ObjectiveTo compare the contents and relative molecular weight distributions of proteins and polypeptides in Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules of Hirudo single medicinal preparations， to evaluate the in vitro anticoagulant， antiplatelet and fibrinolytic activities of the three preparations， and to investigate the effects of temperature， pH and digestive enzymes on the anticoagulant activities of the three preparations. MethodsThe contents of soluble proteins and polypeptides in the three preparations were determined by bicinchoninic acid assay（BCA） and Bradford method， and the relative molecular weight distributions of the three preparations were determined by electrophoresis combined with gel chromatography. The antithrombin activity of the three preparations was evaluated by fibrinogen-thrombin time（Fibg-TT） method， and their anticoagulant activities were further assessed by the elongations of activated partial thromboplastin time（APTT）， prothrombin time（PT） and thrombin time（TT）. The antiplatelet aggregation activities of the three preparations were measured by turbidimetry and the fibrinolytic activities were measured by fibrin plate method. Relative TT was used as index to investigate the effects of temperature， pH and digestive enzyme buffer on anticoagulant activities of the three preparations. ResultsAt the lowest single dosage， the contents of proteins and polypeptides were in the order of Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. Both Huoxue Tongmai capsules and Maixuekang capsules had 11 electrophoretic bands between 4.0 kDa and 90 kDa， the bands of Maixuekang capsules were more clear in the range of >25 kDa， and there was 1 obvious band at 14 kDa for the two capsules. Huoxue Tongmai capsules had one specific band at 9.0 kDa and Maixuekang capsules had one specific band at 48.0 kDa. Naoxuekang dropping pills only had 2 electrophoretic bands at 6.5 kDa and 8.5 kDa， primarily containing peptides below 2 kDa， most of which were oligopeptides. The anticoagulant activity concentrations of the three preparations exhibited a certain dose-dependent effect. At the lowest single dosage， The anticoagulant activity concentrations were ranked as Naoxuekang dropping pills>Huoxue Tongmai capsules>Maixuekang capsules. The prolongation effect of the three preparations on coagulation time was dose-dependent. At the same concentration， the prolongation effect of Naoxuekang dropping pills and Huoxue Tongmai capsules was APTT prolongation rate>TT prolongation rate>PT prolongation rate， whereas for Maixuekang capsules， the sequence was TT prolongation rate>APTT prolongation rate>PT lengthening rate. At the single minimum dosage， the order of APTT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules≈Naoxuekang dropping pills， the order of PT prolongation rate was Naoxuekang dropping pills≈Maixuekang capsules>Huoxue Tongmai capsules， and the order of TT prolongation rate was Maixuekang capsules>Huoxue Tongmai capsules>Naoxuekang dropping pills. The three preparations showed dose-dependent effects on platelet aggregation induced by adenosine diphosphate（ADP） and arachidonic acid（AA）， and the effect induced by ADP was stronger than that induced by AA. The anti-platelet aggregation effect of Naoxuekang dropping pills was significantly stronger than that of Maixuekang capsules（P<0.01）， whereas Huoxue Tongmai capsules had the effect of promoting platelet aggregation. None of the three preparations had the ability to dissolve fibrin. The anticoagulant activity of Naoxuekang dropping pills was least affected by heating， while the activities of the two capsules decreased significantly within 5 min above 80 ℃， and continued to decrease within 2 h. Compared with pure water， the anticoagulant activities of the three preparations could be increased by 1-3 times under strong acidity（pH 1-3）. In the pepsin buffer， the anticoagulant activity of Naoxuekang dropping pills could be increased by 1-3 times， while the anticoagulant activities of Huoxue Tongmai capsules and Maxuekang capsules were significantly decreased， the lowest levels were about 60% and 20%， respectively. In trypsin buffer， the anticoagulant activities of Naoxuekang dropping pills， Huoxue Tongmai capsules and Maixuekang capsules decreased significantly， and the lowest levels decreased to about 41%， 41% and 35%， respectively. ConclusionThe contents of proteins and polypeptides and relative molecular weights of the preparations derived from lyophilized fresh Hirudo powder， dried Hirudo powder and reflux extract of Hirudo decrease sequentially， and the anticoagulant activity decrease gradually， but the anticoagulant pathway is different. And the anti-platelet aggregation activity of the reflux extract is significantly enhanced. The heat resistance and gastrointestinal stability of the three preparations increase successively， and the first two are suitable for enteric-soluble preparations， while the latter is suitable for routine oral administration. The above results can provide data reference for the rationality of different preparation methods， active substances， pharmacodynamics and mechanism of Hirudo preparations.

