OBJECTIVE To investigate the types of neurons that influence the head twitch response(HTR)induced by 5-hydroxytryptaminergic(5-HTergic)psychedelic mescaline in mice.METHODS①Adult male C57BL/6J mice were randomly divided into the normal control group and mescaline(1.56,3.125,6.25,12.5,25 and 50 mg·kg-1)groups,with 15 mice in each group.The drugs of the corresponding groups were ip given,and the HTR frequency of mice was recorded for 30 min.② 5-HT 2A receptor(5-HT2AR)gene bilateral LoxP homozygous mice(5-HT2A flox/flox)were hybridized with calmodulin depen-dent protein kinaseⅡα cyclization recombination enzyme positive(CaMKⅡαcre/+),parvalbumin(PV)cre/+,somatostatin(SOM)cre/+or vasoactive intestinal peptide(VIP)cre/+mice to obtain 5-HT2A R conditional knockout(cKO)mice(5-HT2AΔCaMKⅡα,5-HT2AΔPV,5-HT2AΔSOM and 5-HT2AΔVIP).Each type of cKO mice was randomly divided into the normal control group and mescaline 12.5 mg·kg-1 group,with 15 mice in each group.The drugs of the corresponding groups were ip given before the HTR frequency of mice within 30 min was recorded.③ Each type of cKO mice was randomly divided into the normal control group and mescaline 12.5 mg·kg-1 group,with 12 mice in each group.After receiving the corresponding drug via ip,they were placed in a spontaneous activity test box for 30 minutes and their activity levels were recorded.RESULTS ① Compared with the normal control group,mescaline 3.125,6.25,12.5 and 25 mg·kg-1 significantly increased the HTRs of mice(P<0.05,P<0.01).② Among the different neuronal types of 5-HT2AR cKO mice,only 5-HT2A ΔCaMKⅡα mice had no difference in HTR frequency between the normal control group and the mescaline 12.5 mg·kg-1 group.In 5-HT2AΔPV,5-HT2AΔSOM and 5-HT2AΔVIP mice,the HTRs of mice in the mescaline 12.5 mg·kg-1 group were significantly increased(P<0.01)compared with the normal control group.③ There was no difference in spontaneous activity between the normal control group and the mescaline 12.5 mg·kg-1 group of all cKO mice.CONCLU-SION Pyramidal neurons are involved in mediating the induction of mescaline on HTRs in mice.

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.

Objective To study the application of walking combined with Buerger exercise in the treatment of grade 0 diabetic foot.Methods Eighty patients with grade 0 diabetic foot were randomly divided into observation group and control group.The control group was trained by walking exercise .The observation group was trained by walking combined with Buerger exercise through 12 months of training.The changes of self-sensory symptoms,ABI and DAWV were observed.Results The self-sensory symptoms of the two groups were significantly decreased (observation group: markedly effective 15 cases,effective 19 cases;control group: markedly effective 5 cases,effective 23 cases).The effective rate of the observation group was higher than that of the control group (85% vs.70%,χ2 =7.831,P <0.05).The ABI ratio of the two groups were higher than those before treatment ,and the ABI ratio of the observation group was higher than that of the control group [(0.889 ±0.113) vs.(0.842 ±0.124),t =1.772 P <0.05].The ratio of DAWV in the observation group was higher than that in the control group [(6.772 ±0.435)cm/s vs.(6.543 ±0.552)cm/s,t =2.061,P <0.05].Conclusion Walking combined with Buerger exercise can improve the blood flow velocity and blood flow of the collateral circulation of the lower limbs of patients with grade 0 diabetic foot,and improve the self-sensory symptoms of peripheral neuropathy .

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 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.

Hypoxia refers to the reduction in tissue oxygen supply or utilization. It occurs in various pathological symptoms like embolism,anthracemia,and chronic obstructive sleep. At high altitude, lower partial pressure of oxygen compromises the supply of adequate oxygen to the tissues and leads to many clinical syndromes,such as acute mountain sickness,high-altitude cerebral edema,and high-altitude pulmonary edema. Histamine H3 receptor, primarily as a presynaptic receptor, is widely expressed in the central and peripheral systems. Histamine,dopamine,acetylcholine and many other neurotransmitters are regulated by histamine H3 receptor. Studies have shown that histamine H3 receptor is involved in the hypoxic response of the respiratory network. In addition,histamine,espe?cially histamine H3 receptor,participates in the regulation of cerebral ischemia in the central nervous system. In this paper,we reviewed the structure and functions of histamine H3 receptor and explained its role in the regulation of hypoxia so as to evaluate the possibility of histamine H3 receptor as a drug target for the therapy of hypoxia-induced injuries.

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.

Opioid receptors, as an important member of G protein coupled receptors (GPCR), are the binding targets of endogenous opioid peptides and exogenous opiates. The activation of opioid receptors influences the nervous system, immune physiology and endocrine system. However, prolonged activation of opioid receptors is likely to produce opioid tolerance, leading to opioid addiction. Receptor endocytosis and sorting into the recycling pathway contribute to recovery of cellular opioid responsiveness. Recent studies have revealed that GPCR can be modulated by ubiquitination which plays a unique roles in governing GPCR trafficking. Moreover, ubiquitination of the opioid receptors (μ, κand δ) is increased after stimulation of most opioid agonists. Mutation of the ubiquitin sites affects the internalization and degradation of opioid receptors, which contributes to changes in signal pathways and regulation of opioid receptors. ln this paper, ubiquitination of opioid receptors and the fundamental role of ubiquitination in trafficking of opioid receptors are reviewed.

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.