OBJECTIVETo investigate the effect of Xianxiong decoction on the mice with acute lung injury induced by lipopolysaccharide.

METHODEighty female ICR mice were randomly divided into 8 groups: model group, Xianxiong decoction group, Daxianxiong decoction group, Xianxiong decoction group without Kansui Radix group, Xianxiong decoction group without Glycyrrhizae Radix et Rhizoma group, Glycyrrhizae Radix et Rhizoma and Kansui Radix group, normal group and control group. Animals of each group, except normal group, were undertaken intraperitoneal injection and intranasal inhalation of lipopolysaccharide (LPS) on day 1, 2, 3 to establish acute lung injury (ALI) model. 30 min after modeling, 0.2 mL corresponding drugs were administrated to each mice, dexam ethasone and normal saline were given to the mice of control group and normal group respectively. White blood cell in blood, neutrophil percentage of blood and bronchoalveolar lavage fluid (BALF) supernatant, the ratio of wet and dry lung tissue ( W/D), histopathological changes of lung tissue were estimated. Sixty ICR mice were randomly divided into normal, model, control, high, middle and low dose Xianxiong decoction groups and were modeled in the same way. ELISA was applied to detect the level of NF-kappaB, TNF-alpha and IL-6 in BALF, PCR for NF-kappaB and TNF-alpha mRNA in lung tissue, and Western blot for NF-kappaB and TNF-alpha. Half of 20 ICR mice were administrated with Xianxiong decoction of its maximum tolerant normal saline.

RESULTCompared with model group, the number of WBC in blood of Xianxiong decoction group mice decreased (P < 0.01), percentage of neutrophils in both blood and BALF decreased as well (P < 0.01, P < 0.05); it also significantly reduced the ratio of W/D (P < 0.01); and found the alveolar wall, the number of inflammatory cells infiltrating improved, compared with model group. Xianxiong decoction reduced the level of NF-kappaB, TNF-alpha and IL-6 in BALF (P < 0.01, P < 0.01, P < 0.05); its high and low dose groups only found TNF-alpha level declined. Five mice died 24 h after administration of Xianxiong decoction which indicated its toxicity when other influential factors were considered.

CONCLUSIONXianxiong decoction is effective on the ALI mice induced by LPS, but it is of toxicity at 3 g x mL(-1).

Acute Lung Injury ; drug therapy ; genetics ; metabolism ; pathology ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Interleukin-6 ; genetics ; metabolism ; Lipopolysaccharides ; adverse effects ; Lung ; drug effects ; metabolism ; pathology ; Mice ; Mice, Inbred ICR ; NF-kappa B ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Background Hypertension is a multigenetic inheritable disease.Gene therapy with long-term effects and less side effects by regulating gene expression has been shown to be a potential and exciting prospect. Objective To investigate the effects of RNA interference(RNAi)targeting angiotensin-converting enzyme(ACE)on the blood pressure and ACE expression in kidney of spontaneously hypertensive rats(SHR).Methods SHR were randomly to receive placebo(n=12)or control adenovirus Ad5-EGFP)or a single injection of recombinant adenovi- ral vectors,Ad5-EGFP-ACE-shRNA(n=12,iv).Normotensive Wistar-Kyoto rats(WKY)were served as normal control group.SBP was measured before and after the intervention.Aorta,lung,myocardium and kidney were studied using fluorescence microscope to identify the sites of Ad5-EGFP-ACE-shRNA.Expressions of ACE mRNA and protein in kidney were evaluated by RT-PCR and Western blot.Results SBP of the treat group was effectively reduced by 19.0?3.2 mmHg at the 3rd day,and 22.1?3.3 mmHg at the 13th day of the experiment.The anti- hypertensive effect significant remained at least for 14 days.On the contrary,increase in BP was shown in placebo and the adenovirus control group.Compared with placebo or adenovirus control rats,ACE mRNA expression level in kidney of the treated rats was lower by 61.1% and 62.3% respectively,with ACE protein expression level lower- ing by 56.2% and 53.30% as well(ail P0.05). Conclusion RNA interference targeting ACE gene inhibits the expressions of ACE mRNA and protein.A single dose injection resulted in a prolonged decrease in BP.The evidence of strong antihypertensive effect by genetic therapy justifies efforts for further investigation.

