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 explore the effects of Buyang Huanwu Decoction on neuronal cell apoptosis after cerebral ischemia based on mediating Cav1 in regulating Wnt pathway.Methods Male wild-type(WT)and Cav1-/-(KO)C57BL/6 mice were randomly divided into sham-operation group,model group and Buyang Huanwu Decoction group(18.5 g/kg).Cerebral ischemia model was prepared using middle cerebral artery occlusion method,and drug intervention was given for 14 days.Neurobehavioral score was performed,HE staining was used to observe the morphology of ischemic cortical area of brain tissue,TUNEL staining was used to detect neuronal apoptosis in ischemic cortical area,immunohistochemistry was used to detect the expressions of apoptosis related proteins and Wnt1,glycogen synthase kinase 3β(GSK3β)and β-catenin protein in ischemic cortical area.Results Compared with the same genotype sham-operation group,the neurobehavioral score of the model group mice significantly increased,neuronal cells in the ischemic cortical area showed vacuolar changes,with nuclear condensation and widened intercellular spaces,the apoptosis rate of nerve cells significantly increased,with increased expressions of Bax,GSK3β and decreased expressions of Bcl-2,Wnt1 and β-catenin(P<0.01).Compared with the same genotype model group,the neurobehavioral score of mice in Buyang Huanwu Decoction group were significantly decreased,the pathological damage of the ischemic cortical area improved,the apoptosis rate of nerve cells decreased,the expressions of Bax and GSK3β decreased,and the expressions of Bcl-2,Wnt1 and β-catenin increased(P<0.01).Compared with the WT model group,the KO model group showed an increase in neurobehavioral score,aggravated damage in ischemic cortical area,significantly increased neuronal apoptosis rate,and increased expression of GSK3β(P<0.05).Compared with the WT Buyang Huanwu Decoction group,the KO Buyang Huanwu Decoction group showed an increase in neurobehavioral score,aggravated damage in ischemic cortical area,significantly increased neuronal apoptosis rate,increased expressions of Bax and GSK3β,and decreased expressions of Bcl-2,Wnt1 and β-catenin(P<0.01).Conclusion Buyang Huanwu Decoction can inhibit neuronal cell apoptosis after cerebral ischemia,and its mechanism may be related to regulating the expressions of apoptosis-related proteins by mediating Cav1 to regulate the Wnt signaling pathway.

Objective To explore the mechanism of Buyang Huanwu Decoction against cerebral ischemia-reperfusion injury in rats by regulating lipid metabolism through the cAMP/PKA/PPARγ pathway.Methods 60 rats were randomly divided into sham operation group(Sham),model group(Model),Buyang Huanwu Decoction low-dose group(BHD-L),Buyang Huanwu Decoction medium-dose group(BHD-M),Buyang Huanwu Decoction high-dose group(BHD-H)and Butylphthalide group(NBP).The cerebral ischemia-reperfusion model was prepared by transient middle cerebral artery embolization.The BHD low-,medium-and high-groups were given different doses of Buyang Huanwu Decoction(6.413,12.825,25.65 g·kg-1)by intragastric administration.The NBP group was administered with Butylphthalide(54 mg·kg-1).The sham operation group and the model group were administered with an equal volume of distilled water,all given for 14 days.The rats were subjected to neurobehavioral scoring.HE staining was used to observe brain pathological changes,and the kit was used to detect the levels of phosphocholine(PC),phosphatidylethanolamine(PE),diacylglycerol(DAG),and free fatty acid(FFA)on the ischemic side.RT-qPCR and Western Blot were applied to detect the mRNA and protein expressions of cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),and peroxisome proliferator-activated receptor γ(PPARγ).Results Compared with the sham group,the neurological deficit score was significantly increased(P＜0.01),pathomorphological damage in ischemic cortex was found,the contents of PC and PE were reduced,the contents of DAG and FFA were increased(P＜0.01),and cAMP mRNA expression increased(P＜0.05)in the model group.Compared with the model group,the neurological deficit score of the BHD-L group was decreased(P＜0.05),and the neurological deficit score of the BHD-M,BHD-H and NBP groups was significantly decreased(P＜0.01),the cells in each treatment group were regularly arranged,the intercellular spaces were reduced,and the normal cells were increased.PC and PE were significantly increased,DAG and FFA were significantly decreased(P＜0.01)in the BHD-M,BHD-H and NBP groups.PC was increased,FFA and DAG were decreased in the BHD-L group(P＜0.05,P＜0.01).The mRNA level of PPARγ was increased in the BHD-L group(P＜0.05),and the mRNA and protein levels of cAMP,PKA,and PPARγ were increased in the other treatment groups(P＜0.05,P＜0.01).Conclusion Buyang Huanwu Decoction has a neuroprotective effect on cerebral ischemia-reperfusion injury rats,and its mechanism may be related to regulating the expression of key factors in the cAMP/PKA/PPARγ signaling pathway and lipid metabolism.

