Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.

The design of home care platform and its application in the management of clinical nurse specialists in orthopedics in Jiangsu Province / 中华护理杂志

We constructed a home care platform for orthopedics,and clinical nurse specialists in orthopedics in Jiangsu Province opened online clinics on it.Patients with knee or hip joint replacement could be added to the platform,and the clinical nurse specialists provided patients with professional home care service when discharged.In the interviews of clinical nurse specialists,they said that the application of the platform was conducive to enhance their own values,and expressed their willingness to continue to use it.The joint function and quality of life scores of the intervention group were significantly higher than those of the control group (P＜0.001).The application of the home care platform is conducive to give full play to the role of clinical nurse specialists to provide professional home care services for patients.

Expression of Wnt and integrin pathways in colorectal laterally spreading tumors and their correlation with endoscopic subtypes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of Wnt and integrin pathways in colorectal laterally spreading tumors (LSTs) and their correlation with the different endoscopic subtypes of LSTs to better understand the special growth mechanism of LSTs.</p><p><b>METHODS</b>Fifty-two patients with colorectal LSTs were randomly selected from the cases diagnosed between January 1, 2010 and June 10, 2015 in our hospital, including 37 of nodular mixed type (LST-G-M), 60 of homogeneous type (LST-G-H), 5 of flat elevated type (LST-NG-FE), and 4 of pseudodepressed type (LST-NG-PD). The expression of β-catenin, phospho- GSK-3β, paxillin and ILK in 52 colorectal LSTs and 15 protruded adenomas (PAs) were investigated by immunohistochemical staining. The correlation of β-catenin, phospho-GSK-3β, paxillin and ILK expressions among the endoscopic subtypes of LSTs were analyzed.</p><p><b>RESULTS</b>β-catenin expression was significantly higher in LSTs than in Pas (P<0.05). β-catenin, phospho-GSK-3β, paxillin and ILK expressions were significantly higher in LST-NG-PD than in Pas (P<0.05). The expressions of β-catenin, phospho-GSK-3β and ILK expression were significantly correlated in LSTs (P<0.05) but not in PAs (P>0.05).</p><p><b>CONCLUSION</b>The macroscopic feature of LST-NG-PD may result from a special mechanism of development distinct from other endoscopic subtypes; ILK may play a role in regulating Wnt signaling in LSTs.</p>

Hemolytic Analysis of Two Kinds of Placement of Leukoreduced Suspension Red Blood Cells Storage Bag / 现代检验医学杂志

Objective Discussion leukoreduction of red blood cells suspended in two different storage bag placement and he-molysis rate impact on the supernatant free hemoglobin (FHb),to ensure the clinical transfusion is safe and effective.Meth-ods Selected 20 donors to sample 400 ml whole blood per person to make leukodepleted red blood cells,which were evenly divided into 10 bags.The 10 bags were randomly divided into two groups,one to the upright position,one group of horizon-tal.The two groups were stored under the same conditions.Respectively,in the 7,14,21,28,35 day,randomly removed one storage bag from each group,FHb and red blood cell hemolysis rate were measured and analyzed statistically.Results FHb and hemolysis rate results stored in the first 21 days of testing,uprightgroup were (217.310±48.477)mg/L and (0.250± 0.056)%,respectively horizontal group (173.972±39.027)mg/L and (0.189±0.045)%,the results set upright than hori-zontal group,the results were statistically(t=3.114,P =0.003<0.05 and t=3.798,P =0.001<0.05),the difference was statistically significant.Conclusion In the blood storage period,storage bags can be placed horizontally to reduce the de-struction of red blood cells,blood storage is more favorable.

5-aza-2'-deoxycytidine-induced inhibition of CDH13 expression and its inhibitory effect on methylation status in human colon cancer cells in vitro and on growth of xenograft in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human colon carcinoma cells and xenografts in nude mice, to observe its effect on CDH13 gene expression and methylation in the xenografts, and to explore the possible mechanisms.</p><p><b>METHODS</b>Human colon carcinoma cell line HCT116 cells were treated with 5-Aza-CdR, and the cell morphology was observe by phase contrast microscopy. The cell growth was assessed by MTT assay. A tumor-bearing mouse model was generated by subcutaneous inoculation of human colon carcinoma HCT116 cells into nude mice. The tumor growth in the nude mice was observed, the CDH13 gene expression and its methylation status in the tumors were detected using methylation specific PCR (MSP), RT-PCR, Western blotting and immunohistochemistry.</p><p><b>RESULTS</b>After treatment with 5-Aza-CdR, the inhibition rate of the growth of cultured HCT116 cells was increased as the concentration was increasing. The growth of the xenografts in nude mice was significantly inhibited, and the methylated CDH13 gene was reactivated. After 4 weeks of 5-Aza-CdR treatment, no significant difference was found between the body weights of nude mice in the 5-Aza-CdR group [(18.06 ± 1.29) g] and control group [(17.07 ± 0.84) g], (P > 0.10), and the average volume of xenografts of the 5-Aza-CdR group was (907.00 ± 87.29) mm(3), significantly smaller than the (1370.93 ± 130.20) mm(3) in the control group (P < 0.005). No expression of CDH13 gene was found in the control group. The expression of CDH13 gene in the 5-Aza-CdR group was increased along with the increasing concentration of 5-Aza-CdR.</p><p><b>CONCLUSIONS</b>5-Aza-CdR inhibits the growth of human colon cancer cells in culture and in nude mice, and induces the cancer cells to re-express CDH13 in nude mice. Its mechanism may be that demethylation of the methylated CDH13 promoter induced by 5-Aza-CdR restores CDH13 expression and thus inhibits the tumor growth in nude mice.</p>

