OBJECTIVE To establish the cells stably co-expressing α1A-adrenergic receptor(α1A-AR)and enhanced green fluorescent protein(EGFP)tagged nuclear factor of activated T cells 2(NFAT2)(EGFP-NFAT2)in U2OS cells.METHODS ① The pcDNA3.1-α1-AR-3×FLAG recombinant plasmid was transfected into U2OS-EGFP-NFAT2 cells.The transfected cells were selected by hygromycin B(Hygro-B,200 mg·L-1),and screened by EGFP-NFAT2 nuclear translocation assay after α1A-AR agonist norepinephrine(NE)treatment of 30 min.② The mRNA and protein expression levels of α1A-AR in the selected U2OS-EGFP-NFAT2-α1A-AR cells were examined by real-time quantitative PCR(RT-qPCR)and Western blotting.③ U2OS-EGFP-NFAT2-α1A-AR cells were treated with NE(10-8-10-5 mol·L-1)or dexmedetomidine(DMED,10-8.8-10-5 mol·L-1),respectively,for 30 min.EGFP-NFAT2 nuclear translo-cation was detected by high throughout screening assay.④ The U2OS-EGFP-NFAT2-α1A-AR cells were divided into the solvent control group,α1-AR antagonist naftopidill(1 μmol·L-1)group,NE(1 μmol·L-1)group and naftopidill+NE(co-incubation with naftopidill 1 μmol·L-1 and NE 1 μmol·L-1)group,α2-AR antagonist atipamezole(ATI,0.1 μmol·L-1)group,α 2-AR agonist DMED(0.1 μmol·L-1)group,and ATI+DMED(co-incubation with ATI 1 μmol·L-1 and DMED 0.1 μmol·L-1)group.The drug incubation time was 30 min.EGFP-NFAT2 nuclear translocation was abserved via a high throughout screening system to validate the α1A-AR function in U2OS-EGFP-NFAT2-α1A-AR cells.RESULTS ① There were 58 cell strains expressing α1A-AR in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay.Among these cells,cells No 50 had the highest nuclear translocation function.The α1A-AR mRNA expression of cells No 50 in 5-20 generations were detected by RT-qPCR and were about 500-800 times that of U2OS-EGFP-NFAT2 cells.② The protein band of α1A-AR was also detected in cells No 50,but no band of α1A-AR was detected in U2OS-EGFP-NFAT2 cells by Western blotting.③ NE and DMED increased the relative translocation nuclear index in U2OS-EGFP-NFAT2-α1A-AR cells with ED50 5.94×10-7 and 6.15×10-8 mol·L-1,respectively.④ EGFP-NFAT2 nuclear translocation was significant in U2OS-EGFP-NFAT2-α1A-AR cells after NE addition compared with the solvent control or the naftopidill groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the naftopidill+NE group was significantly decreased compared with the NE group(P<0.01).DMED significantly increased the EGFP-NFAT2 nuclear translocation compared with solvent control or the ATI groups(P<0.01).The EGFP-NFAT2 nuclear translocation in the ATI+DMED group was similar to that of the DMED group.The EGFP-NFAT2 nuclear translocation in the naftopidill+DMED group was decreased significantly compared with the DMED group(P<0.01).CONCLUSION U2OS-EGFP-NFAT2-α1A-AR cells stably co-exrepssing α1A-AR and EGFP-NFAT2 are established,which can be used for high throughout screening of biased chemicals and studies on the mechanism of α1A-AR.

Most α2-AR agonists derived from dexme-detomidine have few structure differences between them and have no selectivity for α2A/2B-AR or Gi/Gs,that can lead to the side effect of drugs.To get novel and potent α2A-AR agonists,we built the homology model for human α 2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity.Compound P300-2342 and its 3 analogs sig-nificantly decreased the locomotor activity of mice(P＜0.05).Furthermore,P300-2342 and its 3 analogs inhibited the binding of[3H]rauwolscine to α 2A-AR and α 2B-AR respectively.In α2A-AR-HEK293 cells,P300-2342 decre-ased forskolinstimulatedcAMPpruductionwithoutincreas-ing cAMP pruduction,that indicated the P300-2342 acti-vating α2A-AR coupling with Gαi/o pathway without Gαs coupling.P300-2342 had no agonistic and antagonistic activities on α 2B-AR.Similar results were shown in 3 analogs of P300-2342.The docking results showed that P300-2342 formed the π-hydrogen bonds with Y394,V114 of α2A-AR,and with V93 of α2B-AR.3 analogs of P300-2342 formed several π-hydrogen bonds with V114,Y196,F390 of α 2A-AR and with V93 of α 2B-AR.We believe that these molecules can serve as leads for fur-ther optimization of α2A-AR agonists with potentially few side effects.

