BACKGROUND: Apelin/APJ system has a wide range of physiological functions,but its intracellular signal transduction,in particular,apelin receptor desensitization,internalization,resensitization degradation,have still no consistent opinion.OBJECTIVE: To construct eukaryotic expression vector expressing human apelin receptor(APJ)tagged to red fluorescent protein(pDsRED-express-C1),and to determine the expression in human embryo kidney 293 cells.METHODS: The plasmid pcDNA3.1-hAPJ was used as a template for PCR amplification of human APJ.Following PCR amplification the PCR product were removed and enzymatic digestion with EcoR I and BamH I.Same enzymes were used to cut vector pDsRED-express-C1.The digestive product was ligated by conventional methods of connection,then transfected into Competent E.coli TOP10.Single clones were picked plasmid extraction,followed by restriction enzyme digestion and finally DNA sequencing.The recombinant plasmid with correct sequencing was transfected into human embryonic kidney cells,PI staining,followed by the observation under a confocal microscope.RESULTS AND CONCLUSION: PCR amplified a 1.2-kb fragment,which was consistent with the expected size of the human APJ.The pDsRed-hAPJ recombinant plasmid was cut into two fragments,one corresponded to the pDsRED-express-C1 vector size,and the other fragment corresponded to APJ target fragment.Confocal microscopy analysis showed that,APJ was expressed mainly in the membrane of human embryo kidney 293 cells.The pDeRed-hAPJ eukaryotic plasmid expression vector was successfully constructed and effective expression of this fusion protein is achieved,which might be instrumental in the study of displacement and intracellular localization of human APJ.

Objective To investigate the effect of sevoflurane anaesthesia on the cognitive function and phosphorylation of tau protein in hippocampal neurons in amyloid precursor protein (APP) transgenic mice.Methods Male APP gene mutation mice,weighing 18-22 g and aged 8-12 weeks,were used in this study.Forty-four APP positive mice were randomly divided into two groups:sevoflurane group (group AS,n =28) and control group (group AC,n =16).And other forty-four APP negative mice were randomly divided into two groups:sevoflurane group (group S,n =28) and control group (group C,n =16).The animals in groups S and AS inhaled 3％ sevoflurane for 4 hours.While in groups C and AC,the animals inhaled pure oxygen for 4 hours.Morris water maze was performed 24 hours after sevoflurane or pure oxygen inhalation.The phosphorylation of tau protein at Ser262 and Ser396 sites was detected by Western blotting on 1 day after pure oxygen inhalation in groups AC and C,and on 1,3 and 7 days after sevoflurane inhalation in groups AS and S.Results Compared with group C,the escape latency was significantly prolonged and the duration of staying at the original platform quadrant was shortened in groups S and AC,and the phosphorylation of tau protein at Ser262 site in group S and phosphorylation of tau protein at Ser262 and Ser396 sites in group AS were increased (P ＜ 0.05).Compared with group S,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened,and the phosphorylation of tau protein at Ser262 and Ser396 sites was increased in group AS (P ＜ 0.05).Compared with group AC,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened,and the phosphorylation of tau protein at Ser262 and Ser396 sites was increased in group AS (P＜0.05).Conclusion Sevoflurane anesthesia can aggravate the impairment of cognitive function in APP positive mice and the increase in the phosphorylation of tau protein at Ser262 and Ser396 sites is involved in the mechanism.

Objective To investigate the effect of sevoflurane anesthesia on the cognitive function and phosphorylation of tau in hippocampal neurons in amyloid precursor protein (APP) transgenic mice.Methods Male APP gene mutation mice,weighing 18-22 g,aged 8-12 weeks,were used in the study.Forty-four APP positive mice were randomly divided into 2 groups (n =10 each):sevoflurane group (group AS,n =28) and control group (group AC,n =16).Forty-four APP negative mice were randomly divided into 2 groups:sevoflurane group (group S,n =28) and control group (group C,n =16).Animals in groups S and AS inhaled 3％ sevoflurane for 4 h.While in groups C and AC,animals inhaled pure oxygen for 4 h.Morris water maze was performed 24 h after sevoflurane or pure oxygen inhalation.The phosphorylation of tau at Ser262 and Ser396 was detected by Western blot on 1 day after pure oxygen inhalation (T1) in groups AC and C,and on 1,3 and 7 days after sevoflurane inhalation in groups AS and S.Results Compared with group C,the escape latency was significantly prolonged and the duration of staying at the original platform quadrant was shortened in groups S and AC,and the phosphorylation of tau at Ser262 in group S and phosphorylation of tau at Ser262 and Ser396 in group AS were increased (P ＜0.05).Compared with group S,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened and the phosphorylation of tau at Ser262 and Ser396 was increased in group AS (P ＜ 0.05).Compared with group AC,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened and the phosphorylation of tau at Ser262 and Ser396 was increased in group AS (P ＜ 0.05).Conclusion Sevoflurane anesthesia can aggravate the impairment of cognitive function in APP positive mice and the increase in the phosphorylation of tau at Ser262 and Ser396 is involved in the mechanism.

