Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective:To evaluate the effect of extracorporeal shock wave on the phosphoproteomics of the spinal cord in rats with diabetic neuralgia.Methods:Thirty-six healthy male SPF-grade Sprague-Dawley rats, aged 2 months, weighing 200-250 g, were divided into 3 groups ( n=12 each) using the random number table method: control group (group C), diabetic neuralgia group (group D), and extracorporeal shock wave + diabetic neuralgia group (group E). The rats were continuously fed a common diet in group C, while the rats were fed a high-sugar and high-fat diet for 8 weeks in D and E groups. Streptozotocin 35 mg/kg was intraperitoneally injected, and the successful induction of diabetic neuralgia was defined as the blood glucose >14.6 mmol/L and the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) ≤85% of baseline values. Group E received extracorporeal shock wave treatment after developing the model, with 1, 000 shocks per session at a frequency of 10 Hz and an energy of 1.0 bar, once per week for a total of 4 sessions. The MWT and TWL were measured before developing the model (T 0) and at 1, 2, 3 and 4 weeks after developing the model (T 1-T 4). After the last extracorporeal shock wave treatment, the rats were anesthetized and sacrificed, and lumbar spinal cord tissues were obtained for proteomic analysis and for detection of the expression of glial fibrillary acidic protein (GFAP), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) (by immunohistochemistry). Results:Compared with group C, the MWT and TWL were significantly decreased at T 1-T 4 in D and E groups ( P<0.05). Compared with group D, the MWT and TWL were significantly increased at T 1-T 4 in group E ( P<0.05). The results of phosphoproteomics screening revealed 284 differentially phosphorylated proteins in D and C groups, 282 in E and C groups, and 303 in E and D groups ( P<0.05). The results of immunohistochemistry showed that the expression of GFAP, IL-1β and TNF-α was significantly up-regulated in group D compared with group C ( P<0.05); the expression of GFAP, IL-1β and TNF-α was significantly down-regulated in group E compared with group D ( P<0.05). Conclusions:The mechanism by which extracorporeal shock wave alleviates diabetic neuralgia is related to inhibition of astrocyte activation and excessive phosphorylation of mGluR5 in rats.

Objective:To determine the median effective dose (ED 50) of ciprofol inhibiting responses to insertion of laryngeal mask airway in the patients when combined with alfentanil. Methods:American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients of either sex, aged 40-64 yr, with body mass index of 20-30 kg/m 2, undergoing elective general anesthesia, were enrolled. Midazolam 0.025 mg/kg was intravenously injected for anesthesia induction, the baseline mean arterial pressure and heart rate were recorded 5 min later, and the average value of three times was considered as the baseline value. Ciprofol and alfentanil 10 μg/kg were intravenously injected in sequence, rocuronium 0.6 mg/kg was intravenously injected when BIS value < 60, 2 min later a laryngeal mask airway was placed, and mechanical ventilation was performed. Positive response was defined as increase in the maximum mean arterial blood pressure or heart rate more than or equal to 20% of the baseline value within 3 min after placement of the laryngeal mask airway or as the occurrence of body movement, bucking, frowning, mouth and face twitching, tearing, laryngospasm or the BIS value failing to drop below 60. The study was performed by the Dixon′s up-and-down method. The initial dose of ciprofol was 0.4 mg/kg, and the ratio between the two successive doses was 1.1. If a positive response occurred, the dose was increased in the next patient, otherwise the dose was reduced. The ED 50 and 95% confidence interval of ciprofol inhibiting responses to insertion of laryngeal mask airway were calculated by the probit method. Results:The ED 50(95% confidence interval) of ciprofol inhibiting responses to insertion of laryngeal mask airway was 0.291(0.231-0.318) mg/kg when combined with alfentanil 10 μg/kg. Conclusions:The ED 50 of ciprofol inhibiting responses to insertion of laryngeal mask airway is 0.291 mg/kg in the patients when combined with alfentanil 10 μg/kg.

