Effects of transplantation of peripheral blood mesenchymal stem cells with hypoxia preconditioning on postangioplasty restenosis in rabbits

AIM: To investigate the function and the mechanism of transplanting bone marrow derived peripheral blood mesenchymal stem cells (PBMSCs) on restenosis after carotid balloon angioplasty in the model of carotid atherosclerosis rabbits, and to determine if the functions of PBMSCs are enhanced after hypoxia preconditioning. METHODS: Bone marrow cells were mobilized by granulocyte colony-stimulating factor (G-CSF), and PBMSCs were collected through density gradient centrifugation and adherent culture, labeled with enhancement type green fluorescent protein (EGFP) genes. All animals with carotid atherosclerosis stenosis were randomly divided into three groups: hypoxia preconditioning group (n=24, received intravenous transplantation of PBMSCs with hypoxia preconditioning), non-hypoxia preconditioning group (n=24, received normal culture of PBMSCs) and control group (n=24, only received equal-volume of culture medium). Vascular endothelial growth factor (VEGF) was determined by enzyme linked immunosorbent assay (ELISA) at 7 d, 14 d and 28 d post-angioplasty, respectively. The vessel morphology, the homing of MSCs and the reendothelialization were analyzed with Weigert staining and immunohistochemistry. RESULTS: Compared to control group, the level of VEGF significantly increased in both hypoxia preconditioning group and non-hypoxia preconditioning group at all time points (P<0.01). The level of VEGF in hypoxia preconditioning group was higher than that in non-hypoxia preconditioning group (P<0.05) at 7 d and 14 d, but no difference at 28 d post-angioplasty was observed. At 7 d, GFP-positive cells were found both in hypoxia preconditioning group and non-hypoxia preconditioning group. Neointima thickening and the rate of restenosis were lower in hypoxia preconditioning group than those in non-hypoxia preconditioning group at 28 d (P<0.05), but both hypoxia preconditioning group and non-hypoxia preconditioning group were markedly lower than that in control group (P<0.01). The reendothelialization in hypoxia preconditioning group was outweigh than that in non-hypoxia preconditioning group (P<0.05), but both two groups were lower than that in control group (P<0.01). CONCLUSION: Intravenous transplantation of PBMSCs contributes to the reendothelialization, and attenuates neointima thickening after carotid balloon-induced injury in the rabbit model. Further, hypoxia preconditioning may strengthen the above function of MSCs, which is corelated with the increase in cytokines induced by hypoxia preconditioning to MSCs.     

Supportive effects of human aorta-gonad-mesonephero stromal cells on the directed differentiation of embryonic stem cells into hematopoietic stem cells

AIM: To direct embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) in vitro by simulating the hematogenic microenvironment in human early embryonic aorta-gonad-mesonephero (AGM) region.METHODS: Murine E14 embronic stem cell line was used for two-step differentiation.In the first step of primary differentiation,E14 ESCs were seeded into semisolid methylcellulose-based medium containing bone morphogenesis protein 4 (BMP4) and vascular endothelial growth factor (VEGF) for embryoid body (EB) formation.On days 3,6,9,12 and 15,single EB cells were analyzed for Flk-1+ cells amount through flow cytometry.In the second step,single cell from EB containing most Flk-1+ cells was further co-cultured with human AGM stromal cells in non-contact system.On co-culture days of 3,6,9 and 12 days,cells were collected for cell count,flow cytometry for Sca-1+c-kit+ cells analysis,and colony forming cell assay.RESULTS: During the EB formation,BMP4+VEGF promoted Flk-1+ cell genesis on day 9 at peak pencentage value of 27.53%±2.84％,which was statistically higher than that in control group as 8.77±1.10 (P<0.05).Collagenase-disassociated single cell from day 9 EB was co-cultured with human AGM stromal cells of hAGMS3 or hAGMS4 for further hematopoietic differentiation.On day 6 Sca-1+c-kit+ cells got to peak value as 7.31%±1.21％ ［(2.57±0.48) folds］ and 7.62%±1.52％ ［(2.35±0.36) folds］ in hAGMS3 and hAGMS4 feeder systems,respectively,both of which were greater than those values of no-stroma groups at the same culture duration (P<0.05).Colonogenic cell assay showed that these Sca-1+c-kit+ cells had ability of forming multiple lineage hematopoietic colonies.CONCLUSION: BMP4 in combination with VEGF promotes Flk-1+ cell genesis during EB formation in vitro.Stromal cells from early human embryonic AGM region further enhance the directed differentiation of these primitive cells into HSCs.This two-step induction differentiation model can be used for molecular mechanism study of ESCs hematopoietic differentiation.     

