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

BACKGROUND:Spinal cord injury is a neurological disorder caused by traumatic or non-traumatic events,often leading to severe functional impairment below the injured segment.In recent years,neural stem cell transplantation has been considered to have significant therapeutic potential in regulating the inflammatory response after spinal cord injury,inhibiting excessive proliferation of glial scars,and promoting nerve regeneration. OBJECTIVE:To review and discuss the potential mechanism of action of acupuncture and neural stem cell transplantation therapy in inhibiting spinal cord injury-induced secondary injury,and to delve into the scientific basis for its treatment of spinal cord injury. METHODS:PubMed,Elsevier,WanFang,and CNKI databases were searched using"spinal cord injury,acupuncture,neural stem cells,SDF-1α/CXCR4 axis"as Chinese and English search terms.Totally 96 articles were finally included.The research findings of acupuncture combined with neural stem cells in the treatment of spinal cord injury were summarized and analyzed,and the mechanism of this combination therapy in the treatment of secondary injury after spinal cord injury was summarized. RESULTS AND CONCLUSION:(1)The stromal-derived factor 1α(SDF-1α)/chemokine receptor 4(CXCR4)axis plays a crucial role in neural stem cell transplantation for spinal cord injury.This signaling mechanism not only affects neural stem cell migration,proliferation,and differentiation,but is also a key factor in determining the efficiency of stem cell homing to the injury site.Therefore,the regulation of targeting this axis is of great significance in enhancing the therapeutic effect of spinal cord injury.(2)Acupuncture,as a traditional Chinese medicine therapy,shows unique advantages in the regulation of secondary injury in spinal cord injury.It can effectively reduce secondary injury after spinal cord injury by regulating inflammatory response,inhibiting apoptosis,improving microcirculation,reducing glial scar formation,and counteracting oxidative stress.(3)Acupuncture was also able to influence the expression and function of the SDF-1α/CXCR4 axis,thereby enhancing the homing and survival ability of neural stem cells and promoting nerve regeneration and functional recovery.(4)The therapy combining acupuncture and stem cell transplantation is an innovative treatment strategy for spinal cord injury and suitable for repairing neural circuits.It combines the wisdom of traditional Chinese medicine with the advantages of modern biotechnology,providing a new treatment option for spinal cord injury patients.However,this combination therapy is still in the research and exploration stage,and its long-term efficacy and safety need to be further verified.(5)Taken together,acupuncture and neural stem cell transplantation for the treatment of spinal cord injury has great potential for clinical application,but in-depth research and optimization of treatment options are still needed.In the future,we look forward to further revealing the efficacy mechanism and optimal indications of this therapy through more clinical trials and mechanism studies,so as to bring better hope of recovery and more efficient therapeutic effects to spinal cord injury patients.

Objective To investigate the efficacy of the therapy of soothing liver and strengthening spleen(shortened as Shugan Jianpi therapy)in the treatment of active thyroid-associated ophthalmopathy(TAO)complicated with dry eye,and to provide a reference basis for clinical treatment.Methods A total of 108 patients with active TAO complicated with dry eye of liver depression and qi stagnation type were randomly divided into observation group and control group,54 patients in each group.Both groups were given conventional treatment for intervention of Graves'disease,and additionally the control group was given hormone shock therapy by intravenous injection of Methylprednisolone Sodium Succinate,and the observation group was treated with Chinese medicine prescription for soothing liver and strengthening spleen orally and intravenous injection of Methylprednisolone Sodium Succinate.The treatment period lasted for 12 weeks,and then the patients were followed up till to the 6th month.The changes of clinical activity score(CAS),proptosis,ocular surface disease index(OSDI),corneal fluorescein staining(FL),Schirmer I test(SIT)and tear film break-up time(BUT)in the two groups were observed before and after the treatment.After treatment,the clinical efficacy of the two groups was evaluated.Results(1)After 6 months of treatment,the total effective rate in the observation group was 94.44%(51/54)and that in the control group was 74.07%(40/54),and the intergroup comparison(tested by chi-square test)showed that the efficacy of the observation group was significantly superior to that of the control group(P＜0.05).(2)After treatment,the CAS,OSDI score and proptosis of the patients in the two groups were all lower than those before treatment(P＜0.01),and the reduction in the observation group was significantly superior to that in the control group(P＜0.01).(3)After treatment,the indicators of tear secretion function such as SIT,FL score and BUT of patients in the two groups were improved compared with those before treatment(P＜0.01),and the improvement in the observation group was significantly superior to that in the control group,the differences being all statistically significant(P＜0.01).Conclusion Shugan Jianpi therapy exerts certain clinical efficacy in treating patients with active TAO complicated with dry eye of liver depression and qi stagnation type,which can effectively relieve the proptosis,prolong the tear film break-up time,promote the secretion of tears and the repair of corneal epithelium,improve the visual function,and enhance the quality of life of the patients.

