Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of ?-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1—40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1—40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP_1—40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP_1—40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1—40-treated rats. The results show that the injection of β-AP_1—40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

Alterations of cardiac and lymphocyte β-adrenoceptors in rat with chronic heart failure

The alterations of cardiac and lymphocyte β-adrenoceptors were observed in the rats with chronic heart failure produced by constriction of both abdominal aorta and renal artery. The results showed that β1-adrenocep-tor density and mRNA levels were increased, whereas these levels remained unchanged for β2 The concentration-contractile response curve for isoproterenol was shifted to the right in cardiac atrium, whereas the concentration-cAMP accumulation response curve for isoproterenol in myocardium was not changed. The number of β-adrenoceptors in blood lymphocyte was markedly reduced. Thus in the heart-failure rats the density of cardiac β-adrenoceptor was increased accompanying reduced β-adrenoceptor-mediated positive inotropic response, suggesting a post adenylate cyclase dys-function or impaired contractile components. In contrast, the alteration of β-adrenoceptor in lymphocyte is consistent with the reduced β-adrenoceptor-mediated inotropic response in heart.     

β-adrenergic modulation of in vivo long-term potentiation in area CA1 and its role in spatial learning in rats

Activation of β-adrenoceptors in area CA1 of the hippocampus facilitates in vitro long-term potentiation (LTP) in this region. However, it is unclear if in vivo LTP in area CA1 and hippocampus-dependent learning are subjected to β-adrenergic regulation. To address this question, we investigated the effects of the β-adrenergic agonist L-isoproterenol or antagonist DL-propranolol on in vivo LTP of area CA1 and the spatial learning in Morris water maze. In the presence of L-isoproterenol (through local infusion into area CA1), the theta-pulse stimulation with the parameter of 10 Hz, 150 pulses/train, 1 train, a frequency weakly modifying synaptic strength, induced a robust LTP, and this effect was blocked when DL-propranolol was co-administered. By contrast, the theta-pulse stimulation with the parameter of 5 Hz, 150 pulses/train, 3 trains, a frequency strongly modifying synaptic strength, induced a significantly smaller LTP when DL-propranolol was administered into area CA1. Accordingly, DL-propranolol impaired the spatial learning in the water maze when infused into area CA1 20 min pretraining. Compared with control rats, the DL-propranolol-treated rats showed significantly slower learning in the water maze and subsequently exhibited poor memory retention at 24-h test. These results suggest that β-adrenoceptors in area CA1 are involved in regulating in vivo synaptic plasticity of this area and are important for spatial learning.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β1 （TGF-β1） can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and fight ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of α-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hyp0Xic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological featureof hypoxic pulmonary vascular remodeling.However,the origin of the cells involved in this process is stillnot well understood.The present study was undertaken to test the hypothesis that transforming growthfactor-β1 (TGF-β1) can induce transdifferentiation of fibroblasts into myofibroblasts,which might play akey role in the muscularization of non-muscular pulmonary arterioles.It was found that mean pulmonaryarterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling index and rightventricular hypertrophy became evident after 14 d of hypoxia.The distribution of nonmuscular,partiallymuscular,and muscular vessels was significantly different after 7 d of hypoxia.Immunocytochemistryresults demonstrated that the expression of co-smooth muscle actin was increased in intra-acinar pulmonaryarteries with increasing hypoxic time.TGF-β1 mRNA expression in pulmonary arterial walls was increasedsignificantly after 14 d of hypoxia,but showed no obvious changes after 3 or 7 d of hypoxia.In pulmonarytunica adventitia and tunica media,TGF-β1 protein staining was poorly positive in control rats,but wasmarkedly enhanced after 3 d of hypoxia,reaching its peak after 7 d Of hypoxia.The myofibroblast phenotypewas confirmed by electron microscopy,which revealed microfilaments and a well-developed rough endo-plasmic reticulum.Taken together,our results suggested that TGF-β1 induces transdifferentiation of fibro-blasts into myofibroblasts,which is important in hypoxic pulmonary vascular remodeling.     