Effect of 5-Aza-CdR on expression and methylation of E-cadherin gene in human colon carcinoma cells / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Colon cancer is one of the most common malignant tumors, and its pathogenesis is not fully understood. Transcriptional silencing by DNA methylation is believed to be an important mechanism of carcinogenesis. E-cadherin can suppress tumor cell invasion and metastasis, and is considered as an invasion/metastasis suppressor gene. Inactivation of E-cadherin gene often occurs in colon carcinoma. This study was to investigate the correlation between E-cadherin gene expression and the methylation status of E-cadherin 5' CpG islands in human colon carcinoma cell line HT-29, and to explore the mechanism of carcinogenesis of colon cancer.</p><p><b>METHODS</b>Immunocytochemical dicho-step method and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of E-cadherin protein and mRNA in HT-29 cells after 5-Aza-CdR treatment; methylation specific PCR was used to analyze the methylation status at promoter of E-cadherin gene.</p><p><b>RESULTS</b>The expression of E-cadherin gene could be restored by 5-Aza-CdR treatment, immunocytochemical staining showed the positive expression ratio of E-cadherin increased from (21+/-7)% (1 micromol/L) to (39+/-13)% (5 micromol/L); E-cadherin genes were methylated and not expressed in HT-29 cells in the colon carcinoma.</p><p><b>CONCLUSIONS</b>E-cadherin methylation plays an important role in the carcinogenesis of colon carcinoma cells and can re-express after the treatment with 5-Aza-CdR.</p>

Preparation of silymarin-loaded amphiphilic chitosan micelle and its in situ absorption in rat intestine / 药学学报

To improve the oral bioavailability of silymarin, the silymarin-loaded amphiphilic chitosan micelles (SM-OGC) were prepared. The absorption of SM-OGC in rat intestine was investigated. SM-OGC was prepared by dialysis method. The size and zeta potential of SM-OGC were investigated. Compared to silymarin suspension, the absorption of SM-OGC was investigated using in situ single pass perfusion model. The diameters and zeta potential SM-OGC were (162.4 +/- 3.0) nm and (+32.6 +/- 0.98) mV, respectively. The encapsulation efficiency was (39.17 +/- 0.98)% and the drug loading of SM-OGC was (28.15 +/- 0.43)%. The absorption of SM-OGC at different segments of intestine was significantly higher than that of silymarin suspension (P < 0.05). The apparent absorption rate (K(a)) and effective permeation coefficient (P(eff)) at the duodenum were the largest. K(a) and P(eff) had no significant difference between jejunum, ileum and colon. OGC micelles might significantly promote the absorption of silymarin in the intestine tract.

Effects of nuclear factor-kappa B p65 ASODN on transforming growth beta-1 and intercellular adhesion molecule-1 of rat hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of nuclear factor (NF)-kappa B p65 ASODN on transforming growth factor beta-1 (TGF beta 1) and intercellular adhesion molecule-1 (ICAM-1) of rat hepatic stellate cells (HSC) and the mechanisms of NF-kappa B p65 ASODN in treating liver fibrosis.</p><p><b>METHODS</b>Type IV collagen enzyme digestion and density centrifugation methods were used to separate rat hepatic stellate cells. NF-kappa B p65 ASODN was manually synthesized and completely phosphorothioate-modified. The changes of TGF beta 1 and ICAM-1 mRNA were detected by RT-PCR and albumen of TGF beta 1 and ICAM-1 were detected by ELISA. The changes of NF-kappa B activity were determined by ELISA.</p><p><b>RESULTS</b>NF-kappa B activity and the expressions of ICAM-1 and TGF beta 1 increased after the HSC were treated by TNF alpha. NF-kappa B activity weakened after being treated with NF-kappa B p65 ASODN (0.001-1.000 micromol/L), P less than 0.05 in a dose dependent manner. Transferring NF-kappa B p65 ASODN (0.001-1.000 micromol/L) also weakened the expression of ICAM-1 and TGF beta 1 mRNA and the protein induced by TNF alpha in HSC. It was also in a dose dependent manner, P less than 0.05.</p><p><b>CONCLUSIONS</b>After transferring NF-kappa B p65 ASODN into HSC, their NF-kappa B activity decreased, and their mRNA and protein expressions of ICAM-1 and TGF beta 1 also decreased. This may serve as a new way in treating hepatic fibrosis.</p>

