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.

Hyperkalemia is one of the common ion metabolism disorders in clinical practice. Hyperkalemia is defined as serum potassium higher than 5.0 mmol/L according to the guidelines at home and abroad. Acute severe hyperkalemia can cause serious consequences, such as flaccid paralysis, fatal arrhythmia, and even cardiac arrest. The use of renin-angiotensin- aldosterone system inhibitors, β-blockers and diuretics, low-sodium and high-potassium diets, and the presence of related comorbidities increase the occurrence of hyperkalemia. Hyperkalemia risk exist in all clinical departments, but there is a lack of a standardization in the management of multi- department cooperation in hospital. Therefore, a number of domestic nephrology and cardiology department experts have discussed a management model for multi-department cooperation in hyperkalemia, formulating the management standard on hospital evaluation, early warning, diagnosis and treatment, and process. This can promote each department to more effectively participate in nosocomial hyperkalemia diagnosis and treatment, as well as the long-term management of chronic hyperkalemia, improving the quality of hyperkalemia management in hospital.

Objective To observe the feasibility of 5.0T MR for cardiac imaging.Methods Three patients with heart diseases and 17 healthy volunteers were prospectively enrolled.Cardiac MR(CMR)cine sequence and black blood sequence imaging were performed using 5.0T and 3.0T MR scanner,respectively.The image quality and artifacts degrees were compared between 5.0T and 3.0T CMR images,and the consistency of left ventricular parameters obtained using 5.0T and 3.0T scanners was analyzed.Results No significant difference of image quality nor artifacts degrees was found between 5.0T and 3.0T CMR images(all P＞0.05).The left ventricular end diastolic volume(EDV),end systolic volume(ESV),ejection fraction(EF),stroke volume(SV)and end diastolic mass(EDM)derived from cine images acquired at different fields were in a good agreement(all ICC＞0.75,all P＜0.001).Conclusion 5.0T MR could be used for cardiac imaging,with image quality of cine and black blood sequences comparable to that of 3.0T MR.

Objective:To evaluate the therapeutic effects of Yufu ointment for acute radiation-induced skin injury. Methods:We enrolled a total of 104 patients with malignant tumors who developed acute skin injury of grade 2-3 during or after the first course of radiotherapy from June 2019 to February 2023. The observation group ( n=53) and control group ( n=51) received external application of Yufu ointment and Shirun Shaoshang ointment, respectively. We recorded the changes in the grade of skin injury and associated symptoms (such as pain, itching, and burning sensation) and healing speed and time for the two groups. Results:A total of 102 patients completed the study, with 53 cases in the observation group and 49 cases in the control group. The incidence rate of moisture skin lesions in the observation group was significantly lower than that in the control group (62.26% vs. 83.67%, P<0.05). The healing speed of moisture skin ulcerations was significantly faster in the observation group than in the control group (1.67 cm 2/d vs. 0.55 cm 2/d, P<0.05). The observation group had a significantly shorter time to skin injury healing than the control group (10.40 d vs. 14.41 d, P<0.05), significantly different for skin injury of grade 2 (10.21 d vs. 17.57 d, P<0.05) but not for skin injury of grades 2.5 and 3 ( P>0.05). Regarding clinical symptoms, both groups experienced significant reductions in pain and burning sensation scores after treatment ( P<0.05); the grade of itching was significantly decreased for the observation group ( P<0.05), but had no significant change for the control group after treatment ( P>0.05). Conclusions:Yufu ointment can significantly alleviate secondary damage for patients with acute radiation-induced skin injury, by accelerating the healing process and relieving symptoms such as pain, itching, and burning sensation with good tolerability, which deserves further promotion.

Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.
Creatine/metabolism* ; Extracellular Vesicles ; Muscle, Skeletal/metabolism* ; Myoblasts/metabolism* ; Regeneration ; Connexins/metabolism*

Objective:To observe the effect of large pushing Tianheshui manipulation intervention on the body temperature of young rabbits with endotoxin-induced fever and discuss its antipyretic mechanism.Methods:Thirty-two young rabbits meeting the standards were selected from 40 ordinary young male New Zealand rabbits after being adapted for 7 d,and randomly divided into a normal group,a model group,a large pushing Tianheshui group,and an ibuprofen group according to the random number table method,with 8 rabbits in each group.Rabbits in the model group,the large pushing Tianheshui group,and the ibuprofen group were subjected to preparing the endotoxin-induced fever model by intravenous lipopolysaccharide from the marginal ear vein.Rabbits in the large pushing Tianheshui group received Tuina(Chinese therapeutic massage)manipulation intervention 1.5 h and 2.5 h after modeling,respectively.Rabbits in the ibuprofen group were intragastrically given ibuprofen suspension 1.5 h after modeling.The dynamic changes in body temperature were observed for the young rabbits after fever modeling.Enzyme-linked immunosorbent assay was used to determine the content changes in positive mediators of hypothalamic body temperature,including prostaglandin(PG)E2 and cyclic adenosine monophosphate(cAMP),as well as negative mediators of hypothalamic body temperature,including arginine vasopressin(AVP)and α-melanocyte stimulating hormone(α-MSH).Results:The body temperature of the young rabbits in the model group was significantly higher than that in the normal group at 0.5-4.0 h,5.0 h,and 5.5 h after modeling(P＜0.01),showing two obvious fever peaks in the model group at 1.5 h and 3.0 h after modeling,respectively,with the highest peak at 1.5 h.Compared with the model group,body temperatures of the large pushing Tianheshui group and the ibuprofen group decreased significantly after 0.5 h of intervention(P＜0.05).Compared with the normal group,the contents of PGE2 and cAMP were significantly increased(P＜0.05),and the contents of AVP and α-MSH were significantly decreased(P＜0.01)in the hypothalamus of the model group.Compared with the model group,the contents of PGE2 and cAMP were significantly decreased(P＜0.01),and the levels of AVP and α-MSH were significantly increased(P＜0.01)in the hypothalamus in the large pushing Tianheshui group and the ibuprofen group.There were no significant differences in the PGE2,cAMP,AVP,and α-MSH contents in the hypothalamus between the ibuprofen group and the large pushing Tianheshui group(P＞0.05).Conclusion:Large pushing Tianheshui manipulation has a significant antipyretic effect on endotoxin-induced fever in young rabbits.The mechanism may be related to inhibiting the positive regulators(PGE2 and cAMP)and promoting the negative regulators(AVP and α-MSH)of hypothalamic control of body temperature.

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

Objective:To analyze the efficacy and safety of enzyme replacement therapy (ERT) in Chinese patients with Fabry disease.Methods:A retrospective analysis of the clinical manifestations, genetic variations, family screening, treatments and adverse reactions was conducted in five patients with Fabry disease admitted to the First Affiliated Hospital of Zhejiang University College of Medicine from July 2020 to May 2021. The dosage of agalsidase β was 1 mg/kg by intravenous pump once every 2 weeks.Results:Five male patients with median age of 37 years old (29-51 years old) were diagnosed based on clinical features, family history, α-galactosidase A (α-Gal A) activity, genetic analysis results and kidney biopsy. The clinical manifestations varied in these five patients. All patients had abnormal electrocardiogram, abnormal cardiac ultrasonography and abnormal urinalysis results, three experienced acroparaesthesia during childhood (one patient had persistent pain until adulthood), three had cutaneous angiokeratoma, four had renal insufficiency, four had hypohidrosis, four had diarrheas, four had cornea verticillata and two had high-frequency hearing loss. Two missense mutations of the GLA gene were identified: c.272T>C(p.I91T) and c.868A>G(p.Met290Val). Two nonsense mutations were c.1024C>T(p.Arg342*) and c.838C>T(p.Gln280*). Furthermore, the frameshift mutation c.348del p.(Ile117Phefs*4) was detected, which was not included in the known database, presented with classical Fabry disease. There was no serious adverse reaction during agalsidase β infusion in 5 patients. ERT reduced the plasma globotriaosylsphingosine (lyso-GL-3) levels after treatment of 2-10 months ( P<0.05), and the long-term diarrhea symptom were significantly improved. Conclusions:The clinical manifestations of Fabry disease are varied. Severe adverse events rarely occur in patients treated with short-term ERT. Plasma lyso-GL-3 levels decrease significantly after treatment.