Effects of glutamine and recombinant human growth hormone on intestinal mucosal barrier and proliferating cell nuclear antigen in postoperative portal hypertension patients

AIM:To investigate morphologic and functional changes of small intestinal mucosa and proliferating cell nuclear antigen in postoperative portal hypertension patients with single or combined administration of Gln and rhGH.METHODS:Twenty-nine portal hypertension patients with surgical treatment were prospectively randomized to four groups as follows: ① Gln group (n=6);② rhGH group (n=8);③ Gln+rhGH group (n=7) and ④ control group (n=8).A standard solution for TPN was given three days after operation for a week.The concentration ratio of urinary lactulose and mannitol (L/M),the villus height and crypt depth and PCNA index of small intestinal mucosa were compared.RESULTS:A week after TPN postoperation,the increased ratios of L/M in Gln+rhGH group were less than those in control group (P<0.05).The villus height and crypt depth increased in Gln+rhGH group compared with preoperation (P<0.05) or control group (P<0.05).PCNA index increased in Gln+rhGH group compared with preoperation (P<0.05) or other three groups (P<0.05).The villus height of control group decreased (P<0.05),whereas the crypt depth had no significant difference (P＞0.05).CONCLUSION:This study suggest that Gln together with rhGH reduce the intestinal permeability and protect the mucosa integrality in postoperative portal hypertension patients,but not in single treatment.     

Jagged1 knocking down in endothelial cells accelerates PDGF induced proliferation and migration of vascular smooth muscle cells in rats

AIM: To investigate the effect of Jagged1 expression in endothelial cells (EC) on platelet derived growth factor (PDGF) induced proliferation and migration of vascular smooth muscle cells (VSMC) in rat.METHODS: Rat aorta EC was inoculated in the lower chamber and VSMC were in the upper chamber of the cell coculture system. Three groups were divided: control, sicontrol and siJagged1. The EC Jagged1 protein expression was assayed by Western blotting to evaluate small RNA interfering (RNAi) efficiency. After the cells were cocultured with PDGF for 24 h, the proliferation and migration of VSMC were respectively evaluated by ［3H］-TdR incorporation and migrating cells counting. Protein expression of α-SM-actin in VSMC was assayed by Western blotting. RESULTS: The Jagged1 protein expression in EC was significantly lower in siJagged1 group than that in control group (0.26±0.02 vs 0.67±0.02, P<0.05), and no statistic significance was observed between control and sicontrol groups. The VSMC ［3H］-TdR incorporation and migration were higher in PDGF +siJagged1 group than those in PDGF group {［3H］-TdR incorporation (23 074±2 702) counts·min-1·well-1 vs (16 442±1 803)counts·min-1·well-1, n=5, P<0.05; migration (27±4) cells/field vs (15±3)cells/field, n=5, P<0.05}. The α-SM-actin protein in VSMC was lower in PDGF + siJagged1 group than that in PDGF group (0.25±0.06 vs 0.49±0.04, n=3, P<0.05).CONCLUSION: Jagged1 knock down in rat EC accelerates PDGF induced proliferation and migration of VSMC. These results suggest that Jagged1 expression in EC plays an important role in maintaining VSMC contract phenotype and inhibiting VSMC overgrowth after arterial injury.     