Preparation of doxorubicin-loaded chitosan polymeric micelle and study on its tissue biodistribution in mice / 药学学报

To prepare doxorubicin-loaded N-octyl-N'-succinyl chitosan polymeric micelle (DOX-OSC) and study the biodistribution of DOX-OSC in mice, DOX-OSC was prepared by dialysis method. By using doxorubicin injection (DOX-INJ) as control, DOX-OSC and DOX-INJ were administered to mice through caudal vein at a dose of 5 mg x kg(-1) body weight. The RP-HPLC method was established to determine the DOX levels in the plasma and other tissues of mice. The tissues distribution and targeting efficiency were evaluated by pharmacokinetic parameters (AUC, MRT) and targeting parameters (Re, Ce and Te). The drug loading and entrapment efficiency of DOX-OSC were (35.8 +/- 0.4)% and (75.3 +/- 1.1)%, respectively. The diameter and zeta potential of DOX-OSC were (174 +/- 12) nm and (-37.1 +/- 3.0) mV, respectively. The transmission electron microscope result showed DOX-OSC with spherical shape. The biodistribution results showed that the concentration of DOX of both DOX-OSC and DOX-INJ decreased rapidly in blood after iv administration. While free DOX levels in blood at 12-96 h were not detectable for DOX-INJ, in contrast, DOX level in blood at 96 h was still found for DOX-OSC. In contrast to DOX-INJ group, DOX-OSC showed a higher targeting efficiency in the liver and spleen. The AUCs of DOX in the liver and spleen were 20.0 and 47.4 times and the MRT were 11.2 and 37.2 times, respectively. And the levels of DOX-OSC in the heart and kidney tissues were significantly reduced. And the drug distribution of DOX-OSC in the heart and kidney tissues were 17.0% and 11.4%, respectively. Hence, DOX-OSC shows an excellent drug loading capabilities and a higher targeting efficiency in the liver and spleen. That the levels of DOX-OSC in the heart and kidney tissues are significantly reduced, might improve the treatment efficacy of DOX and decrease the side effects.

Solubilizing and sustained-releasing abilities and safety preliminary evaluation for paclitaxel based on N-octyl-O, N-carboxymethyl chitosan polymeric micelles / 药学学报

A series of novel self-assembled polymeric micelles based on carboxymethyl chitosan bearing long chain alkyl chains (N-octyl-O, N-carboxymethyl chitosan, OCC) was synthesized. PTX loaded OCC polymeric micelles (PTX-OCC) were prepared by dialysis method. The effects of the degree of substitutions (DS) of octyl groups on the solubilizing abilities of OCC for paclitaxel were studied. The PTX-OCC were characterized using drug loading content, drug encapsulation efficiency, dynamic light scattering, zeta potential and transmission electron microscopy (TEM). Take PTX injection (PTX-INJ) as control, the safety of PTX-OCC including hemolysis, hypersensitiveness in guinea pigs and acute toxicity in mice were also evaluated. OCC showed excellent loading capacities for paclitaxel with the DS of octyl groups in the range of 37.9% - 58.6%. Drug loading contents were up to 24.9% - 34.4% with drug encapsulation efficiency 56.3% - 89.3%, which both increased with the increasing of DS of octyl groups. The mean size of PTX-OCC was 186.4 - 201.1 nm which decreased with the increasing of DS of octyl groups. The zeta potential was -47.5 to -50.9 mV, which had no obvious relation with the DS of octyl groups. The TEM images showed a spherical shape. No burst release phenomena were observed and drug cumulative release was in the range of 60% -95% in 15 days. PTX-OCC with higher DS of octyl groups showed stronger sustained releasing ability. In terms of the induction of membrane damage and hypersensitiveness, PTX-OCC was superior to PTX-INJ. The LD50 and its 95% confidence interval of PTX-OCC were 134.4 (125.0 - 144.6) mg x kg(-1), which was 2.7 fold of PTX-INJ. The present PTX-OCC could be potentially useful as safety carriers for intravenous delivery.