Although a single nucleotide polymorphism for N-acetyltransferase 10 (NAT10) has been identified in patients with early-onset stroke, the role of NAT10 in ischemic injury and the related underlying mechanisms remains elusive. Here, we provide evidence that NAT10, the only known RNA N4-acetylcytidine (ac4C) modification "writer", is increased in the damaged cortex of patients with acute ischemic stroke and the peri-infarct cortex of mice subjected to photothrombotic (PT) stroke. Pharmacological inhibition of NAT10 with remodelin on Days 3-7 post-stroke or astrocytic depletion of NAT10 via targeted virus attenuates ischemia-induced infarction and improves functional recovery in PT mice. Mechanistically, NAT10 enhances ac4C acetylation of the inflammatory cytokine tissue inhibitor of metalloproteinase 1 (Timp1) mRNA transcript, which increases TIMP1 expression and results in the accumulation of microtubule-associated protein 1 light chain 3 (LC3) and progression of astrocyte autophagy. These findings demonstrate that NAT10 regulates astrocyte autophagy by targeting Timp1 ac4C after stroke. This study highlights the critical role of ac4C in the regulation of astrocyte autophagy and proposes a promising strategy to improve post-stroke outcomes via NAT10 inhibition.

ObjectiveTo evaluate the effectiveness of stone needle thermocompression and massage compared to flurbiprofen gel patch in relieving chronic musculoskeletal pain in the shoulder and back， and to explore the potential mediating mechanism through muscle elasticity. MethodsA total of 120 patients with chronic musculoskeletal pain in the shoulder and back were randomly assigned to either stone needle group or flurbiprofen group， with 60 patients in each. The stone needle group received stone needle thermocompression and massage for 30 minutes， three times per week； the flurbiprofen group received flurbiprofen gel patch twice daily. Both groups were treated for 2 weeks. Pain improvement， as the primary outcome， was assessed using the Global Pain Scale （GPS） at baseline， after 2 weeks of treatment， and again 2 weeks post-treatment. To explore potential mechanisms， a mediator analysis was conducted by measuring changes in superficial and deep muscle elasticity using musculoskeletal ultrasound at baseline and after the 2-week treatment period. ResultsThe stone needle group showed significantly greater pain relief than the flurbiprofen group 2 weeks post-treatment. After adjusting for confounders related to pain duration， the between-group mean difference was -8.8 ［95% CI （-18.2， -0.7）， P＜0.05］. Part of the therapeutic effect was mediated by changes in deep muscle elasticity， with a mediation effect size of -1.5 ［95% CI （-2.0， -0.9）， P = 0.024］， accounting for 17.9% of the total effect. ConclusionStone needle thermocompression and massage can effectively relieve chronic musculoskeletal pain in the shoulder and back， partly through a mediating effect of improved deep muscle elasticity.

Objective To examine the anti-central-fatigue function of maca and its underlying mechanism.Methods Forty Sprague-Dawley rats were divided randomly into a negative control group,model control group,and low-,medium-,and high-dose maca groups(0.6,1.2,and 2.4 g/kg·body weight).Rats in all groups except the negative control group were subjected to multi-factor stimulation,including cold-water swimming,sleep deprivation,restraining,and tail-clamping,to establish central fatigue rat models.Rats in the low-,medium-,and high-dose maca groups received 0.6,1.2,or 2.4 g/kg maca,respectively,by gavage for 35 days.Behavioral testing was carried out using the Morris water-maze,sucrose-preference,and tail-suspension tests.Markers of oxidative stress in the hippocampus,including superoxide dismutase(SOD),malondialdehyde(MDA),and catalase(CAT),were detected using test kits.Proteins connected with the AMP-activated protein kinase(AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor γ coactivator 1-α(PGC-1α)signaling pathway in the hippocampus were detected by Western blot.Results Rats in the low-,medium-,and high-dose maca groups spent significantly more time in the target quadrant compared with the model control group(P＜0.05 or P＜0.01),but there was no significant dose-effect relationship.Rats in the medium-and high-dose maca groups showed decreased escape latency(P＜0.05),increased time crossing the platform location(P＜0.05),increased sucrose preference(P＜0.05),decreased tail suspension time(P＜0.05),increased the activities of CAT(P＜0.01)and SOD(P＜0.05),and decreased MDA content(P＜0.01).Rats in the low-,medium-,and high-dose maca groups also showed significantly increased protein expression levels of AMPK and nuclear respiratory factor 1(P＜0.01 or P＜0.05),but no significant dose-effect relationship was observed.Rats in the medium-and high-dose maca groups showed increased protein expression of PGC-1α(P＜0.05 or P＜0.01),and rats in the high-dose maca group showed increased protein expression of SIRT1 and mitochondrial transcription factor A(P＜0.05 or P＜0.01).Conclusions Maca can improve the indicators of central fatigue in rats,determined by behavioral testing and oxidative stress-related factors.The underlying mechanism may be related to its regulatory effects on the AMPK/SIRT1/PGC-1α signaling pathway.