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.

In the original publication the bands in Fig. 1J and Fig. 2B were not visible. The correct versions of Fig. 1J and Fig. 2B are provided in this correction.

[Abstract] Objective: To explore the clinical significance and in vitro biological effect of ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) expression in thyroid carcinoma (TC) tissues. Methods: TCGAdata were used to analyze the expression of UCHL5 in thyroid carcinoma tissues and its relationship with the prognosis of patients. 82 pairs of TC tissues and corresponding adjacent tissues were collected in the Department of Vascular and Thyroid Surgery, Sichuan Provincial People's Hospital from May 2018 to July 2019; TC cell lines (KTC-1 and WRO) were cultured in vitro, and transfected with UCHL5 overexpression vectors or their control vectors via lentivirus. The mRNAand protein expressions of UCHL5 and B-Raf proto-oncogene serine/threonine-protein kinase (BRAF) in tissues and cells were detected by qPCR and Western blotting, respectively. Cell proliferation was detected by CCK-8, and cell invasion and migration were detected by Transwell and Wound-healing experiments. Results: The expression of UCHL5 was low in TC tissues (P<0.01), and its expression was upregulated in tumor tissues with high TNM stage (P<0.01). The expression of UCHL5 was significantly correlated with BRAF expression and TNM stage of patients (all P<0.01), but not significantly related with patient's age, gender, pathological type and BRAF mutation (all P>0.05). In vitro overexpression of UCHL5 in KTC-1 and WRO cells could significantly promote BRAF expression, cell proliferation and metastasis (all P<0.01). Conclusion: The expression of UCHL5 is low in TC tissue, but upregulated with tumor progression. The high expression of UCHL5 in TC patients suggests poor prognosis. Meanwhile, UCHL5 can promote the malignant behaviors of TC cells in vitro.

To explore the effect of Naoshuming decoction on cerebral ischemic rats.
 Methods: The model of cerebral ischemia in rats was established via middle cerebral artery occlusion (MCAO). The MCAO model rats were randomly divided into a model group (n=36), a Naoshuming decoction at high dose group (n=36), a Naoshuming decoction at middle dose group (n=36) and a Naoshuming decoction at low dose group (n=36). In addition, a normal group (n=12) and a sham operation group (n=12) were included. Rats in each group were killed on the 3rd, 7th, and 14th day to detect relevant indicators. The Ayelet Levy 14 method was used to score the neurological function. Immunohistochemical method was used to detect the protein expression of nuclear factor kappa-B (NF-κB)/p50, NF-κB/p65, tumor necrosis factor-α (TNF-α), and IL-1β. The quantitative real-time PCR were used to detect the mRNA expression of NF-κB, TNF-α and IL-1β. 
 Results: Compared with the sham group, at each time point, the inflammation indexes in the model group and different dose of Naoshuming decoction groups were significantly enhanced, and all of them showed neurological dysfunction. But the inflammatory indexes and neurological function scores would were gradually improved with the pass of time. Compared with the model group, the neurological dysfunction, the protein levels of NF-κB/p50, NF-κB/p65, TNF-α and IL-1β, and the mRNA of NF-κB, TNF-α and IL-1β in the high, middle and low dose of Naoshuming decoction groups were reduced at 3, 7 and 14 d, with statistical difference (all P<0.05 or P<0.01). 
 Conclusion: Naoshuming decoction can alleviate the cerebral ischemic injury in rats.
Animals ; Brain Ischemia ; Infarction, Middle Cerebral Artery ; Inflammation ; Interleukin-1beta ; NF-kappa B ; Rats ; Tumor Necrosis Factor-alpha