Resveratrol restored the structural and functional association between M3 receptor and connexin 43 gap junction proteins in ischemia-reperfusion injury of isolated rat heart / 药学学报

This study is to explore whether the protective effect of resveratrol on ischemia-reperfusion injury is correlated with the structural and functional association between M3 receptor (M3 subtype of muscarinic acetylcholine receptor) and Cx43 (connexin 43 gap junction proteins). Immunoprecipitation, immunoblotting and immunofluorescence were applied to investigate whether resveratrol has an effect on structural and functional association between M3 and Cx43. The effect of resveratrol on electrocardiogram Lead II ex vivo in rats, SOD (superoxide dismutase) activity and MDA (malondialdehyde) content was also observed in order to evaluate the protective effect of resveratrol on ischemia-reperfusion injury. Resveratrol could restore the structural and functional association between M3 receptor and Cx43 gap junction proteins that was partially destroyed under ischemia-reperfusion injury. The phosphorylation and spatial distribution disturbances in Cx43 expression caused by ischemia-reperfusion injury were also restored. Also, the QRS duration, SOD activity and MDA content were restored. Resveratrol could restore the structural and functional association between M3 receptor and Cx43 gap junction proteins.

Preparation of paclitaxel-loaded chitosan polymeric micelles and influence of surface charges on their tissue biodistribution in mice / 药学学报

<p><b>AIM</b>To prepare paclitaxel-loaded cationic chitosan micelles (PTX-CCM) and paclitaxel-loaded anionic chitosan micelles (PTX-ACM) and study the influence of surface charges on the biodistribution of paclitaxel-loaded chitosan polymeric micelles in mice.</p><p><b>METHODS</b>PTX-CCM and PTX-ACM were prepared by dialysis method and were administered to mice by caudal vein at a dose of 20 mg x kg(-1) body weight. The RP-HPLC method was established to determine the PTX concentrations in the plasma and other tissues of mice. The tissues distribution of PTX-CCM and PTX-ACM were evaluated by the pharmacokinetic parameters (AUC, MRT).</p><p><b>RESULTS</b>The diameter and zeta potential of PTX-CCM were 164 nm and +23.7 mV, while those of PTX-ACM were 180 nm and -28.0 mV, respectively. The drug loading and drug encapsulation efficiency for PTX-CCM were 26.4% (w/w) and 76.2% , while those of PTX-ACM were 34.6% (w/w) and 89.9%, respectively. The highest uptake of PTX-CCM and PTX-ACM in liver were 64.72% and 91.84% of dose, respectively. Meanwhile, MRT of both were 5.50 h and 51.39 h prolonged. The highest uptake of PTX-CCM and PTX-ACM in spleen were 7.08% and 5.16% of dose, respectively. Meanwhile, MRT of both were 9.04 h and 26.82 h. For PTX-CCM group, the AUC and C(max) of PTX in the lung were 2.71 times and 5.87 times of those of PTX-ACM group respectively. While in both PTX-CCM and PTX-ACM groups, the highest uptake of PTX in the heart were only 0.36% and 0.24% of dose, respectively and PTX in the kidney were only 0.75% and 0.33% of dose respectively.</p><p><b>CONCLUSION</b>PTX-CCM and PTX-ACM showed excellent drug loading capabilities with amount of cationic charges and anionic charges on their surface, respectively. Both PTX-CCM and PTX-ACM groups showed a higher targeting efficiency in the liver and spleen in vivo and accumulated in both tissues for relatively long time, especially in PTX-ACM group. In contrast to PTX-ACM, PTX-CCM showed a higher lung targeting efficiency in vivo while PTX-ACM had a stronger retention ability in the lung. Meanwhile in both groups the levels of PTX in the heart and kidney tissues were significantly lower which might decrease the side effects of PTX.</p>

Culture of vascular smooth muscle cells from coronary arteries of diabetic rats / 中国药理学通报

Aim To investigate the method of culturing vascular smooth muscle cells (VSMC) of coronary arteries derived from experimental diabetic rats and to establish the model of VSMC for the study of chronic cardiovascular diseases of diabetes. Method After the establishment of the model of diabetic rats, the coronary arteries of rats were separated carefully to culture VSMC using the method of explanting culture. Results The primary culture of VSMC was performed successfully by the method mentioned above. The cells could be isolated by different kinds of digestive enzyme in three steps. 7 ~10 days afterward, the cells overlapped in layers and displayed the typical ‘hill and valley’ pattern and positive immunochemical staining for smooth muscle actin. Conclusion VSMC from experimental diabetes proliferates faster than normal VSMC and its cultivating condition is strictly limited. The morphology of diabetic VSMC is as same as that of normal rats.