Pyrrolidine dithiocarbamate protects pancreatic β-cells from oxidative damage through regulation of FoxO1 activity in type 2 diabetes rats

Pyrrolidine dithiocarbamate （PDTC） can lower the bloot glucose level and improve the insulin sensitivity in diabeti, rats. However, the mechanisms underlying this effect o PDTC treatment in diabetic rats remained uncertain, h this study, we evaluated the mechanisms by which PDT（ conferred protection against oxidative damage to pancreat ic islet β-cells in rats with experimental type 2 diabete mellitus （DM）. DM in the rats was elicited by long-tern high-fat diet accompanied with a single intraperitonea （i.p.） injection of a low dose of streptozotocin. After a 7-da1 administration of PDTC （50 mg/kg/day i.p.）, blood glucos levels were measured and pancreatic tissues were collecte / for the determination of various biochemical and enzyma 1 ic activities using immunohistochemistry, immunofluoresI cence, and western blot techniques. The percentage o 1 apoptotic pancreatic islet β-cells was detected by flow cyto metry. The results showed that diabetic rats had elevate blood glucose levels and insulin resistance, accompanieq with an increase in malondialdehyde content, nitrotyrosin production, and inducible nitric oxide synthase expression A decrease in superoxide dismutase and glutathione pero idase activities was also observed in DM rats, culminatin with elevated β-cell apoptosis. PDTC treatment significantl reduced the oxidative damage and the β-cell apoptosi and also increased the insulin production through down-reg lating FoxO1 acetylation and up-regulating nuclear PDX- level. These data suggested that PDTC can protect islet βcells from oxidative damage and improve insulin productio through regulation of PDX-1 and FoxO1 in a DM rat model.     

Variations of thioredoxin system contributes to increased susceptibility to apoptosis in cardiomyocytes of type 2 diabetic rats

Cardiac complications are the leading cause of death in diabetes. However, the mechanism of diabetes in inducing myocardial injury and apoptosis, and whether the thioredoxin （Trx） system is involved remain unclear. In this study, male Sprague-Dawley rats were randomly divided into two groups： the control and the diabetes groups, and then were randomly divided into five different timepoints （the 1st, 2nd, 4th, 12th, and 24th week）. The results showed that diabetes-induced cardiac injury was enhanced in the type 2 diabetes rats, as evidenced by aggravated cardiac dysfunction, biochemical indicators, and increased myocardial apoptosis （TUNEL and caspase-3 activity）. The activity of myocardial Trx and Trx reductase （TR） in diabetic rats was significantly decreased from the second week and continually aggravated with the disease progression. In diabetic rats, the mRNA expression of Trxl, Trx2, TR1, and TR2 was decreased first and then increased after the fourth week. Meanwhile, the protein expression of these Trx system members was significantly increased at the 12th week. Trx nitration was cleared, the Trx/ASK1 interaction was significantly decreased, and the activity of p38 was significantly enhanced in cardiac tissues at the 12th week. These results demonstrated that diabetes may cause myocardial injury and apoptosis, and the extent of which was accompanied with the development of the disease. The mechanism is associated with the development of diabetes and the decreased activity of Trx and TR. The reasons for decreased Trx activity may include： decrease of Trx and TR protein expression; nitration modification of Trx; and up-regulation of TXNIP expression.     

The kinetics of IL—4 and IFN—γ gene expression in Mice after Trichosansin immunization

Trichosanthin (TCS) is a potent allergen to mice.According to our previous experiments,it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization,while OVA alone could not induce IgE to it.In this work,the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method.It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response.In contrast,the IFN-γ gene expression was suppressed.Furthermor,the IL-4 gene expression in the secondary response was lower than that in the primary response.Thus the presence of IgE memory B cells were studied.Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation,while in the MLN of the mice primed 30 days before and without boost,it was almost as the same amount of the unimmunized control.These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.     

Hypoxia Induces Transforming Growth Factor-β1 Gene Expression in the Pulmonary Artery of Rats via Hypoxia-inducible Factor-1α

The present study was undertaken to investigate the dynamic expression of hypoxia induciblefactor-1 α (HIF-1α) and transforming growth factor-β1 (TGF-β1) in hypoxia-induced pulmonary hypertensionof rats.It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d ofhypoxia.Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d ofhypoxia.HIF-1α mRNA staining was less positive in the control,hypoxia for 3 d and hypoxia for 7 d,butbegan to enhance significantly after 14 d of hypoxia,then remained stable.Expression of HIF-1 α protein inthe control was less positive,but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats.TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, butshowed no obvious changes after 3 or 7 d of hypoxia.In pulmonary tunica adventitia and tunica media,TGF-β1 protein staining was less positive in control rats,but was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d of hypoxia,and then weakening after 14 and 21 d of hypoxia.Western blottingshowed that HIF- 1α protein levels increased significantly after 7 d of hypoxia and then remained at a highlevel. TGF-β1 protein level was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d ofhypoxia,and then decreasing after 14 and 21 d of hypoxia.Linear correlation analysis showed that HIF-1αmRNA, TGF-β1 mRNA, TGF-β1 protein were positively correlated with mPAP,vessel morphometry andright ventricular hypertrophy index.TGF-β1 protein (tunica adventitia) was negatively correlated withHIF-lα mRNA.Taken together,our results suggest that changes in HIF-lα and TGF-β1 expression afterhypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.     

