Compared to the control group,the TH expression level decreased in the SN in the model group(P<0.01),while α-syn increased(P<0.01).Compared to the model group,the TH expression level in the EA group increased(P<0.05),while α-syn decreased(P<0.05).Compared to the control group,the protein expressions of NLRP3 and Caspase-1 in the SN of the model group increased(P<0.01);compared to the model group,the expressions of NLRP3 and Caspase-1 in the SN of the midbrain of mice decreased after EA treatment(P<0.01).Compared to the control group,IL-1β in the SN of the mouse midbrain increased in the model group(P<0.01).Compared to the model group,IL-1β decreased in the EA group(P<0.05).Conclusion This experiment shows that stimulation of EA in"Fengfu","Taichong",and"Zusanli"can effectively reduce abnormal aggregation of the PD marker α-syn,increase TH expression,and enhance the motor dysfunction of PD model mice.The molecular mechanism is related to the regulation of the expression of NLRP3,Caspase-1,and IL-1β of inflammasome-related pathways.

Objective To investigate the effects of electroacupuncture on the intestinal NIMA-related kinase 7(NEK7)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway in mice with Parkinson's disease.Methods According to the randomized number table method,36 C57BL/6 mice were randomly divided into the control group,the model group,and the electroacupuncture group,with 12 mice per group.The Parkinson's disease mouse model was established by gavage of rotenone solution(10 mg/kg)for 28 d.After molding,the electroacupuncture group was stimulated with"Fengfu"(DU17),"Taichong"(LR3),and"Zusanli"(ST36)for 14 d,while the control group and the model group were only treated with immobilization.The motor ability of mice was detected by pole climbing test and hindlimb rating score,the positive expressions of nigral tyrosine hydroxylase(TH)and colon occludin were detected by immunohistochemistry,the histological morphology of colon was observed by hematoxylin-eosin staining,and Western blotting was used to detect the protein expressions of NEK7,NLRP3,Caspase-1,and interleukin-1β(IL-1β).Results Compared with the control group,mice in the model group had lower score on the pole climbing test and a higher hindlimb rating score(P＜0.01);the average optical densities of nigral TH and colon occludin were decreased(P＜0.01);significant inflammatory infiltration was observed in the colonic tissue,and the muscularis propria was thinned;and the protein expressions of NEK7,NLRP3,Caspase-1,and IL-1β in the colonic tissue were elevated(P＜0.01).Compared with the model group,mice in the electroacupuncture group had higher score on the pole climbing test and a lower hindlimb rating score(P＜0.01);the average optical densities of nigral TH and colon occludin were increased(P＜0.01);the degree of inflammatory infiltration of colonic tissues decreased,and the muscularis propria was thickened;and the protein expressions of NEK7,NLRP3,Caspase-1,and IL-1β of colonic tissues were decreased(P＜0.01).Conclusion Electroacupuncture at"Fengfu"(DU17),"Taichong"(LR3),and"Zusanli"(ST36)can improve motor functional impairments in mice with Parkinson's disease,and the mechanism may be through the inhibition of intestinal NEK7/NLRP3 pathway,improving the intestinal barrier damage,relieving the intestinal inflammation,and improving the dopaminergic neuron injury.