Cryoablation therapy with explicit anti-tumor mechanisms and histopathological manifestations has a long history.A large number of clinical practice has shown that cryoablation therapy is safe and effective,making it an ideal tumor treatment method in theory.Previously,its efficacy and clinical application were constrained by the limitations of refrigerants and refrigeration equipment.With the development of the new generation of cryoablation equipment represented by argon helium knives,significant progress has been made in refrigeration efficien-cy,ablation range,and precise temperature measurement,greatly promoting the progression of tumor cryoablation technology.This consensus systematically summarizes the mechanism of cryoablation technology,indications for oral mucosal melanoma(OMM)cryotherapy,clinical treatment process,adverse reactions and management,cryotherapy combination therapy,etc.,aiming to provide reference for carrying out the standardized cryoablation therapy of OMM.

BACKGROUND:Cell death and neuroinflammation are two important targets in the treatment of spinal cord injury.Pyroptosis is a programmed cell death closely related to neuroinflammation and targeted inhibition of pyroptosis after spinal cord injury is a promising therapeutic strategy. OBJECTIVE:To summarize the molecular mechanism,positive and negative regulatory factors and therapeutic strategies of pyroptosis in spinal cord injury. METHODS:The search terms were"spinal cord injury,pyroptosis,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),Caspase,Gasdermin D(GSDMD),IL-1β,IL-18"and 93 English literatures included in PubMed and Web of Science were finally selected for review. RESULTS AND CONCLUSION:As a newly discovered programmed cell death,pyroptosis has been shown to play an important role in the secondary injury stage after spinal cord injury.Among the regulatory factors of pyroptosis after spinal cord injury,CD73,NRF2,GDF-11,dopamine,FANCC and miR-423-5P could inhibit pyroptosis,while TLR4 and Aopps could promote pyroptosis.In terms of treatment,the active ingredients of traditional Chinese medicine(paeonol,tripterine,betulinic acid,piperine,kaempferol,and camptothecin),exosomes of various cell origins,and some drugs(metformin,topotecan,lithium,zinc,and carbon monoxide-releasing molecule 3)can effectively inhibit pyroptosis and reduce secondary spinal cord injury,but the toxicity and specific dose of these drugs need to be further studied.The specific molecular mechanism by which pyroptosis aggravates spinal cord injury is still poorly understood.The role of non-classical pathways and other inflammasomes is worth further exploration.At present,the research on pyroptosis after spinal cord injury only stays at the animal experiment stage.There are no related clinical studies and no approved targeted therapeutic drugs.(6)The application of pyroptosis after spinal cord injury has great potential,and its specific regulatory mechanism should be further studied in the future to provide a new target for the treatment of spinal cord injury.

Objective To investigate the effect and mechanism of Yiyuan moxibustion on urinary storage function in rats with neurogenic bladder(NB)after suprasacral spinal cord injury(SCI)based on adenosine activated protein kinase(AMPK)-connexin 43(Cx43)pathway.Methods Twelve female Sprague-Dawley rats were selected as the sham operation group using the random number table method,and 68 rats were used to prepare the suprasacral SCI model according to the modified Hassan Shaker spinal cord transection method.After the SCI model was stable,the standard NB model after SCI was screened out.The rats with successful modeling were divided into the model group(n=14),the Yiyuan moxibustion group(n=14),the inhibitor group(n=14),and the Yiyuan moxibustion+inhibitor group(n=14)according to the random number table method.The rats in the Yiyuan moxibustion group were treated with Yiyuan moxibustion.The rats in the inhibitor group were injected with AMPK inhibitor dorsomorphin(0.2 mg/kg)by tail vein.The rats in the Yiyuan moxibustion+inhibitor group were treated with Yiyuan moxibustion combined with tail vein injection of dorsomorphin(0.2 mg/kg).The sham operation group and the model group did not receive any intervention for 14 days.After the intervention,the bladder function of rats was evaluated by urodynamics.HE staining was used to observe morphological changes of the bladder tissue;the content of adenosine triphosphate(ATP)in bladder tissue was detected by colorimetry.The positive expressions of phosphorylated AMPK(p-AMPK),Cx43 and tyrosine protein kinase receptor(C-kit)in bladder tissue were detected by immunohistochemistry.The protein expressions of AMPK,p-AMPK,Cx43 and C-kit in bladder tissue were detected by Western blotting.Results Compared with the model group,the maximum bladder capacity and bladder compliance of the rats in the Yiyuan moxibustion group were increased,the leak point pressure was decreased,the cells in the Yiyuan moxibustion group were relatively regular and orderly,the vacuolar cells were reduced,and the tissue edema was reduced;the ATP content in the bladder tissue was decreased,the positive expression rate of p-AMPK was increased,the positive expression rate of Cx43 and C-kit were decreased,the protein expressions of p-AMPK protein was increased,the expression of Cx43 and C-kit were decreased,and the mRNA expression of Cx43 and C-kit were decreased(P<0.05).Compared with the Yiyuan moxibustion+inhibitor group,the maximum bladder capacity and bladder compliance of the rats in the Yiyuan moxibustion group increased,the leak point pressure decreased,the cells in the Yiyuan moxibustion group were arranged more closely and the tissue edema was reduced;the ATP content in the bladder tissue decreased,the positive expression rate of p-AMPK increased,the positive expression rates of Cx43 and C-kit decreased,the protein expression of p-AMPK increased,the protein expressions of Cx43 and C-kit decreased,and the mRNA expressions of Cx43 and C-kit decreased(P<0.05).Conclusion Yiyuan moxibustion can improve the urinary storage function of NB rats after suprasacral SCI,and its mechanism may be related to activating the AMPK-Cx43 pathway in bladder tissue,reducing the excitation transmission between bladder detrusor cells,thereby reducing the frequency of smooth muscle contraction.