Role of NF-κB in pentetrazole-induced repeated seizure in developing rats

AIM:To investigate the role of NF-κB in pentetrazole-induced repeated seizure in developing rats with the inhibitor of NF-κB pyrrolidine dithiocarbamate (PDTC). METHODS:10-day-old Wistar rats (n=72) were prepared for epilepsy model and divided into three groups at random: the PTZ group, the PDTC+PTZ group and the control group. The behavioral changes, the cells morphology and neurons counts in hippocampus, the expression of NF-κB, BrdU (5-bromo, 2-deoxyuridine) immunoreactive cells in hippocampus and the mossy fiber sprouting were observed.RESULTS:(1) The NF-κB expressed in PTZ group was significantly higher than that in PDTC+PTZ group and control group (P<0.01). (2) The dentate gyrus granule cell count in PTZ group was significantly higher than that in control group (P<0.05). In PDTC+PTZ group cell counts in CA1, CA3 and hilar region were significantly lower than those in PTZ group (P<0.05). (3) The BrdU-immunoreactive cells counts in dentate gyrus in PTZ group and PDTC+PTZ group were significantly higher than those in control group (P<0.01), but in PDTC+PTZ group BrdU-immunoreactive cell count was significantly lower than that in PTZ group (P<0.01). Correlate analyzes between NF-κB expression and BrdU-immunoreactive cell counts/granule cell counts showed positive correlation (P<0.01). (4) The mossy fiber sprouting in both PTZ and PDTC+PTZ group was observed. However, the degrees of sprouting showed no significant differences between two groups. CONCLUSION:NF-κB plays a crucial role in epilepsy of developing rats. It encourages neurogenesis and protects neurons in hippocampus, but has no significant effect on mossy fiber sprouting.     

Response of human triple-negative breast cancer to paclitaxel after vascular normalization in nude mice

AIM: To explore whether there is synergistic effect of recombinant human endostatin (rh-Endo) and paclitaxel (Pac) in the time window of vascular normalization and the role of magnetic resonance imaging (MRI) in early assessment of chemotherapy by observing the response of human triple-negative breast cancer (TNBC) to Pac after vascular normalization in nude mice. METHODS: The human TNBC MDA-MB-231 cells were planted in the subcutaneous region of right lower abdomen of BALB/c-nu female nude mice. These nude mice were randomly divided into 4 groups (n=7). rh-Endo was given for 17 consecutive days in rh-Endo group and rh-Endo+Pac group. Pac was given on the 6th and 12th days in Pac group and rh-Endo+Pac group. The dosage of both drugs was 10 mg·kg^-1·d^-1 (ip). On the day before the treatment and the 5th, 11th and 17th days after treatment, all the transplanted tumors were examined by MRI. All the mice were killed by cervical dislocation and their transplanted tumors were taken down for examinations after the last MRI on the 17th day. The changes of pathology, immunohistochemisty, microvessel density (MVD) and Ki67 expression were measured. RESULTS: On the 17th day, the volume of transplanted tumor in rh-Endo+Pac group was smaller than that in model group and rh-Endo group (P<0.05), and no difference between rh-Endo+Pac group and Pac group was found. On the 17th day, the tumor inhibitory rates in rh-Endo group, Pac group and rh-Endo+Pac group were 14.61%, 39.08% and 54.79%, respectively. The slow diffusion coefficient in Pac group was increased compared with model group, while it was decreased compared with rh-Endo+Pac group (P<0.05). No distant metastatic lesion in the tumor-bearing mice was observed. The necrotic rates in rh-Endo+Pac group and Pac group were higher than those in model group and rh-Endo group. The MVD in model group was higher than that in the other 3 groups. The MVD in rh-Endo+Pac group was decreased compared with Pac group and rh-Endo group. The Ki67 level in rh-Endo+Pac group was decreased compared with rh-Endo group, and no difference between rh-Endo+Pac group and Pac group was detected.CONCLUSION: In the time window of vascular normalization, the combination of Pac and rh-Endo has a significant antitumor effect on TNBC, but this study did not observe a significant synergistic effect of the 2 drugs. The change of slow diffusion coefficient can predict the therapeutic effect in advance.     