Objective:To evaluate the modified efficacy of thoracic paravertebral block (TPVB) combined with general anesthesia in the patients undergoing laparoscopic radical nephrectomy.Methods:Eighty patients, aged 38-64 yr, with body mass index of 18-24 kg/m 2, of American Society of Anesthesiologists physical status Ⅰ or Ⅱ, scheduled for elective laparoscopic radical nephrectomy, were selected and randomly divided into 2 groups ( n=40 each) using a random number table method: general anesthesia group (group GA) and TPVB combined with general anesthesia group (group TPVB+ GA). A paravertebral catheter was placed at T 8 and T 10 under ultrasound guidance before induction of anesthesia, and 0.5% ropivacaine 10 ml was administered via the catheter in group TPVB+ GA.Anesthesia was induced with propofol, sufentanil, etomidate and rocuronium and maintained by intravenous infusion of propofol and remifentanil.Patient-controlled intravenous analgesia was performed with sufentanil, ketorolac tromethamine and tropisetron at the end of surgery.When postoperative visual analog scale score≥4, tramadol 50 mg was intravenously injected as rescue analgesic.Immediately before anesthesia induction (T 0), at 5 min after establishing pneumoperitoneum (T 1), at 2 h of pneumoperitoneum (T 2), and immediately after the end of pneumoperitoneum (T 3), and at 24 h after operation (T 4), venous blood samples were collected for determination of plasma norepinephrine concentrations (by enzyme-linked immunosorbent assay), plasma cortisol level (using radioimmunoassay), and blood glucose concentrations were measured.The intraoperative consumption of sufentanil and remifentanil was recorded.The intraoperative hypertension, hypotension, and bradycardia were recorded, and the nausea and vomiting, pruritus, and requirement for rescue analgesia occurred within 24 h after surgery were recorded. Results:Compared with group GA, the plasma concentrations of norepinephrine, cortisol and blood glucose were significantly decreased at T 1-4, the intraoperative consumption of sufentanil and remifentanil was reduced, and the postoperative requirement for rescue analgesia was decreased in group TPVB+ GA ( P<0.05). There was no significant difference in the incidence of intraoperative and postoperative adverse reactions between the two groups ( P>0.05). Conclusion:TPVB combined with general anesthesia is helpful in carrying out the anesthetic model of low-consumption opioids and is more helpful in inhibiting intraoperative and postoperative stress responses and postoperative pain responses than general anesthesia alone when used for laparoscopic radical nephrectomy.

Objective To evaluate the effect of sevoflurane preconditioning on high-mobility group box 1 protein ( HMGB1) ∕Toll-like receptor 4 ( TLR4) ∕nuclear factor kappa B ( NF-κB) signaling pathway during lung ischemia-reperfusion ( I∕R) in rats. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-250 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group ( group S) , lung I∕R group ( group I∕R) and sevoflu-rane preconditioning group ( group SP ) . The right pulmonary hilum was only isolated but not ligated in group S. Lung I∕R was induced by clamping the right pulmonary hilum for 60 min followed by 120 min of reperfusion in anesthetized rats in group I∕R. In group SP, 2. 1％ sevoflurane was inhaled for 30 min to per-form sevoflurane preconditioning, and the lung I∕R model was established at 10 min after the end of inhala-tion. The rats were sacrificed at 120 min of reperfusion, and the lungs were removed for examination of the pathological changes which were scored and for determination of wet to dry weight ratio ( W∕D ratio) , con-tent of tumor necrosis factor-alpha ( TNF-α) in lung tissues ( by enzyme-linked immunosorbent assay) and expression of HMGB1, TLR4 and NF-κB protein in lung tissues (by Western blot). Results Compared with group S, the pathological scores, W∕D ratio and content of TNF-α were significantly increased, and the expression of HMGB1, TLR4 and NF-κB was up-regulated in I∕R and SP groups ( P<0. 05) . Compared with group I∕R, the pathological scores, W∕D ratio and content of TNF-αwere significantly decreased, and the expression of HMGB1, TLR4 and NF-κB was down-regulated ( P<0. 05) , and the pathological changes of lung tissues were significantly attenuated in group SP . Conclusion Sevoflurane preconditioning reduces lung I∕R injury probably through inhibiting HMGB1∕TLR4∕NF-κB signaling pathway in rats.