Objective:To evaluate the dose-response relationship of alfentanil in combination with midazolam-etomidate inhibiting cardiovascular responses to laryngeal mask airway implantation in elderly patients.Methods:American Society of Anesthesiologists Physical Status Ⅰ or Ⅱ patients of either sex, aged 65-85 yr, with body mass index of 20-30 kg/m 2, undergoing elective operation under general anesthesia, were enrolled in this study.Midazolam 0.025 mg/kg was intravenously injected for adequate sedation, 5 min later mean arterial pressure and heart rate were recorded for 3 consecutive times at 3-min interval, the mean value was collected and considered as the baseline value.Etomidate 0.2 mg/kg was intravenously injected, and alfentanil and rocuronium 0.6 mg/kg were intravenously injected when bispectral index value < 60.A laryngeal mask airway was inserted at 1.4 min after intravenous injection of alfentanil, and mechanical ventilation was performed.The dose of alfentanil was determined by the Dixon′s up-and-down method.The initial dose of alfentanil was set at 6.83 μg/kg.The dose of alfentanil in the next patient was determined according to the development of cardiovascular response to laryngeal mask airway placement.If the cardiovascular response to laryngeal mask airway placement occurred, the dose was increased for the next patient, and if cardiovascular response to laryngeal mask airway placement did not occur, the dose was decreased, and the ratio between the two successive doses was 1.0∶1.1.The cardiovascular response to laryngeal mask airway placement was defined as increase in maximum mean arterial pressure or maximum heart rate by≥20% of baseline values within 2 min after laryngeal mask airway placement.The median effective dose (ED 50), 95% effective dose (ED 95) and 95% confidence interval (95% CI) of alfentanil inhibiting cardiovascular responses to laryngeal mask airway placement in elderly patients were calculated by the Probit method. Results:When combined with midazolam and etomidate, the ED 50 (95% CI) of alfentanil inhibiting the cardiovascular responses to laryngeal mask airway placement in elderly patients were 5.605 (5.036-6.082) μg/kg, and the ED 95 (95% CI) were 6.625 (6.125-9.763) μg/kg. Conclusions:When combined with midazolam and etomidate, the ED 50 and ED 95 of alfentanil inhibiting the cardiovascular responses to laryngeal mask airway placement are 5.605 and 6.625 μg/kg, respectively, in elderly patients.

Objective:To investigate the dose-effect relationship of alfentanil inhibiting cardiovascular responses to tracheal intubation when combined with midazolam and etomidate.Methods:American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients of either sex, aged 18-64 yr, with body mass index<32 kg/m 2, undergoing elective operation under general anesthesia with endotracheal intubation, were enrolled in this study.Midazolam 0.025 mg/kg was intravenously injected for adequate sedation, and 5 min later mean arterial pressure and heart rate were recorded for 3 consecutive times at an interval of 3 min, and the mean value was calculated and served as the baseline value.Etomidate 0.3 mg/kg was intravenously injected, and alfentanil and rocuronium 0.6 mg/kg were intravenously injected when bispectral index value < 60, and then 1.4 min later tracheal intubation was performed.The dose of alfentanil was determined by the Dixon′s up-and-down method.The initial dose of alfentanil was set at 20 μg/kg.The dose of alfentanil in the next patient was determined according to the development of cardiovascular responses to tracheal intubation, and the ratio between the two successive doses was 1.0∶1.1.The cardiovascular response was defined as as positive when the maximum value of mean arterial pressure or heart rate increased by ≥20% of the baseline value within 2 min after endotracheal intubation.Probit method was used to determine the ED 50, ED 95 and 95% confidence interval of alfentanil inhibiting cardiovascular responses to tracheal intubation. Results:When combined with midazolam and etomidate, the ED 50 (95% confidence interval) of alfentanil inhibiting cardiovascular responses to tracheal intubation was 21.343 (19.105-24.516) μg/kg, and the ED 95 (95% confidence interval) was 25.043 (22.983-48.983) μg/kg. Conclusions:When combined with midazolam and etomidate, the ED 50 and ED 95 of alfentanil inhibiting cardiovascular responses to tracheal intubation are 21.343 and 25.043 μg/kg, respectively.