Objective To prepare a red blood cells based multifunctional nanoscaled drug delivery system,and to study its in vitro photothermal and photodynamic effects.Methods The indocyanine green (ICG)/doxorubicin (DOX) co-loaded nanoscaled red blood cells (DIRAs) were prepared using an extrusion method.The morphology,particle size,encapsulation efficiency,and stability were determined.The heating related change of particle size was studied using a size and potential tester.The in vitro photothermal effect was studied using an infrared imaging device.The uptake of DIRAs to 4T1 cells was studied using a CLSM examination.The in vitro photodynamic effect was studied using a fluorescence probe and CLSM examination.Results DIRAs were successfully prepared with a uniform and homogeneous size which was about (97.0±20.1) nm.The Zeta potential was about-21.6 mV and the encapsulation efficiency of ICG and DOX were 93.5％ and 95.2％,respectively.The DIRAs had excellent stability within 28 days.This nanoscaled drug delivery system had identical photothermal effect compared to free ICG.The cellular uptake of DOX was significantly improved after the laser irradiation and the photodynamic effect was enhanced.Conclusions The prepared DIRAs have regular shape,suitable particle size,high encapsulation efficiency and high photothermal conversion efficiency.DIRAs can improve the cellular uptake of DOX and enhance the photodynamic efficiency.This biomimetic muhifunctional nano-system could facilitate breast cancer treatment by combining PTT7PDT and chemotherapy.

Aim To observe the effect of antibody NCX-3F10 on the main ion current of rat ventricular myocytes and its effect on arrhythmias induced by ischemia/reperfusion(I/R).Methods ① The whole-cell patch clamp technique was employed to record the Na+/Ca2+ exchange current(INa/Ca) and other major ion currents in rat ventricular myocytes.② The rat models of arrhythmia induced by ischemia/reperfusion were established by ligating the left coronary artery to in vivo and in vitro.Then the effects of antibody on the arrhythmia were observed.③ The IonOptix ion imaging system was used to observe the effect of antibody on calcium transients in single ventricular myocytes.Results ① The antibody NCX-3F10 dose-dependently inhibited INa/Ca from 5 to 40 mg·L-1.The IC50 for outward and inward currents was 11.15 and 11.69 mg·L-1, and the maximum inhibitory rates were 61% and 62%, respectively.The antibody also had an inhibitory effect on calcium current(ICa-L), and had no significant effect on inward rectifier potassium current(IK1), transient outward potassium current(Ito) and sodium current(INa).② In the isolated rat heart group I/R, 100% rats showed ventricular tachycardia, and 88.89% rats had ventricular fibrillation.After administration of antibody NCX-3F10(10 mg·L-1) 5 min before reperfusion, the incidence of ventricular tachycardia decreased to 44.43%(P<0.05), and the duration of ventricular tachycardia and ventricular fibrillation was also shortened remarkably(P<0.05).③ In the anesthetized rats after administration of antibody NCX-3F10(50 μg·kg-1) 5 min before reperfusion, the incidence and duration of ventricular tachycardia,the incidence and duration of ventricular fibrillation, and total number of ventricular premature beats were significantly decreased(P<0.05).④ From 5 to 40 mg·L-1, NCX-3F10 antibody decreased calcium transient amplitude in rat single ventricular myocytes dose-dependently(P<0.05).Conclusions The NCX-3F10 antibody shows significant arrhythmic effects on ischemia-reperfusion induced arrhythmia in rats both in vitro and in vivo, the underlying mechanism of which is related to NCX and L-type calcium current inhibition and calcium overload reduction by the NCX antibody.

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.