Aim:To construct the short hairpin small interfering RNA(shRNA) eukaryotic expression vector specific to mouse lectin like oxidized low density lipoprotein receptor 1(LOX-1) gene and to observe its silencing effect on LOX-1 in RAW264.7 cells.Methods:(1)The pLOX-1-shRNA expression vector was constructed by gene recombination,Then transfected into the cultured RAW264.7 cells.At 48 h after Transfection,the expression of LOX-1 mRNA in RAW264.7 cells were determined by semi-quantitative RT-PCR,the expression of LOX-1 proteins examined by Western blot.(2) Oil Red O Dyeing experiment was used to show the cellular lipid droplets in lipid-loaded cells.The method of cholesterol oxidase analysis was performed to determine the content of cellular cholesterol.The ability of uptake Dil-ox-LDL in RAW264.7 cells was assayed by fluorescence microscopy.Results: pLOX-1-shRNA expression vector was successfully constructed.Transfection of pLOX-1-shRNA expression vector into RAW264.7 cells down regulaled the expression level of LOX-1 gene,as compared with the control group,transfection of the RAW264.7 cells with LOX 1-shRNA led to a remarkable reduction of the number macrophages transformed into foam cell,and could suppress the uptake of ox-LDL.Conclusion:The pLOX-1-shRNA expression vector can inhibit the expression of LOX 1 in RAW264.7 cells and the transformation of the macrophages into foam,which may he beneficial in searching new gene therapy of atherosclerosis.

The aim of the present study is to investigate the effects of adiponectin (APN) on hypoxia/reoxygenation (H/R) injury in cultured cardiomyocytes. Primary cardiomyocytes were obtained from neonatal rats by enzymatic digestion method and identified by immunofluorescent technique. Primary cells cultured for 72 h were used in experiment and divided into 5 groups randomly: Control group, H/R group, H/R+APN group, H/R+APN+adenine 9-beta-D-arabinfuranoside (AraA, AMPK inhibitor) group, and H/R+AraA group. The cardiocyte morphology and beating rate were observed under inverted microscope. The DNA ladder was examined by agarose gel electrophoresis, and the cell apoptosis was determined by flow cytometry. Moreover, the malondialchehyche (MDA) content in myocardial cells and the superoxide dismutase (SOD) activity in the supernatant were measured using kits, the fluorescence intensity of intracellular Ca2+ was observed by laser scanning confocal microscope, and the phosphorylation of AMPK was determined by Western blotting. Compared with control group, H/R group showed increased apoptotic rate, oxidative stress level, intracellular Ca2+ concentration and phosphorylation level of AMPK (P<0.05), while significant ameliorations in the above indices were seen in H/R+APN group. On the contrast, AraA attenuated the protective effect of APN and decreased the phosphorylation of AMPK. These results suggest that adiponectin can protect cardiomyocytes from H/R-induced oxidative stress and apoptosis through AMPK pathway.
AMP-Activated Protein Kinases ; metabolism ; Adiponectin ; pharmacology ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cardiotonic Agents ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Female ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Myocytes, Cardiac ; cytology ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