Ulcerative colitis (UC) manifests as an etiologically complicated and relapsing gastrointestinal disease. The enteric nervous system (ENS) plays a pivotal role in rectifying and orchestrating the inflammatory responses in gut tract. Berberine, an isoquinoline alkaloid, is known as its anti-inflammatory and therapeutic effects in experimental colitis. However, little research focused on its regulatory function on ENS. Therefore, we set out to explore the pathological role of neurogenic inflammation in UC and the modulating effects of berberine on neuro-immune interactions. Functional defects of enteric glial cells (EGCs), with decreased glial fibrillary acidic protein (GFAP) and increased substance P expression, were observed in DSS-induced murine UC. Administration of berberine can obviously ameliorate the disease severity and restore the mucosal barrier homeostasis of UC, closely accompanying by maintaining the residence of EGCs and attenuating inflammatory infiltrations and immune cells overactivation. , berberine showed direct protective effects on monoculture of EGCs, bone marrow-derived dendritic cells (BMDCs), T cells, and intestinal epithelial cells (IECs) in the simulated inflammatory conditions. Furthermore, berberine could modulate gut EGCs-IECs-immune cell interactions in the co-culture systems. In summary, our study indicated the EGCs-IECs-immune cell interactions might function as a crucial paradigm in mucosal inflammation and provided an infusive mechanism of berberine in regulating enteric neurogenic inflammation.

Objective: To evaluate the occupational health risk of decorative coating manufacturing enterprises and to explore the applicability of occupational hazard risk index model in the health risk assessment, so as to provide basis for the health management of enterprises. Methods: A decorative coating manufacturing enterprise in Hebei Province was chosen as research object, following the types of occupational hazards and contact patterns, the occupational hazard risk index model was used to evaluate occupational health risk factors of occupational hazards in the key positions of the decorative coating manufacturing enterprise, and measured with workplace test results and occupational health examination. Results: The positions of oily painters, water-borne painters, filling workers and packers who contacted noise were moderate harm. And positions of color workers who contacted chromic acid salts, oily painters who contacted butyl acetate were mild harm. Other positions were harmless. The abnormal rate of contacting noise in physical examination results was 6.25%, and the abnormality was not checked by other risk factors. Conclusion The occupational hazard risk index model can be used in the occupational health risk assessment of decorative coating manufacturing enterprises, and noise was the key harzard among occupational harzards in this enterprise.

Objective To investigate the role of Calcineurin binding protein 1(Cabin1)in renal tubular epithelial cells(RTECs)injury. Methods The male Sprague-Dawley rats were randomly divided into Sham-oper-ated and 5/6 nephrectomized group. Nephrectomized rats were further divided into two groups ,which were 4 and 8 weeks after operation,including 6 rats in each group. Rats were sacrificed at 4 or 8 weeks after nephrectomy,then control or remnant kidneys were harvested. 2μm sections of kidney tissues were collected and stained with Masson's trichrome and were graded for tubulointerstitial lesion score (TILS). RTECs mitochondrial morphology changes were detected by electron microscope. Western blot was applied to detect Cabin1 protein level in the renal tissue. Results At 8 weeks after the operation,plenty of RTECs fell off from the basement membrane,accompanied with interstitial fibrosis and the infiltration of inflammatory cells. Moreover ,TILS were significantly increased in rats at 8 weeks after operation while compared to sham-operated rats(7.16 ± 0.52 vs. 0.00 ± 0.00,P<0.05). RTECs mi-tochondria begun to swell at 4 weeks after 5/6 nephrectomy,while the disruption of cristae could be found in rats at 8 weeks. Cabin1 protein expression apparently increased in the remnant kidney. Cabin1 protein obviously increased in rats at 8 weeks after the surgery compared to sham-operated rats(0.97 ± 0.09 vs. 0.22 ± 0.07,P<0.05)and rats at 4 weeks after nephrectomy(0.97 ± 0.09 vs. 0.45 ± 0.03,P<0.05). Conclusions Cabin1 is overexpressed during RTECs injury in 5/6 nephrectomized rats. It can be a crucial factor regulating the damage of RTECs.

Objective To investigate the function and molecular mechanism of tacrolimus in podocyte injury and restoration.Methods Cultured podocytes were stimulated by Angiotensin Ⅱ (Ang Ⅱ) or Ang Ⅱ plus tacrolimus.Cells were collected at different time points (0 h,12 h and 24 h).The distribution of F-actin was observed after immunofluorescence staining,and the protein expression of nephrin and podocin were detected by Western Blot (WB).Results In normal control podocytes,F-actin was arranged in cytoplasm powerfully.Ang Ⅱ induced the disruption and discontinuity of F-actin.Tacrolimus inhibited the effect of Ang Ⅱ,stabilized the regular arrangement the F-actin.Compared to normal cells,the protein expression of nephrin in Ang Ⅱ group significantly decreased at 24 h after stimulation (0.76 ± 0.32 in AngⅡ group vs.1.18 ± 0.40 in normal group,P ＜ 0.05).And tacrolimus stabilized the expression of nephrin protein (1.00 ± 0.19 in treatment group vs.0.76 ± 0.32 in Ang Ⅱ group,P ＜ 0.05).Ang Ⅱ and tacrolimus did not affect the expression of podocin protein.Conclusion Tacrolimus inhibits podocyte injury induced by Ang Ⅱ,stabilizes the regular arrangement of cytoskeleton and protein expression of nephrin.