Mitochondria-targeted antioxidant peptide SS-31 mediates neuroprotection in a rat experimental glaucoma model

To investigate the neuroprotective effects of the mitochondria-targeted antioxidant Szeto-Schiller peptide 31 (SS-31) in a rat experimental glaucoma model, SS-31 was intraperitoneally (IP) injected into Sprague-Dawley rats, followed by intracameral injection of polystyrene microspheres to induce elevated intraocular pressure (IOP). After 6 weeks, electroretinography (ERG) and flash visual-evoked potentials (F-VEPs) were recorded to assess retinal function. Hematoxylin-eosin staining was performed on retinal cross-sections to measure ganglion cell complex (GCC) thickness. Apoptotic retinal cells were assessed by TUNEL staining. Brn3a-positive retinal ganglion cells (RGCs) were counted in retinal flat mounts via immunofluorescence. The retinal total SOD, SOD2, and MDA expression levels were assessed in retinal tissue homogenates. The cyt c, Baxz and Bcl-2 protein levels in rat retinas were detected by western blot analysis. Bax and Bcl-2 expressions were also evaluated using immunohistochemistry in paraffinized sections. Our results showed that the rats that received microsphere injection developed elevated IOP. SS-31 ameliorated the reductions in the a- and b-wave amplitudes on ERG and the F-VEP amplitude in glaucomatous eyes. GCC thickness was preserved, TUNEL-positive cells were decreased in the retina, and Brn3a-positive RGCs were in creased in the SS-31-treated glaucoma group compared with those in the non-treated glaucoma group. SS-31 significantly reduced MDA levels and increased SOD2 levels after glaucoma induction. Significant suppression of cyt c release, upregulation of Bcl-2, and downregulation of Bax were observed following SS-31 administration. In summary, SS-31 exerts neuroprotective effects in this experimental glaucoma model by inhibiting mitochondrial dysfunction and therefore represents a promising therapeutic agent for glaucoma.     

Infusion of methylphenidate into the basolateral nucleus of amygdala or anterior cingulate cortex enhances fear memory consolidation in rats

The psychostimulant methylphenidate (MPD; also called Ritalin) is a blocker of dopamine and norepi-nephrine transporter. It has been clinically used for treatment of Attention Deficit and Hyperactivity Disorder (ADHD). There have been inconsistent reports regarding the effects of systemically adminis-tered MPD on learning and memory, either in animals or humans. In the present study, we investigated the effect of direct infusion of MPD into the basolateral nucleus of amygdala (BLA) or the anterior cin-gulate cortex (ACC) on conditioned fear memory. Rats were trained on a one-trial step-through inhibi-tory avoidance task. MPD was infused bilaterally into the BLA or the ACC, either at ‘0’ or 6 h post-training. Saline was administered as control. Memory retention was tested 48 h post-training. In-tra-BLA or intra-ACC infusion of MPD ‘0’ h but not 6 h post-training significantly improved 48-h memory retention: the MPD-treated rats had significant longer step-through latency than controls. The present results indicate that action of MPD in the BLA or the ACC produces a beneficial effect on the consoli-dation of inhibitory avoidance memory.     

Ectopic discharges trigger sympathetic sprouting in rat dorsal root ganglia following peripheral nerve injury

Experimental backgrounds of ectopic discharges were made by i.p. administrating of 4-aninopyriding (4-AP), a K+ channel blocker, or anisodamine, a muscarinic receptor blocker, in CCI rats, and the sympathetic sprouting in the dorsal root ganglia (DRG) as well as the heat-hyperalgesia was observed. It was demonstrated that the increased ectopic discharges induced by 4-AP promote sympathetic sprouting in the DRG and a greater number of sympathetic basket cells were developed, causing exacerbation of heat-hyperalgesia in CCI rats. On the contrary, the sympathetic sprouting in the DRG and heat-hyperalgesia are evidently diminished after anisodamine injection. Our results suggest that ectopic discharges may be an immediate factor in triggering sympathetic sprouting in DRG following peripheral nerve injury.     

Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model

AIM:To investigate the effect of human umbilical cord stem cells,both mesenchymal and hematopoietic(CD34+),in the treatment of arthritis.METHODS:Mesenchymal stem cells(MSCs) and hematopoietic(CD34+) stem cells(HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing fullterm normal vaginal delivery.MSC,HSC,methotrexate(MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant(CFA) induced arthritis after the onset of disease(day 34),when arthritis had become well established(arthritis score ≥ 2).Arthritic indices were evaluated and the levels of interleukin(IL)-1,tumor necrosis factor(TNF)-α and interferon(IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay.Animals of all groups were sacrificed 34 d after beginning treatment,except positive control(PC) which was sacrificed at 10,21 and 34 d for microscopic observation of disease progression.We used hematoxylin,eosin and Masson's trichrome stains for histopathological examination of cartilage and synovium.RESULTS:The mean arthritis scores were similar in all groups at 12 and 34 d post immunization,with no statistical significant difference.Upon the injection of stem cells(hematopoietic and mesenchymal),the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group.Mean hindpaw diameter(mm) in the MSC rats was about half that of the PC and MTX groups(P = 0.007 and P = 0.021,respectively) and 0.6 mm less than the HSC group(P = 0.047),as indicated by paw swelling.Associated with these findings,serum levels of TNF-α,IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups(P 0.05),while the expression of IL-10 was increased.Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity.Stem cells treated groups(both hematopoietic CD34+ and mesenchymal),showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity.With Masson's trichrome,stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats.Vacuoles were also visible in the outer vessel wall.The vessel became hemorrhagic and finally necrotic.In addition,there was extensive fibrosis completely obliterating the joint cavity.The mean color area percentage of collagen in this group was 0.324 ± 0.096,which was significantly increased when compared to the negative control group.The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively,which showed a marked decrement compared to the PC group,denoting a mild increase in synovial tissue collagen fibers.CONCLUSION:MSC enhance the efficacy of CFAinduced arthritis treatment,most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.     

鸡垂体前叶提取物诱导鸡超数排卵

Chicken anterior pituitary extract(CAPE) and acetone dried chicken anterior pituitary (ACAPE) were injected intraperitoneally into normal laying hens (‘ovulation suppressed’ following pretreatment with daily subcutaneous injection of PMSG) to induce multiple ovulations. The dose of PMSG, the effect of CAPE and ACAPE and the time required for induction of ovulation following injection of ovulation inducing hormone were determined. The results revealed that (1) when 75 IU PMSG was administered daily, egg laying stopped in 33% of the treated hens within 6 days after the first injection. However, the percentage of hens showing the same effects changed significantly (over 95%) within 3 to 6 days when the amount of PMSG was increased to 100 IU; (2) the number of ovulated ova was 1 00±0 00, 2 33±0 26,2 20±0 20 respectively after receiving 100 mg, 200 mg and 300 mg; the number of ovulated ova was 2 00±0 00, 2 86±0 48, 3 00±1 50 respectively after receiving 10 mg, 15 mg and 20 mg ACAPE; (3) The time from injection to ovulation in almost all hens was about 7 5 h except one hen ovulated about 6 5 h after receiving ACAPE .     

Excessive hepatic copper accumulation in jaundiced rats fed a high-copper diet

The response of copper metabolism to dietary copper challenge was investigated in jaundiced rats with elevated plasma concentrations of conjugated bilirubin as a result of impaired canicular transport of bilirubin glucuronides. Control and jaundiced rats were fed purified diets with either normal (64 μmol Cu/kg) or high (640 μmol Cu/kg) concentration of added copper. Copper loading produced a greater increase in hepatic copper concentrations in the jaundiced than in control rats. The greater dietary-copper-induced increase in hepatic copper in the jaundiced rats can be explained by the observed smaller rise in biliary copper excretion and a greater efficiency of dietary copper absorption. In individual rats, there was a positive relationship between hepatic copper concentrations and biliary copper concentrations. It is suggested that not the transport of copper from liver cells to bile but that from plasma to bile is diminished in the jaundiced rats. The elevated plasma copper concentrations in the jaundiced rats may support this suggestion.     

Dynamic changes of cardiovascular regulating factors in rats after aerobic exhaustive exercise

Objective To study the changes of cardiovascular regulating factors in rats during recovery of aerobic exhaustive exercise. Methods Sixty male Wistar rats were randomly divided into control group, 1 h-exercise group, 3 h-exercise group, exhausted group, 2 h-recovery group and 12 h-recovery group. The rats were killed at corresponding times for each group after an 8-week-long treadmill training, and the levels of NO, El-, ANP and TXB2 in plasma were measured in each group. Results NO/ET ratio of 1 h-exercise group was significantly higher than that in control group （P〈0.01）, while it was significantly decreased in 3 h-exercise group and exhausted group （P〈0.05）. ANP contents in rat plasma were significantly higher in 3 h-exercise group, exhausted group and 2 h-recovery group than that in control group （P〈0.05 or P〈0.01）. The concentration of TXB2 in plasma was significantly increased in 3 h-exercise group, exhausted group and 2 h-recovery group （P〈0.05）. Conclusion Changes in cardiovascular regulating factors after exhaustive exercise may lead to deficiency of coronary circulation blood/oxygen supply, which may cause exercise- induced fatigue.     