BACKGROUND:Perinatal hypoxic-ischemic brain injury is one of the most common causes of cerebral palsy.Shujin Jiannao Prescription is an experienced formula for treating cerebral palsy and improving blood supply to the brain developed by the Dongzhimen Hospital,Beijing University of Chinese Medicine. OBJECTIVE:To explore the possible mechanism of Shujin Jiannao Prescription in treating hypoxic-ischemic cerebral palsy. METHODS:Sixty-four 7-day-old Sprague-Dawley rats were randomly divided into six groups.There were 12 rats in each of the control and model groups as well as 10 animals in each of the minocycline group,and the low-,medium-,and high-dose groups of Shujin Jiannao Prescription.The neonatal rat ischemic-hypoxic cerebral palsy model was established in all groups except for the control group.After successful modeling,rats in each drug group were respectively gavaged with minocycline and Shujin Jiannao Prescription at a dose of 4,8,and 16 g/kg per day for 1 week.Body mass of rats was measured and behavioral changes were detected before and after drug administration.Hematoxylin-eosin staining was used to observe the histomorphology of hippocampal CA1 region of rat brain tissue,and immunohistochemistry and western blot were used to detect the expression levels of Bcl-2,Bax,and Caspase-3 in the brain tissue of rats. RESULTS AND CONCLUSION:Compared with the model group,medium-and high-dose Shujin Jiannao Prescription significantly increased the body mass of rats(P<0.05).Compared with the model group,minocycline effectively prolonged the suspension time of ischemic-hypoxic cerebral palsy rats(P<0.05),while medium-and high-dose Shujin Jiannao Prescription significantly prolonged the suspension time,shortened the inclined plane test time,and increased the Longa score of rats(P<0.05).The pathological results showed that after drug intervention,only a small number of neuronal cells in the brain tissue of rats were necrotic,the cells were more neatly arranged,the cell structure was more complete,and only part of the cell nuclei became smaller.Compared with the model group,minocycline and medium-and high-dose Shujin Jiannao Prescription reduced the expression of Bax Caspase-3(P<0.05),medium-and high-dose Shujin Jiannao Prescription increased the expression of Bcl-2(P<0.05),and Bcl-2/Bax protein expression was increased in minocycline and three Shujin Jiannao Prescription groups(P<0.05).In addition,the protein expression was increased in a dose-dependent manner after intervention with Shujin Jiannao Prescription,and there was no significant difference between the minocycline and three Shujin Jiannao Prescription groups(P>0.05).To conclude,the mechanism by which Shujin Jiannao Prescription treats ischemic-hypoxic cerebral palsy in rats may be to enhance the expression of anti-apoptotic protein Bcl-2,inhibit the expression of pro-apoptotic protein Bax,and reduce the expression of Caspase-3,ultimately inhibiting the apoptosis of hippocampal neuronal cells in rats with cerebral palsy.Within a certain range,the higher dose of Shujin Jiannao Prescription indicates the better therapeutic effect,and the high-dose Shujin Jiannao Prescription is as effective as minocycline.

Objective:To investigate influences of ginsenoside Rg1 regulating miR-144-3p on neuroinflammation and blood-brain barrier damage in rats with experimental cerebral hemorrhage,and its regulation on formyl peptide receptor 2(FPR2)/p38 path-way.Methods:Ninety SD rats were randomly divided into control group,cerebral hemorrhage group,ginsenoside Rg1 low-dose group(10 mg/kg),ginsenoside Rg1 high-dose group(40 mg/kg),ginsenoside Rg1 high-dose+ago-miR-144-3p group(40 mg/kg ginseno-side Rg1+ago-miR-144-3p),with 18 mice in each group.Except for control group,experimental intracerebral hemorrhage rat model was constructed by injecting collagenase Ⅱ into right caudate nucleus,and then each group was given intraperitoneal administration and intracerebral injection as required.Neurological damage in rats was scored;rat brain water content was determined by dry-wet spe-cific gravity method;levels of TNF-α,IL-6 and IL-1β in rat brain tissues homogenate were determined by ELISA;ultrastructure around cerebral edema was observed by electron microscope;permeability of blood-brain barrier in rats was determined by Evans blue(EB)method;expressions of miR-144-3p/FPR2/p38 pathway were determined by qRT-PCR and Western blot.Results:Compared with control group,blood-brain barrier damage was aggravated in cerebral hemorrhage group,neurological function damage score,brain water content,miR-144-3p,TNF-α,IL-6,IL-1β,p38 mRNA,p-p38/p38 expressions in brain homogenate were increased(P<0.05),FPR2 mRNA and protein expressions were decreased(P<0.05);compared with cerebral hemorrhage group,blood-brain barrier damage was reduced in ginsenoside Rg1 low-dose group and ginsenoside Rg1 high-dose group,neurological function damage score,brain water content,miR-144-3p,TNF-α,IL-6,IL-1β,p38 mRNA,p-p38/p38 expressions in brain homogenate were decreased(P<0.05),FPR2 mRNA and protein expressions were increased(P<0.05);ago-miR-144-3p was able to reverse protective effects of gin-senoside Rg1 on blood-brain barrier and neuroinflammation in rats(P<0.05).Conclusion:Ginsenoside Rg1 may inhibit blood-brain barrier damage and neuroinflammation in rats by regulating miR-144-3p/FPR2/p38 axis.

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.
Pregnancy ; Female ; Animals ; Mice ; Tetraploidy ; Blastocyst ; Embryo, Mammalian ; Cell Differentiation ; Embryonic Development