Research progress of NOD2-mediated signaling pathways and relationship with autoinflammatory diseases and its inhibitors / 药学学报

Nucleotide-binding oligomerization domain containing protein 2 (NOD2) is a member of intracellular pattern recognition receptor. After being activated, it will induce the release of inflammatory factors through a series of signal cascade transduction, thus playing an important role in the innate immune response. The abnormal NOD2 signaling pathway is involved in the occurrence and development of many diseases, especially the single nucleotide polymorphisms (SNPs) of the NOD2 gene have been identified to be closely associated with autoinflammatory diseases (AIDs). Therefore, inhibitors targeting NOD2 pathway have great potential in the treatment of inflammatory immune diseases. This review presents the recent progress of NOD2 receptor-mediated signal transduction pathways and its regulation mechanisms, the relationship between NOD2 and AIDs, and the inhibitors of NOD2 pathway.

Inhibitory effects of carboxyamidotriazole on cytokines production by peritoneal macrophages from adjuvant arthritis rats / 基础医学与临床

Objective To explore the effects of carboxyamidotriazole (CAI) on cytokines production by peritoneal macrophages from adjuvant arthritis(AA) rats in vitro. Methods Freund's completed adjuvant was used to induce AA in rats.Peritoneal macrophages were prepared from asepsis and incubated with CAI(10,20,40 μmol/L).The contents of TNF-α,IL-1β and IL-6 in the culture supernatant were measured by ELISA,and mRNA expressions of TNF-α,IL-1β and IL-6 were determined by real-time quantitative PCR.NF-κB p65 DNA binding activity in the nu-clear protein was detected with TransAM kit. Results The level of TNF-α,IL-1β and IL-6 in the culture superna-tant, their intracellular mRNA expression and NF-κB p65 DNA-binding activity in peritoneal macrophages of AA rats were significantly inhibited by CAI (20 and 40 μmol/L) (P<0.05 and P<0.01).Conclusions CAI may de-crease the production of pro-inflammatory cytokines such as TNF-α,IL-1β and IL-6 through inhibiting the activa-tion of NF-κB,which is potentially associated with its anti-arthritic mechanisms.

Attitudes of 8-year program medical students to concept mapping after the pharmacology teaching/learning practice and analysis of the learning readiness for students / 基础医学与临床

Concept mapping is a new method to help learners organizing their knowledge with a visual semantic net-work. In the process of pharmacology teaching concept mapping are used as a teaching tool and a learning tool, respectively. Then a survey was conducted on 8-year program medical students to obtain information regarding students attitude towards concept mapping in pharmacology teaching and learning as well as their learning readiness. The authors hope the information may play a direction role for the teaching activity in the future and make Pharmacology teaching contents and methods meet the knowledge requirement and learning readiness of medical students.

Culture and identification of mouse submandibular epithelial cells in vitro / 基础医学与临床

Objective To establish a culture method of mouse submandibular epithelial cells and to explore the optimal isolation and culture conditions so as to provide an in vitro experimental model for cell biology study and drug evaluation of salivary gland related diseases such as Sjogren's syndrome. Methods Collagenase type Ⅳ was used to digest and isolate the submandibular cells of mice. And the survival rate of cells was determined by trypan blue stai-ning. After purified by differential attachment method, the cells were cultured in F-12/DMEM medium containing10 μg/L epidermal growth factor. Optical microscope was applied to observe the morphology of the cultured cells and the cell proliferation feature was estimated by proliferation curve. In addition, immunofluorescence staining was conducted to identify the cells. Results The cell survival rate obtained by collagenase digestion was 97.5％. The morphology characteristic showed the typical epithelioid with polygon in the arrangement of typical " pebble stone" appearance. The cells were stable in growth with active proliferation according to the proliferation curve and could be subcultured to three passages. Immunofluorescence results showed that the expression of cytokeratin 8 was positive while vimentin was negative, which was consistent with the phenotypic characteristics of salivary gland cells. Conclusions The method of primary culture and subculture of mouse submandibular epithelial cells was successfully established. The method is easy to operate, which provide a potential method basis for further research study.

Beneficial effect of berberine on atherosclerosis based on attenuating vascular inflammation and calcification / 基础医学与临床

Objective To investigate the beneficial effect of berberine (BBR) on atherosclerosisin Apo-/-E mice and explore the underlying mechanisms. Methods Fortyeight Apo-/-E mice were randomly allocated into 4 groups: control, model (fed with highfat diet for 4 weeks), BBR[p.o., 100 mg/(kg · d)]and Atorvastatin [p.o., 5 mg/ (kg · d)]groups with 12 mice in each group. The morphology and inflammation infiltration of aortic were examined with HE staining. The expressions of BMP-2, OPG, OCN, RUNX2 in aortic were examined by immumohisto-chemical staining. Blood lipid levels were examined by automatic biochemical analyzer. The expressions of IL-6, TNF-α and BMP-2 in serum and tissues were detected by ELISA method. The expression of ALP and the content of calcium were detected. HUVECs were stimulated with TNF-a and incubated with various concentrations of BBR for 24 h.The content of ICAM-1, VCAM-1, MMP-9 in the culture supernatant was detected by ELISA method. Results 4-week berberine treatment significantly reduced serum TC and LDL-c levels and improved the plaque stability in Apo-/-E mice fed with a high-fat diet(P＜0.05) which was comparable with the effect of atorvastatin. Berberine also significantly decreased the level of IL-6 and TNF-a in mice serum and aortic tissues (P＜0.05). Berberine tended to decrease ALP, BMP-2 levels and the content of calcium in mice serum and aortic tissues (P＜0.05) which were not observed in atorvastatin group. Berberine significantly reduced the levels of ICAM-1, VCAM-1, and MMP-9 in TNF-a-stimulated HUVECs. Conclusions BBR can profitably regulate the level of blood lipid in mice fed with a high-fat diet, decrease the injury caused by inflammation, and attenuate vascular calcification. It may improve atherosclerosis and make a contribution to cardiovascular protection.