The aim of the present study was to investigate the effects of adiponectin (APN) on the expression of T-cadherin in cultured Sprague-Dawley (SD) rat cardiomyocytes injured by hypoxia/reoxygenation (H/R). Primary myocardial cells from neonatal rats were obtained by enzymatic digestion. The cells were divided into control group, H/R group and H/R+APN (3, 10, 20 and 30 μg/mL) groups. The H/R group was incubated in anoxic environment (anoxic solution saturated with high concentration N2) for 3 h, and then in the reoxygenation environment (the reoxygenation solution saturated with pure oxygen) for 1 h. The H/R+APN group was pretreated with different concentrations of APN for 24 h prior to the initiation of H/R. The content of lactate dehydrogenase (LDH) was measured by chemistry chromatometry. Cellular apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of T-cadherin was detected by RT-PCR and Western blotting. The results showed that, compared with control group, the apoptotic rate and release of LDH were significantly increased in the H/R group, whereas the expressions of T-cad mRNA and protein were decreased. Pretreating with APN significantly and dose-dependently decreased apoptotic rate and LDH release, and up-regulated T-cad mRNA and protein level in rat neonatal cardiomyocytes under H/R conditions. These results suggest that APN may protect cardiomyocytes against H/R-induced injury by up-regulating H/R-decreased T-cad expression.
Adiponectin ; pharmacology ; Animals ; Apoptosis ; Cadherins ; metabolism ; Cell Hypoxia ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxygen ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

The present study was to investigate the effect of glucagon like peptide-1 (GLP-1) on high glucose-induced oxidative stress of cardiomyocytes and the possible role of the PI3K-Akt signal path in this process in the neonatal SD rats. With enzymatic digestion and immunofluorescence identification, cardiomyocytes after 72-96 h of primary culture were used in experiment. The cells were divided into 5 groups: normal control group, high glucose group, high glucose + GLP-1 group, high glucose + GLP-1 + LY294002 group and high osmolarity control group. The content of MDA was detected by TBA colouration method. The content of SOD was detected by xanthine oxidase method. The change of NADPH P47phox subunit mRNA quantity was detected by PCR gel electrophoresis. The level of ROS was detected by flow cytometry, and was also observed by fluorescence microscope. The DNA ladder was examined by agarose gel electrophoresis, and the cell apoptosis was determined by Annexin-V-FITC/PI flow cytometry, and the phosphorylation of Akt was determined by Western blotting. Compared with those in the normal control group, in the high glucose group, the cells grew poorly, and the beating rate was significantly lower (P < 0.05); The apoptotic rate was significantly increased (P < 0.05); The MDA content was increased (P < 0.05); It showed the typical DNA ladder, which is the characteristic of apoptosis; The SOD activity was decreased (P < 0.05); The level of intracellular ROS increased (P < 0.05); And the expression of NADPH P47phox subunit mRNA was increased; However the phosphorylation level of Akt was decreased. Pretreatment with GLP-1 improved the above-mentioned parameters and decreased the expression of NADPH P47phox subunit mRNA (P < 0.05). However, compared with the high glucose + GLP-1 group, LY294002, an inhibitor of PI3K-Akt signal path, attenuated the protective effect of GLP-1 in the high glucose + GLP-1 + LY294002 group. It is suggested that GLP-1 plays a protective role in the high glucose-induced injury and apoptosis of cardiomyocytes, and the PI3K-Akt signal path is involved in this process.
Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cells, Cultured ; Female ; Glucagon-Like Peptide 1 ; pharmacology ; Glucose ; pharmacology ; Male ; Myocytes, Cardiac ; cytology ; pathology ; Oxidative Stress ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

Objective To investigate the efficacy and safety of paroxe-tine combined with low dose of quetiapine in the treatment of generalized anxiety disorder.Methods One hundred and forty patients with gener-alized anxiety disorder were randomly assigned to treatment group ( parox-etine combined with low dose of quetiapine group , n =71 ) and control group ( paroxetine group , n =69 ).The efficacy was assessed by the Hamilton rating scale for anxiety ( HAMA ) , the Treatment Emergent Symptom Scale ( TESS ) for side effects , and Pittsburgh sleep quality in-dex(PSQI)for sleep qualities of all participants at baseline and at the end of 1st, 2nd, 4th, 6th, 8thweek.Results There were no significant differ-ences between two groups at cure rate ( 70.4%/68.1%) , notable im-provement rate ( 15.5%/13.0%) , progress rate ( 8.5%/10.1%) , and ineffective rate ( 5.6%/8.7%) overall the treatment course.Compare with the baseline , the treatment group showed notable decrease of HAMA at the 1 st weekend ( P<0.05 ) , and the control group showed that at the 2nd weekend (P<0.05).The treatment group had a more significant de-crease than the control group at the 1st, 2nd, and 4th week end(P<0.05 or P<0.01 ).The psychic anxiety score of the treatment group at the 1st, 2nd, 4th weekend and the somatic anxiety score at the 2nd, 4th weekend showed significant decrease ,compared with the control group ( P<0.05 or P<0.01 ).Both of the two group had no serious adverse events.Conclusion Paroxe-tine combined with low dose of quetiapine is an effective and safe way to treat generalized anxiety disorder , and patients have good compliance under the treatment.