OBJECTIVE To inv estigate the in vitro inhibitory effects of acteoside on cytochrome P 450（CYP）enzymes in liver microsomes of rats. METHODS Using probe substrates method ，acteoside（0.1，0.3，1，3，10，30 μmol/L）was incubated with probe substrates phenacetin ，mephentoin，diclofenac，coumarin，dextromethorphan and testosterone （substrates of CYP 1A2， CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）in liver microsomes of rats. Another blank control group and positive inhibitor group [ α-naphthoflavone，ticlopidine，sulfabendazole，pilocarpine，quinidine and ketoconazole （inhibitors of CYP 1A2，CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）] were set up. Using indapamide as the internal standard ， the contents of corresponding metabolites （acetaminophen， 4-hydroxymephenytoin， 4-hydroxydiclofenac，7-hydroxycoumarin，dextran，6 β-hydroxytestosterone） were detected by ultra high performance liquid chromatography-tandem mass spectrometry . The IC 50 values were calculated by GraphPad v 8.0 software. By computer molecular docking technology ，acteoside and positive inhibitors were molecularly docked with the CYP enzyme ，and the binding mode and strength of the two molecules were analyzed. RESULTS The IC 50 values of acteoside to CYP 1A2 and CYP 2A6 enzymes were more than 30 μmol/L，and those of acteoside to CYP 2D6，CYP2C19，CYP2C9 and CYP 3A4 enzymes were 24.87，21.52，12.56 and 7.55 μmol/L，respectively. The hydrogen bond and hydrophobic force could form between acteoside and CYP 3A4 enzyme，and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP 3A4 enzyme. The binding free energy of acteoside and ketoconazole to CYP 3A4 enzyme were － 10.2 and － 12.4 kcal/mol （1 kcal/mol＝4.19 kJ），respectively. CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP 3A4 enzyme in liver microsomes of rats ，and its affinity is equivalent to that of positive inhibitor ；the compound shows weak inhibitory effect on other 5 CYP enzymes.

Objective:To investigate the performance of von willebrand factor antigen (vWF:Ag) and D-dimer in predicting thrombotic risk in nonvalvular atrial fibrillation (NVAF) patients with anticoagulant therapy.Methods:From March 2017 to March 2019, 256 patients were enrolled, including 152 males and 104 females, aged (57.9±20.4) years old; according to the end-point events during the follow-up period, the patient group was further divided into 227 cases in the no-event group and 29 cases in the thrombotic event group;50 cases in the control group, including 30 males and 20 females, aged (45.0±5.3) years old. vWF:Ag was detected by blood coagulation instrument and determination of D-dimer was done by fluor-euzyme linked immunoassay Analyzer. Mann-Whitney U test was used for data comparison between any two groups, Kruskal-Wallis H test was used for comparison among multiple groups and multivariate correlation analysis was done by Logistic regression to obtain odds ratio ( OR). The prediction performance with thrombotic events of vWF:Ag and D-dimer was evaluated by ROC curve, Kaplan-Meier curve was used to analyze the survival curve and the hazard ratio ( HR) was obtained by Cox proportional hazard regression model. Results:The levels of vWF:Ag and D-dimer in the control group were 103% (86%-131%) and 249 (90-522) μg/L, 234% (102%-623%) and 744 (100-3 352) μg/L in the patient group; in the patient group, of which 225% (102%-623%) and 650 (100-3 281) μg/L in non-event group, 333% (210%-494%) and 1 325 (487-3 352) μg/L in thrombus event group; compared the healthy control, the levels of vWF:Ag and D-dimer were increased in patients group ( P<0.001), of which non-event groups were higher than healthy controls ( P<0.001), and the thrombotic event group was higher than that of the non-event group ( P<0.001). Plasma vWF:Ag level and D-dimer level in NVAF patients were higher than those in the control group ( P<0.001). Plasma vWF:Ag level and D-dimer level in the non-event group were significantly higher than those in the healthy control group ( P<0.001). The plasma vWF:Ag and D-dimer levels of patients in the thrombotic event