Objective To investigate the protective effect of Schisandrin B on myocardial infarction in rats and its mechanism.Methods Fifty SD rats were randomly divided into sham group,model group,positive control group(10 mg·kg-1 captopril),experimental-L group(30 mg·kg-1 schisandrin B)and experimental-H(60 mg·kg-1 schisandrin B)group,with 10 rats in each group.Cardiac function related indexes were detected,serum myocardial injury markers and serum inflammatory factors were detected by the kit,myocardial apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)method,and myocardial tissue related protein expression levels were detected by Western blot method.Results Left ventricular ejection fraction(LVEF)levels in sham group,model group,positive control group,experimental-L group and experimental-H group were(78.42±4.32)％,(41.65±2.94)％,(59.76±5.35)％,(49.13±3.92)％and(67.04±3.00)％;the contents of creatine kinase-MB(CK-MB)were(33.95±2.68),(100.51±3.92),(48.27±3.70),(70.34±2.93)and(49.13±3.67)U·mL-1;interleukin-1 β(IL-1 β)levels were(1.02±0.12),(3.02±0.20),(1.61±0.13),(2.33±0.26)and(1.34±0.14)ng·mL-1;TUNEL positive cell rates were(3.47±0.82)％,(31.79±3.68)％,(11.22±1.02)％,(19.74±1.42)％and(14.38±1.13)％;the expression levels of Nod-like receptor thermal protein domain associated protein 3(NLRP3)protein were 0.35±0.07,1.04±0.10,0.55±0.05,0.85±0.07 and 0.39±0.06;the protein levels of cysteine aspartic acid specific protease 1(caspase-1)were 0.34±0.05,1.05±0.10,0.50±0.06,0.72±0.05 and 0.46±0.03,respectively.The above indexes were compared between model group and sham group,the differences were statistically significant(all P＜0.05).The positive control group,experimental-L group and experimental-H group were compared with model group,the differences were statistically significant(all P＜0.05).The difference between experimental-H group and experimental-L group were statistically significant(all P＜0.05).Conclusion Schisandrin B can mediate inflammatory response and cardiomyocyte apoptosis,improve cardiac function,and protect rats with myocardial infarction,which is related to NLRP3 inflammatory pathway.

Objective To investigate the effect of Ganoderma lucidum polysaccharide peptide(GLPP)on proliferation,migration and apoptosis of diffuse large B cell lymphoma(DLBCL)cells and its mechanism.Methods OCI-LY19 cells were divided into six groups:control,GLPP,si-NC,si-protein arginine methyltransferase 6(PRMT6),GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups.The si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were transfected with si-NC,si-PRMT6,pcDNA3.1-NC and pcDNA3.1-PRMT6,respectively.After the transfection was completed,control,si-NC and si-PRMT6 groups were treated with RPMI-1640 medium,while the GLPP,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were cultured with RPMI-1640 medium containing with 20 μg·mL-1 GLPP.After administration 24 h,the cell proliferation inhibition rates,mobility rates and apoptosis rates were detected.The expression levels of PRMT6 protein were measured by Western blotting.Results The cell proliferation inhibition rates of si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups were(1.28±0.16)％,(38.61±3.29)％,(52.84±7.74)％and(22.75±3.87)％,respectively.The number of cell migrations in the control,GLPP,si-NC,si-PRMT6,GLPP+pcDNA3.1-NC and GLPP+pcDNA3.1-PRMT6 groups was(252.65±24.65),(136.54±16.46),(231.65±21.24),(142.76±15.34),(140.23±9.84)and(192.38±23.38)cells;the apoptosis rates were(4.36±0.52)％,(28.24±2.36)％,(4.23±0.45)％,(24.54±2.27)％,(28.42±3.85)％and(14.25±2.13)％);the expression levels of PRMT6 protein were 1.82±0.21,0.56±0.05,1.78±0.19,0.54±0.05,0.29±0.02 and 0.32±0.03,respectively.The differences of above indexes were statistically significant between control group and GLPP group,between si-NC group and si-PRMT6 group,between GLPP+pcDNA3.1-NC group and GLPP+pcDNA3.1-PRMT6 group(all P＜0.05).Conclusion GLPP could inhibit proliferation,migration and promote apoptosis of DLBCL cells by down-regulating PRMT6 expression.