Objective:To improve the consistency of test results through reducing inter-laboratory variation in SARS-CoV-2 antibody detection with WHO SARS-CoV-2 antibody candidate international standard (IS, sample G) and antibody reference panel (samples E, F, H, I, J).Methods:Ten WHO samples (A-J) including the candidate IS and reference panel were evaluated using different methods, such as microneutralization tests based on live SARS-CoV-2, pseudovirus neutralization assay and commercial ELISA kits. The test results were compared using statistical analysis.Results:Using IS (sample G) as a reference, the relative concentrations of other samples could be determined with less variation. ELISA and pseudovirus neutralization assay had consistent results with those obtained with the microneutralization test based on SARS-COV-2 strain HB02. Weakly positive samples could be detected only by a certain kit.Conclusions:The availability of an IS for antibodies would facilitate the standardization of SARS-CoV-2 antibody detection methods. The reference panel fitted all the assays based on the SARS-CoV-2 prototype Wuhan strain. Pseudovirus neutralization assay and ELISA could be used as alternatives to live SARS-CoV-2-based neutralization test to some extent.

Objective To investigate the clinical significance of autophagy-related protein 7 (ATG7) in the diagnosis of HBV-related hepatocellular carcinoma (HBV-HCC) by measuring the expression level of serum ATG7 in patients with HBV-HCC. Methods A total of 50 patients with chronic hepatitis B (CHB) and 89 patients with HCC who were hospitalized in Mengchao Hepatobiliary Hospital of Fujian Medical University from June 2018 to December 2020 were enrolled, among whom 67 patients had HBV-HCC (HBV-HCC group) and 22 patients had no HBV-HCC (non-HBV-HCC group), and 20 healthy volunteers who underwent physical examination were enrolled as healthy control (HC) group. Demographic data and laboratory data including alpha-fetoprotein (AFP) were collected from each group, and ELISA was used to measure the serum level of ATG7. The receiver operating curve (ROC) was plotted for ATG7 and AFP used alone or in combination, and the area under the ROC curve (AUC) was compared. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for comparison between two groups; the chi-square test was used for comparison of categorical data between groups; a Spearman correlation analysis was used to investigate correlation. Results The serum level of ATG7 was 22.88(19.79-23.04) ng/mL in the HBV-HCC group, 17.06(14.45-19.40) ng/mL in the non-HBV-HCC group, 19.21(16.65-20.82) ng/mL in the CHB group, and 13.82(8.70-17.82) ng/mL in the HC group, with a significant difference between groups ( χ 2 =65.144, P < 0.001). ATG7 had an AUC of 0.818 (95% confidence interval [ CI ]: 0.743-0.879) and AFP had an AUC of 0.777 (95% CI : 0.698-0.843) in the diagnosis of HBV-HCC, suggesting that ATG7 had a slightly higher AUC than AFP ( Z =0.852, P =0.394). ATG7 combined with AFP had an AUC of 0.859 (95% CI : 0.790-0.913) in the diagnosis of HBV-HCC, which was significantly higher than the AUC of ATG7 alone ( Z =2.192, P =0.028) and AFP alone ( Z =2.076, P =0.038). Conclusion ATG7 is a good marker for the diagnosis of HBV-HCC, and combined measurement of ATG7 and AFP can significantly improve the diagnostic rate for HBV-HCC.