group were significantly higher than those in the non-event group patients ( P<0.001). The result of ROC showed that the critical value of vWF: Ag for predicting thrombosis within 3 months of NVAF patients was 229% and area under the curve (AUC) was 0.839 (95% CI:0.784-0.894); When the critical value of D-dimer was 588 ng/ml, AUC was 0.803 (95% CI:0.745-0.861).While vWF:Ag combined with D-dimer, AUC was 0.868 (95% CI:0.826-0.909). Logistic regression analysis showed that plasma vWF:Ag level in NVAF patients was significantly correlated with age ( OR=10.240, 95%CI 2.773-37.820), congestive heart failure ( OR=34.779, 95%CI 8.010-151.019), hypertension ( OR=0.068, 95%CI 0.023-0.198) and type 2 diabetes ( OR=6.618, 95%CI 2.469-17.734) ( P<0.001), as well as was significantly correlated with vascular disease ( OR=4.801, 95%CI 1.204-19.145) ( P=0.026). Plasma D-dimer level was significantly correlated with congestive heart failure ( OR=0.146, 95%CI 0.036-0.588) and medication compliance ( OR=0.114, 95%CI 0.016-0.832) ( P value was 0.007 and 0.032). Survival analysis showed that the cumulative probability of thrombosis within 3 months was significantly increased (Log-rank χ2 was 11.394, 17.895 and 32.825 respectively, P value<0.001) in the patients with plasma levels above the critical value of vWF:Ag, D-dimer or vWF:Ag combined with D-dimer. Cox proportional regression model showed that neither vWF:Ag nor D-dimer could independently predict thrombotic events during anticoagulant therapy( HR was 0.866 and 0.834, P-value was 0.253 and 0.152, respectively), but it could improve the prediction performance significantly( HR=0.780, P=0.048) for combined application of both vWF:Ag and D-dimer. Conclusion:The changes with plasma vWF:Ag and D-dimer levels in NVAF patients were associated with a variety of clinicopathological factors and closely related to the risk of thrombosis within 3 months. Combined application could provide the effective basis for clinical prediction of the condition.

Objective:To investigate the influence of multilayer alginate chitosan sustained-release microspheres loading vascular endothelial growth factor(VEGF) and vancomycin in the proliferation and osteogenic differentiation of human plaacenta-derived mesen chymal stem cells(HPMSCs),and to provide theoretical basis for its clinical application in the repair of bone defect.Methods:The microspheres were prepared based on the previous research and HPMSCs were co-cultured with drug (VEGF/vancomycin)-loaded microspheres (drug-loaded microspheres+HPMSCs group), non-drug loaded microspheres (microspheres+HPMSCs group) and without any microspheres (HPMSCs group).Then the proliferation rate of HPMSCs was identified by CCK-8 kit.The osteogenic differentiation potential of HPMSCs was detected by Alizarin red staining and alkaline phosphatase (ALP) kit when the HPMSCs had been co-cultured with drug loaded microspheres in osteogenic medium (HPMSCs+drug-loaded microspheres+induction group), non-drug loaded microspheres in osteogenic medium (HPMSCs+microspheres+induction group), without any microspheres in osteogenic medium (HPMSCs+induction group) and without any micropheres in normal medium (HPMSCs+PBS group) for 21 d.Results:Compared with HPMSCs group,the proliferation rates of HPMSCs in drug-loaded microspheres+HPMSCs and microspheres+HPMSCs groups had no significant changes (P>0.05).The calcium deposition in HPMSCs+drug-loaded microspheres+induction group was more than those in microspheress+HPMSCs+indution group and HPMSCs+induction group after Aalizarin red staining;the ALP activity in drug-loaded microspheres+HPMSCs+indution group was higher than those in microspheres+HPMSCs+indution group and HPMSCs+induction group (P<0.05),and the ALP activity in microspheres+HPMSCs+induction group was higher than that in HPMSCs+induction group(P<0.05).Conclusion:The sustained-release microspheres loading VEGF and vancomycin have no significant effect on the proliferation activity of HPMSCs and the microspheres could stimulate the osteogenic differentiation of HPMSCs.