Depression is a common chronic mental illness.Animal models of depression are widely used to study the pathogenesis of depression as well as in the development of new antidepressant drugs.The consistency and reliability of animal models of depression to simulate human depressive symptoms directly affect the study result.However,at present,no animal models have been found that are completely consistent with the onset of human depression,which hinders the in-depth study of depression.By using scientific and reasonable modeling and evaluation method,the pathological state of depression can still be simulated to a large extent,providing a basis for further in-depth research of the pathogenesis of depression and the development of effective antidepressant drugs.This paper aims to provide a reference for researchers by reviewing several commonly used animal models and behavioral tests of depression.

Objective To explore characteristics of time-domain and time-frequency of task-related electroencephalogram(EEG)in patients with post-stroke aphasia(PSA).Methods From January,2018 to December,2021,18 PSA patients(PSA group)were recruited from Dongzhimen Hospi-tal,Beijing University of Chinese Medicine,and nine healthy subjects(control group)were recruited with matched gender,age and education level.EEG data based on the Chinese word-picture matching task were col-lected,and the average amplitude of N400 and event-related synchronization(ERS)/desynchronization(ERD)were analyzed.The PSA group was assessed with Chinese Rehabilitation Research Center Aphasia Examination(CRRCAE).Results The effect of N400 was significant on left frontal,central frontal,right frontal,left central and central areas.The average amplitude of N400 was higher on the frontal area in PSA group than in the control group,and it was low-er on right temporal-parietal area.For word-picture mismatching,average amplitude of N400 at FPZ was moder-ately correlated with auditory comprehension(r=0.483,P<0.05),the average amplitude of N400 at T8 was moderately correlated with repetition(r=0.584,P<0.05)and reading aloud(r=0.556,P<0.05),and the aver-age amplitude of N400 at P6 was moderately correlated with speaking(r=0.476,P<0.05)and reading(r=0.502,P<0.05).For word-picture matching,the average amplitude of N400 at P7 was moderately negatively cor-related with calculation(r=-0.481,P<0.05).Compared with the control group,the energy decreased in θ and αfrequency bands with absence of ERS in PSA group,and it increased in β frequency band with absence of ERD.Conclusion The activities of Chinese word-picture matching task-related EEG have been inhibited on right temporal-pa-rietal area for PSA patients,and it compensatively increases on frontal area,while the energy decreases in θ and αfrequency bands with ERS deficiency,and increases in β frequency band with ERD deficiency.

DNA methylation is an important epigenetic modification in mammals, playing a crucial role in various physiological processes, including cell differentiation and the gene expression regulation. The ten-eleven translocation (TET) protein family of DNA demethylases is integral to the regulation of DNA methylation, as it catalyzes the oxidation of 5-methylcytosine to form 5-hydroxymethylcytosine. During early embryonic development, the genome undergoes extensive DNA demethylation, and any aberration in this reprogramming process can result in abnormal embryonic development and physiological defects in offspring. The TET proteins, due to their unique dynamics and multifaceted roles, facilitate DNA demethylation and are involved in development and maturation of germ cells, the establishment of pluripotency, cell lineage differentiation, and transcriptional processes throughout mammalian embryogenesis. Furthermore, these proteins are closely associated with the maintenance of pregnancy and susceptibility of progeny to disease. Factors such as genetic mutations, maternal health conditions, and exposure to adverse environmental influences can impact TET protein activity, resulting in abnormal patterns of DNA demethylation. A comprehensive investigation of the related mechanisms of TET proteins is essential for enhancing our understanding of epigenetic regulation during early life, diagnosing and treating related diseases such as early fetal development retardation, and informing strategies for the prevention and management of pregnancy.This article reviews the regulatory role of DNA demethylation mediated by TET protein in mammalian embryonic development and pregnancy outcomes.