Therapeutic Effect of Carboxyamidotriazole on Adjuvant Arthritis in Rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of carboxyamidotriazole (CAI) on adjuvant arthritis (AA) in rats.</p><p><b>METHODS</b>The rats were randomly divided into normal group,two vehicle groups (polyethylene glycol 400 control and normal sodium control group), CAI-treated groups (10, 20, and 40 mg/kg) and positive control dexamethasone group. Freund's completed adjuvant was used to induce AA in rats. The arthritis index (AI) was scored, and X-ray check of the hind limbs and histopathological examination were performed. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the inflamed paw tissues were measured.</p><p><b>RESULTS</b>The administration of CAI significantly decreased the AI, restored the body weights, and ameliorated the radiological and histopathological features of joint destruction in AA rats (P<0.05, P<0.01). In addition, CAI reduced the TNF-α, IL-1β, and IL-6 levels in the inflamed paw tissues (P<0.05, P<0.01).</p><p><b>CONCLUSION</b>CAI has therapeutic effect on AA rats, which may be achieved by decreasing the pro-inflammatory cytokines at the site of inflammation.</p>

Anti-inflammatory and analgesic potency of carboxyamidotriazole, a tumoristatic agent / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the potential anti-inflammatory and analgesic activities of carboxyamidotriazole (CAI).</p><p><b>METHODS</b>A variety of animal models, including the croton oil-induced ear edema, the cotton-induced granuloma, the rat adjuvant-induced arthritis, were used to evaluate anti-inflammatory effect of CAI. Vascular endothelial growth factor (VEGF)--or histamine-stimulated local vascular permeability in mouse modulated by CAI was also determined. In addition, we assessed the effect of CAI on the levels of proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-beta) at the site of inflammation and in sera. Moreover, antinociceptive effect of CAI on inflammatory pain was assessed using acetic acid-induced writhing model and the formalin test.</p><p><b>RESULTS</b>CAI significantly inhibited acute and chronic phases of inflammation, reduced VEGF or histamine-induced vascular permeability, and showed marked inhibition of proinflammatory cytokines such as TNF-alpha and IL-1 beta. CAI also showed potential therapeutic effect on peripheral inflammatory pain.</p><p><b>CONCLUSION</b>CAI is a promising anti-inflammatory and analgesic agent.</p>

Effect of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.</p><p><b>METHODS</b>Using whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.</p><p><b>RESULTS</b>(1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.</p><p><b>CONCLUSIONS</b>Dual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.</p>

Dual regulation by delta opioid receptor agonists on the delayed rectified potassium channels in NG108-15 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the dual effects by the delta opioid receptor agonists DPDPE on the delayed rectified potassium channels in NG108-15 cells.</p><p><b>METHODS</b>A series of outward currents were evoked in NG108-15 cells by depolarizing voltage from -50 mV to +80 mV at holding potential of -90 mV. These currents were delayed rectified potassium currents. Relatively selected delta opioid receptor agonists DPDPE of higher and lower concentrations were used to modulate the delayed rectified K+ current in NG108-15 cells. Opioid receptor antagonist Naloxone (NAL) and relatively selected delta opioid receptor antagonist Naltrindole (NTI) were used in the present experiments for the characterization of the actions of opioid receptors.</p><p><b>RESULTS</b>The relatively higher concentrations of delta opioid receptor agonist DPDPE (> or = 10(-6) mol/L) significantly increased the amplitude of the delayed rectified K+ current. On the contrary, the relatively lower concentrations of DPDPE (< or = 10(-12) mol/L) decreased the amplitude of the delayed rectified K+ current (P < 0.05). Furthermore both the increase and decrease were time-dependent.</p><p><b>CONCLUSIONS</b>delta opioid receptor agonist has dual regulatory effects on the delayed rectified potassium channels in NG108-15 cells.</p>

Binding characteristics of new synthesized opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To determine the affinity of new opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell.</p><p><b>METHODS</b>The binding characteristics of the opioid ligand [3H] diprenorphine (3H-dip) were studied by cellular biological techniques and radioligands binding in cloned mu opioid receptors stably expressed in CHO cells in saturation binding experiments, and were followed by competition binding experiments with a variety of new synthesized opioid receptor ligands.</p><p><b>RESULTS</b>The Kd and Bmax of [3H] diprenorphine bound to mu receptors were 1.06 nmol/L and 930 fmol/mg protein, respectively. Competition binding experiments revealed that ligand 3# and 12# displayed much higher affinity than DAMGO and Morphine for the cloned mu opioid receptor. However, the affinities of ligands 2#, 6#, 8# and 9# were lower than DAMGO and Morphine.</p><p><b>CONCLUSION</b>The present results suggest that the new ligands 3# and 12# have higher affinity to mu opioid receptors. However, ligands 2#, 6#, 8# and 9# have lower affinity to mu opioid receptors.</p>