AIM: To investigate the effect of zacopride, an inward rectifier potassium channel agonist, on ouabain-induced arrhythmias in adult rats, and to explore the underlying electrophysiological mechanism.METHODS: Using ouabain to establish in vitro and in vivo arrhythmic rat models, the effects of zacopride on ouabain-induced arrhythmias were observed.The technique of whole-cell patch clamp was used to observe the effects of zacopride on inward rectifier potassium current (IK1), resting membrane potential (RMP) and delayed afterdepolarizations (DADs) in single rat ventricular myocyte.RESULTS: Zacopride at 1 μmol/L significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain in rat hearts in vitro (P<0.05).In anesthetized rats, zacopride at 15 μg/kg significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain (P<0.05).IK1 was significantly inhibited by ouabain (P<0.05), which was partially and even completely reversed by zacopride at 0.1~10 μmol/L.RMP value was significantly reduced by ouabain (P<0.05), and then increased to different levels after treatment with zacopride (0.1~10 μmol/L).Zacopride at 1 μmol/L showed its maximal effect and RMP was restored to normal level.Moreover, zacopride at 1 μmol/L markedly suppressed ouabain-induced DADs in single rat ventricular myocyte.The incidence of DADs decreased from 91.67% to 12.50% after zacopride was applied (P<0.05), and this effect was abolished by 1 μmol/L BaCl2.CONCLUSION: Inward rectifier potassium channel agonist zacopride significantly inhibits ouabain-induced ventricular arrhythmias in adult rats.The mechanism is related to increased RMP level and inhibition of DADs by activation of IK1 channel.

Aim To examine the effect of zacopride,a specific inward rectifier potassium channel(IK1)agonist,on L-thyroxine(T4)-induced ventricular remodeling and the underlying mechanism.Methods SD rats were randomly divided as control,L-thyroxine(L-thy,1 mg·kg-1·d-1,ig,10 d)model,L-thy +zacopride(5,15,50 μg·kg-1,respectively,ip),L-thy+zacopride(15 μg·kg-1)+chloroquine(7.5 μg·kg-1,ip)and L-thy+captopril(100 mg·kg-1·d-1,drinking water)groups.Echocardiography and cardiac hypertrophic indexes were measured to confirm the establishment of the ventricular remodeling model.The changes of IK1 and L-calcium current(ICa-L)were detected by whole cell patch clamp technique.The confocal microscopy and fluorescent indicator Fluo-4 were applied to examine the intracellular Ca2+ concentration([Ca2+]i)of isolated adult rat ventricular myocytes.Results L-thyroxine induced left ventricular hypertrophy with increased ratio of heart weight(HW)to body weight(HW·BW-1),ratio of left ventrical weight(LVW)to body weight(LVW·BW-1),left ventricular dimension in diastole(LVIDd),left ventricular dimension in systole(LVIDs),interventricular septum thickness(IVS)and decreased ejection fraction(EF),fractional shortening(FS)(P<0.01).Patch clamp data suggested IK1 was downregulated,while ICa-L was upregulated(P<0.01).In isolated adult cardiomyocytes,L-thyroxine increased the cell area and [Ca2+]i(P<0.01).Zacopride treatment obviously alleviated cardiac remodeling,improved cardiac function,reversed the changes of IK1 and ICa-L,and significantly attenuated intracellular calcium overload(P<0.01).The optimum dose of zacopride in vivo was 15 μg·kg-1 at which the effect was compared favourably with captopril,a classical anti-remodeling agent.Low-dose IK1 atagonist chloroquine could reverse the effect of zacopride(P<0.01).Conclusion Via activating IK1,zacopride could significantly decrease Ca2+ influx and intracellular calcium overload thereby inhibiting L-thyroxine-induced cardiac ventricular remodeling.