Objective To study the common basis and association between sickness behaviour and occurrence of classical symptoms of kidney yang deficiency of rats with adriamycin nephropathy.Methods The SD rats were given adriamycin by tail-vein injection for 2 times(4.0 and 3.5 mg·kg-1,one week apart)to construct the model of nephrotic syndrome with Chinese medicine symptom of kidney yang deficiency.After successful modeling,the model rats were randomly divided into adriamycin group(ADR group),corticosterone group(CORT group)and hydrocortisone group(HYD group),with 12 rats per group;another 12 normal rats were taken as normal group.In the HYD group,25 mg·kg-1·d-1 HYD was administered for 14 d to establish kidney yang deficiency model with simple hypothalamus-pituitary-adrenal cortex(HPA)axis inhibition.CORT group was adding 25 μg·mL-1 corticosterone to the water for 6 weeks,and the others drinking water supplied.The levels of urinary 17-hydroxy steroid were measured by enzyme linked immunosorbent assay.Glucocorticoid receptor(GR)and nuclear factor-kappa B(NF-κB)protein expression levels in kidney and hypothalamus were detected by Western blotting.Results In the normal,ADR,CORT and HYD groups,the urinary 17-hydroxysteroid levels were(19.14±1.94),(10.07±1.62),(20.30±1.55)and(14.23±2.37)μg·L-1;the relative expression levels of GR protein in hypothalamic were 0.63±0.05,0.11±0.05,0.85±0.08 and 0.35±0.06;the relative expression levels of NF-κB protein in hypothalamic were 0.06±0.03,0.96±0.03,0.59±0.01 and 0.23±0.04;the relative expression levels of GR protein in kidney tissue were 0.94±0.06,0.06±0.02,0.40±0.02 and 0.09±0.08;the relative expression levels of NF-κB protein in kidney tissue were 0.07±0.05,0.81±0.12,0.72±0.07 and 0.49±0.08,respectively.Compared with the ADR and HYD groups,the above indexes in the normal group were statistically significant(P＜0.05,P＜0.01).And compared with the ADR group,the relative expression levels of NF-κB protein in renal tissue with CORT group were not statistically significant(P＞0.05),but the other indexes in CORT group were statistically significant(all P＜0.01).Conclusion HPA axis dysfunction with GR damaged and activated inflammatory levels are the common basis for the combination of typical symptoms of kidney yang deficiency and sickness behaviour which characterised by"deficiency and cold syndrome".

AIM To study the chemical constituents from the leaves of Cyanocarya paliurus(Batalin)Iljinskaja and their α-glucosidase inhibitory activities.METHODS The 95%ethanol extract from the leaves of C.paliurus was isolated and purified by macroporous resin,silica gel,Sephadex LH-20,polyamide,C18 reversed-phase silica gel and semi-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.Their α-glucosidase inhibitory activities were evaluated by PNPG.RESULTS Fifteen compounds were isolated and identified as cyclopaloside C(1),cyclopaloside A(2),juglanosides E(3),vaccinin A(4),ent-murin A(5),kaempferol 3-O-α-L-rhamnopyranoside(6),kaempferol-3-O-β-D-glucopyranoside(7),kaempferol-3-O-β-D-glucuronide methyl ester(8),kaempferol-3-O-β-D-glucuronide ethyl ester(9),kaempferol-3-O-β-D-glucuronide butyl ester(10),quercetin-3-O-α-L-rhamnopyranoside(11)quercetin-3-O-β-D-glucopyranoside(12),quercetin-3-O-β-D-galactopyranoside(13),quercetin-3-O-β-D-glucuronide butyl ester(14),dihydrokaempferol(15).The IC50 value of total extracts ihibited α-glucosidase was(1.83±0.04)μg/mL,and the IC50 values of compounds 1,4-5 were(29.48±1.86),(0.50±0.07),(0.71±0.07)μmol/L,respectively.CONCLUSION Compound 1 is a new tetrahydronaphthalene glycoside.Compounds 4-5,8-10 and 14 are isolated from the leaves of C.paliurus for the first time.Compounds 4-5 are relatively rare flavonoid lignans with potential inhibitory activities against α-glucosidase.