Objective To investigate the effects of RNA interference(RNAi)targeting angiotensinconverting enzynle(ACE)on the blood pressure and myocardial remodeling in spontaneously hypertensive rats(SHRs).Methods Saline(control),adenovirus(Ad5)and recombinant adenoviral vectors(Ad5-ACE-shRNA expressing ACE gene-specific shRN)were randomly administered by caudal intravenation to SHRs(n=12 each group)at day 1 and day 16.Normotensive Wistar-Kyoto rats(WKY)served as normal controls.Systolic blood pressure(SBP)of the caudal artery was measured daily.Expressions of ACE at mRNA and protein levels in myocardium and aorta were evaluated by RT-PCR and Western blot respectively,ACE serum concentration was measured by ELISA at day 3(n=6 each group).The ratio of left ventricular to body weight(LVW/BW),myocardial collagen content were measured and myocardial uhrastrueture observed under transmission electron microscope at the study end.Results Ad5-ACE-shRNA injection significantly reduced SBP(-22 mm Hg,1 mm Hg=0.133 kPa)and the antihypertensive effect could last at least 14 davs post each injection.SBP was not affected by saline and Ad5 injections.ACE expressions at mRNA and Drotein levels at myocardium and aorta as well as serum ACE were significantly decreased in Ad5-ACE-shRNA treated SHRs compared to that in saline and Ad5 groups(all P＜0.05)and was coinparable to that in WKY group(P＞0.05).The LVW/BW ratio(2.24±0.19)and myocardial collagen content[(1.283±0.019)μg/mg]in Ad5-ACE-shRNA treated SHRs were also significantly lower than those in saline treated[3.21±0.13 and (1.686±0.013)μg/mg,both P＜0.05]and Ad5 treated SHRs [3.13 ±0.12,(1.682±0.009)μg/mg,both P＜0.05]but still higher than those of WKY group[2.06±0.11,(1.257±0.019)μg/mg,both P＜0.05].Myocardial uhrastructure was also significantly improved in Ad5-ACE-shRNA treated SHRs compared to saline and Ad5 treated SHRs.Conclusion RNAi targeting ACE gene significantly inhibited the expressions of ACE at mRNA and protein levels and resuhed in prolonged antihypertensive effects and myocardial uhrastructure improvements in this SHR model.