DNA methylation is an important epigenetic modification in mammals, playing a crucial role in various physiological processes, including cell differentiation and the gene expression regulation. The ten-eleven translocation (TET) protein family of DNA demethylases is integral to the regulation of DNA methylation, as it catalyzes the oxidation of 5-methylcytosine to form 5-hydroxymethylcytosine. During early embryonic development, the genome undergoes extensive DNA demethylation, and any aberration in this reprogramming process can result in abnormal embryonic development and physiological defects in offspring. The TET proteins, due to their unique dynamics and multifaceted roles, facilitate DNA demethylation and are involved in development and maturation of germ cells, the establishment of pluripotency, cell lineage differentiation, and transcriptional processes throughout mammalian embryogenesis. Furthermore, these proteins are closely associated with the maintenance of pregnancy and susceptibility of progeny to disease. Factors such as genetic mutations, maternal health conditions, and exposure to adverse environmental influences can impact TET protein activity, resulting in abnormal patterns of DNA demethylation. A comprehensive investigation of the related mechanisms of TET proteins is essential for enhancing our understanding of epigenetic regulation during early life, diagnosing and treating related diseases such as early fetal development retardation, and informing strategies for the prevention and management of pregnancy.This article reviews the regulatory role of DNA demethylation mediated by TET protein in mammalian embryonic development and pregnancy outcomes.

Objective To evaluate the effect of optical body surface monitoring system(OSMS)in setup of intensity modulated radiotherapy(IMRT)for thoracic tumorsand to analyze its relationship with BMI.Methods Thesetup errors of CBCT and location CT with body membrane and the registration errors of OSMS and first body surface reference image without body membrane were obtained in 49 patients with thoracic tumor who received routine intensity modulated radiotherapy.The paired t-test was used to analyze the difference of registration errors between the two image guidance methods.Pearson′s correlation analysis was used to analyze the correlation between CBCT errors and OSMS errors,and Bland-Altman analysis was employed to evaluatethe agreement of the two errors.The correlation and consistency of the two registration errors in patients with different BMI index were analyzed.PTV external marginsby the two registration methods were calculated using Van Herk formula.Results The OSMS and CBCT groups demonstrated significant differences in setup errors in the ventrodorsal direction(P<0.05),while no significant differences were found in the left-right,head-foot translation directions,or RTN rota-tion directions(P>0.05).Although the two methods showed a significant correlation in setup errors(P<0.05),this correlation was only moderate in the head-foot and left-right directions(r=0.500,0.408),weak in the RTN rotation direction(r=0.339),and very weak in the ventrodorsal direction(r=0.152).The limits of agreement(LOA,95％CI)between the two methods were[-0.45,0.45]cm in the left-right direction,[-0.59,0.57]cm in the head-foot direction,and[-0.48,0.40]cm in the ventrodorsal direction,with(-2.08°～2.19°)in the RTN rotation direction.Different BMI levels influenced the results of the two registration methods,particularly in patients with a BMI of 18.5～23.9 kg/m2.In this group,OSMS and CBCT exhibited a strong correlation in the head-foot direction(r=0.731),a moderate correlation in the left-right direction(r=0.512),and weak correlations in the ventrodorsal and RTN rotation directions(r=0.345,0.267).The absolute difference in setup errors between the two imaging systems was 0.4～0.5 cm/2°.Using CBCT and OSMS image guidance,the margins in the left-right,head-foot,and ventrodorsal directions were[0.5 cm,0.7 cm,0.3 cm]for CBCT,and[0.5 cm,0.7 cm,0.5 cm]for OSMS.Conclusion In chest tumor patients with a BMI of 18.5～23.9 kg/m2,OSMS and CBCT image guidance methods show good correlation in the head-foot and left-right directions,but their limits of agreement exceed the clinically acceptable range.OSMS cannot yet replace CBCT for image guidance in chest tumor intensity-modulated radiotherapy.Further improvements to tumor motion surrogates are necessary to enhance the accuracy of OSMS image guidance.