Gender difference regarding selenium penetration into the mouse brain

A sex difference in the penetration of selenium into the brain was observed using lipopolysaccharide (LPS)-injected mice. The selenium concentration increased in the brains of sodium selenite-injected LPS-treated female mice, but not males. The selenium concentration peaked when selenite was injected 3 h after the injection of LPS into female mice. In addition, selenium in the brain increased when a dosage of 30 μmol/kg and more of selenite was injected into LPS-treated female mice. Also, the selenium concentration in the brain increased and peaked 2–3 h after selenite injection; 24 h later, the level was similar to the Se-only group. The penetration of selenium into the brain was inhibited by pretreatment with aminoguanidine, an inhibitor of nitric oxide synthetase. From the present results, selenium more easily penetrated into the brains of female mice compared to males after LPS treatment, and nitric oxide may have affected the penetration. However, the sex difference mechanism for selenium penetration needs further investigation.     

Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.     

Liver X receptor agonist T0901317 reduces athero-sclerotic lesions in apoE-/- mice by up-regulating NPC1 expression

In this study, we studied the effect of liver X receptor (LXR) agonist T0901317 on Niemann-Pick C1 protein (NPC1) expression in apoE-/- mice. Male apoE-/- mice were randomized into 4 groups, baseline group (n=10), control group (n=14), treatment group (n=14) and prevention group (n=14). All of the mice were fed with a high-fat/high-cholesterol (HFHC) diet containing 15% fat and 0.25% cholesterol. The baseline group treated with vehicle was sacrificed after 8 weeks of the diet. The control group and the prevention group were treated with either vehicle or T0901317 daily by oral gavage for 14 weeks. The treatment group was treated with vehicle for 8 weeks, and then was treated with the agonist T0901317 for additional 6 weeks. Gene and protein expression was analyzed by real-time quantitative PCR, immunohistochemistry and Western blotting, respectively. Plasma lipid concentrations were measured by commercially enzymatic methods. We used RNA interference technology to silence NPC1 gene expression in THP-1 macrophage-derived foam cells and then detected the effect of LXR agonist T0901317 on cholesterol efflux. Plasma triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 treatment reduced the aortic atherosclerotic lesion area by 64.2% in the prevention group and 58.3% in the treatment group. LXR agonist treatment increased NPC1 mRNA expression and protein levels in the small intestine, liver and aorta of apoE-/- mice. Compared with the normal cells, cholesterol efflux of siRNA THP-1 macrophage-derived foam cells was significantly decreased, whereas cholesterol efflux of LXR agonist T0901317-treated THP-1 macrophage-derived foam cells was significantly increased. Our results suggest that LXR agonist T0901317 inhibits atherosclerosis development in apoE-/- mice, which is related to up-regulating NPC1 expression.     

A new interaction between Abi.1 and βPIX involved in PDGF.activated actin cytoskeleton reorganisation

Members of the Rho family of GTPases are key regulators of the actin cytoskeleton. In particular, activated Racl stimulates membrane dorsal ruffle formation in response to platelet-derived growth factor （PDGF）. Abl-interactor （Abi）- 1 and βP1X, a guanine nucleotide exchange factor for Racl, localise at these Rac1-induced actin structures and play important roles in the induction of membrane dorsal ruffling in response to PDGF in fibroblasts. Here, we demonstrate a novel interaction between Abi-1 and βPIX using the yeast two-hybrid system, in vitro pull-down assays, and in vivo co-immunoprecipitation experiments. In vitro, the C-terminal fragment of βPIX interacted with Abi-1, while in vivo the N-terminal fragment of βPIX interacted with Abi-1. The biological function of this interaction was investigated in mouse fibroblasts in response to PDGF stimulation. Abi-1 and βPIX co-localised in the cytoplasm and to membrane dorsal ruffles after PDGF treatment. We show that the co-expression of Abi-1 and truncated forms of βPIX in mouse fibroblasts blocked PDGF-induced membrane dorsal ruffles. Together, these results show that the interaction between Abi-1 and βPIX is involved in the formation of growth factor-induced membrane dorsal ruffles.