Objective To investigate the effects of RNA interference (RNAi) targeting angiotensin Ⅱ Type 1 receptor (AT1R) and angiotensin-converting enzyme (ACE) on blood pressure and myocardial remodeling in spontaneous hypertensive rats (SHRs). Methods Saline (control), adenovims (Ad5) and recombinant adenoviral vectors (Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA expressing ACE, AT1R, ACE and AT1R gene-specific shRNA, respectively) were randomly administered by caudal intravenation to SHBs (n = 12 each group) at day 1 and 17. Normotensive Wistar-Kyoto rats (WKY) served as normal controls. Systolic blood pressure (SBP) of the caudal artery was measured daily. Expression of ACE and AT1R at mBNA levels in ventricle and aorta were evaluated by fluorescence quantitative PCR. Angiotension Ⅱ serum concentration was measured by ELISA at day 3 (n = 6 each group). The ratio of left ventricular to body weight (LVW/BW) and myocardial collagen content were measured, myocardial ultrastructure observed under transmission electron microscope at the study end. Results The caudal artery pressure of saline and Ad5 group was equally increased by about 26 nun Hg(1 mm Hg=0.133 kPa) compared to baseline (both P <0.05). Ad5-ACE-shRNA,Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA injection significantly reduced SBP (-24 mm Hg, -22 mm Hg and -26 mm Hg respectively, all P<0.05 vs. baseline) and the antihypertensive effect could last at least 15 days post each injection. SBP was not affected by saline and Ads injections. ACE and AT1 mRNA expressions at ventricle and aorta were significantly decreased in Ad5-ACE-shRNA, Ad5-ACE-AT1R-shRNA and Ad5-AT1R-shRNA, Ad5-ACE-AT1R-shRNA treated SHRs compared to those in saline and Ad5 groups (all P < 0.05) and was comparable to that in WKY group (P> 0.05). The LVW/BW ratio [(2.22±0.18)μg/mg, (2.23 ±0.19)μg/mg, (2.17±0.16) μg/mg] and myocardial collagen content [(1.291±0.019)μg/mg, (1.298±0.019) μg/mg, (1.276±0.019)μg/mg] in Ad5-ACE-shRNA, Ad5-AT1R-shRNA and Ad5-ACE-AT1R-shRNA treated SHBs were also significantly lower than those in saline treated [(3.23±0.13) μg/mg and(1.683±0.013) μg/mg, both P<0.05] and Ad5 treated SHRs [(3.25±0.12) μg/mg and (1.693±0.013) μg/mg, both P<0.05], but still higher than those of WKY group [(2.06±0.12) μg/mg and (1.258±0.019) μg/mg, both P < 0.05]. Myocardial ultrastructure was also significantly improved in all SHRs underwent RNAi treatments compared to saline and Ad5 treated SHRs. Conclusion RNAi targeting ACE and AT1R gene significantly inhibited myocardial and aortic ACE and AT1R mRNA expressions and resulted in prolonged antihypertensive effects and myocardial ultrastructure improvements in SHRsl. The RNAi technology may be a potential new strategy of gene therapy for hypertension.

Aim To explore the improvement effect of Bazi Bushen capsule on thymic degeneration in SAMP6 mice and the possible mechanism.Methods Twenty 12 week old male SAMP6 mice were randomly divided into the model group(SAMP6)and the Bazi Busheng capsule treatment group(SAMP6+BZBS).Ten SAMR1 mice were assigned to a homologous control group(SAMR1).The SAMP6+BZBS group was oral-ly administered Bazi Bushen capsule suspension(2.8 g·kg-1)daily,while the other two groups were orally administered an equal amount of distilled water.After nine weeks of administration,the morphology of the thymus in each group was observed and the thymus in-dex was calculated;HE staining was used to observe the structural changes of thymus tissue;SA-β-gal stai-ning was used to detect thymic aging;flow cytometry was used to detect the proportion of thymic CD3+T cells in each group;Western blot was used to detect the levels of p16,Bax,Bcl-2,and cleaved caspase-3 proteins in thymus;immunofluorescence was applied to detect the proportion of cortical thymic epithelial cells in each group;ELISA was employed to detect IL-7 lev-els in thymus.Results Compared with the SAMP6 group,the thymic index of the SAMP6+BZBS group significantly increased(P＜0.05);the disordered thy-mic structure was significantly improved;the positive proportion of SA-β-gal staining significantly decreased(P＜0.01);the proportion of CD3+T cells apparently increased(P＜0.05);the level of p16 protein signifi-cantly decreased(P＜0.05);the level of Bcl-2 pro-tein significantly increased(P＜0.05),while the lev-el of cleaved caspase-3 protein markedly decreased(P＜0.05);the proportion of cortical thymic epithelial cells evidently increased;the level of IL-7 significantly increased(P＜0.01).Conclusions Bazi Bushen capsule can delay thymic degeneration,inhibit cell ap-optosis in thymus and promote thymic cell development in SAMP6 mice,which may be related to increasing the proportion of cortical thymic epithelial cells and promoting IL-7 secretion.