OBJECTIVE To establish a new cell line that can stably express humanα2A-adrenoceptor (α2A- AR). METHODS Recombinant plasmid of α2A- AR with hygromycin B (Hygro) resistance (pcDNA3.1/Hygro-HA-α2A-AR)was stably transfected into Chinese hamster ovary(CHO)cells which had expressed protein kinase A catalytic subunits(PKAcat) with labeling of enhanced green fluorescent protein(EGFP)by a Lipofectamine based method. A single positive clone expressingα2A-AR was selected through cultivation in the presence of 200 mg · L-1 hygromycin B followed by PKA redistribution assay. The transcriptional expression ofα2A-AR was detected by quantitative real-time PCR(qRT-PCR). Time-resolved fluorescence resonance energy transfer immunoassay was used to identify the function of inhibiting cAMP accumulation of α2A-AR. RESULTS The CHO-PKAcat-α2A-AR cell line No.7 exhibited stable response in PKA redistribution assay. qRT-PCR analysis demonstrated that the high expression ofα2A-AR in the cell line remained stable after a few generations compared with CHO-PKAcat-EGFP cells (P<0.01). The cAMP accumulation caused by forskolin was significantly inhibited by α2A-AR agonist in CHO-PKAcat-α2A-AR cells(P<0.01). CONCLUSION CHO-PKAcat-α2A-AR cell line is constructed successfully, which provides an effective model for drug screening and studies of mechanisms.

Objective To explore the impact of intensive follow-up visit in patients with nonvalvular atrial fibrillation taking Warfarin in process of anticoagulant therapy. Methods A total of 100 patients with persistent nonvalvular atrial fibrillation were randomly divided them into intensive follow-up group and regular follow-up group on average. We compared the treatment compliance, INR value, embolism and hemorrhage between two groups. Results The INR qualified rate was 86. 0% after 6 months taking medication in the intensive follow-up group and 72. 0% in the regular follow-up group (χ2 =4. 570,P<0. 05). The incidence rate of hemorrhage was 12% in the intensive follow-up group and 28% in the regular follow-up group (χ2 =4. 000,P<0. 05). Conclusions With the function of propaganda, education, supervision, promotion and attention on patients, follow-up visit makes patients taking Warfarin receive normative, effective anticoagulant treatment and improves the safety of medicine use, thus the effect of preventing stroke can be achieved.

OBJECTIVE Toestablisharecombinantcelllinethatcanstablyexpressratpurinergic P2receptorP2X4(rP2X4R).METHODS ToconstructgreenfluorescentproteinandrP2X4recombinant plasmid (pEGFP-N1-rP2X4),lipofectamine was used to transfect pEGFP-N1-rP2X4 into human embry-onic kidney (HEK293)cells that were screened with G41 8 (1 g·L-1 ).The quantitative expression of rP2X4 receptor was verified by qRT-PCR and Western blotting analysis.Whole-cell patch clamp record-ing was used to investigate the function of the stably expressed rP2X4 receptor. RESULTS The sequence of plasmid pEGFP-N1-rP2X4 was verified by PubMed Blastn comparison.qRT-PCR and Western blotting analysis demonstrated that the expression of rP2X4 receptor in HEK293-pEGFP-N1-rP2X4 cell lines remained stable after 25 generations (P1 ,P3,P5,P10,P15,P20 and P25).Whole-cell patch clamp recording experiments showed that the rP2X4 receptor agonist,purine-5′-triphosphate (ATP,3.0 μmol·L-1 ),could activate rP2X4 receptors in HEK293-pEGFP-N1-rP2X4 cell lines.Specific activating current could be blocked by non-selective rP2X4 receptor antagonist TNP-ATP (30.0μmol·L-1).CONCLUSION rP2X4receptorisstablyexpressedinHEK293-pEGFP-N1-rP2X4cell line and maintains stable expression and function within 25 continuous generations.The establish ment of HEK293-pEGFP-N1-rP2X4 cell line can contribute to further investigations of the roles of rP2X4 receptors in neuropathic pain.

Objective To investigate the effects of preoperative parecoxib on the levels of inter-leukin 6 (IL-6)and postoperative analgesia for breast surgery.Methods Sixty breast cancer patients undergoing mastectomy were randomly divided into two groups:parecoxib group (group P)and con-trol group (group C),n=30 in each group.All patients received sevoflurane and fentanyl anesthesia. Group P was injected parecoxib 40 mg at 10 minutes before induction of anesthesia,meanwhile group C was injected saline 5 ml.All patients received postoperative patient-controlled intravenous analgesia (PCIA)with fentanyl.VAS scores for pain were assessed at postoperative 2,4,8,12,24 hours. The serum levels of IL-6 were measured by ELISA at 10 minutes before induction,4 h,8 h,and 24 h after surgery.Results Group P had lower VAS scores than group C at 2-12 h after surgery (P <0.05).Compared with 10 minutes before induction,the levels of IL-6 increased significantly at post-operative 4,8,24 h in two groups (P<0.01),while group P had lower levels of IL-6 than group C (P <0.01).Conclusion Preoperative administration of parecoxib has a stronger analgesic effect in breast cancer patients after mastectomy,and decreases the levels of IL-6.