Effect of inhibiting mTOR signaling pathway on p-AKT1 molecule in lung of juvenile SD rats and its significance

AIM:To investigate the effect of inhibiting mammalian target of rapamycin (mTOR) signaling pathway on phosphorylated AKT1 (p-AKT1) during lung injury induced by hyperoxygen in juvenile SD rats and its significance. METHODS:The SD rats (3 weeks old, n=72) were randomly divided into air + saline group, hyperoxia + saline group, hyperoxia + OSI-027 group, and hyperoxia + rapamycin group (n=18 in each group). The animal model was constructed by continuous intervention with a 90% volume fraction of oxygen, and normal saline, OSI-027 and rapamycin were administered by intraperitoneal injection at 1, 3, 6, 8, 10 and 13 d of the observation period. At 3, 7 and 14 d, the changes of the body weight, wet/drg weight ratio (W/D), lung histopathology, alveolar septal width and lung injury score were measured, and immunohistochemistry and Western blot were used to detect the distribution and protein levels of phosphorylated S6K1 (p-S6K1) and p-AKT1 in the lung tissues. RESULTS:Compared with air group, the body weight of the rats in hyperoxia group was significantly decreased (P<0.05), the lung W/D was increased in the acute phase of lung injury (P<0.05), and the alveolar septal width and lung injury scores were significantly increased (P<0.05). The p-S6K1 positive cells in the lung tissues were increased (P<0.05), p-AKT1 positive cells were decreased (P<0.05), p-S6K1 protein was increased significantly (P<0.01), and p-AKT1 protein was decreased significantly (P<0.01). Compared with hyperoxia group, the lung tissue injury in hyperoxia + OSI-027 group was alleviated (P<0.05), p-S6K1 positive cells in the lung tissues was decreased (P<0.05), p-AKT1 positive cells was increased (P<0.05), p-S6K1 protein level was significantly decreased (P<0.05), and p-AKT1 protein level was increased (P<0.05). Hyperoxia+rapamycin further aggravated lung injury (P<0.05), p-S6K1 positive cells decreased (P<0.05), p-AKT1 positive cells increased (P<0.05), p-S6K1 protein levels decreased significantly (P<0.05), and p-AKT1 protein levels increased significantly (P<0.05). Compared with hyperoxia + rapamycin group, the lung tissue damage was alleviated in hyperoxia + OSI-027 group (P<0.05), p-AKT1 positive cells in the lung tissues were decreased (P<0.05), and p-AKT1 protein level was decreased (P<0.05). CONCLUSION:p-AKT1 may be involved in the development of hyperoxia-induced lung injury, and its regulation mechanism may be related to the mTOR signaling pathway. In hyperoxia-induced lung injury, the protein level of p-AKT1 is decreased, and mTOR inhibitors increase the p-AKT1 protein. However, only the mTORC1/2 dual inhibitor OSI-027 alleviates the hyperoxia-induced fibrosis in juvenile SD rats.     

Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Changes of plasma endostatin levels in nude mice bearing human nasopharyngeal carcinoma

AIM: To approach the changes of endostatin levels in BALB/c nude mice bearing human nasopharyngeal carcinoma(NPC) in different period (5, 10, 20, 30 and 40 days) and the relationship between endostatin and tumor's development. METHODS: BALB/c nude mice bearing NPC was reproduced by hypodermic implantation of human CNE-2 cells into right-side of axillary fossa. The level of plasma endostation was detected, and the weight of isolated tumors was measured. On the basis of the regulation of these changes, their relationships were explored. RESULTS: At 5 days [(137.61±53.41) μg/L] or 10 days [(103.06±17.33) μg/L] endostatin level had no apparent alternation in comparison with control group [(113.56±21.74) μg/L, P>0.05]. At 20, 30 and 40 days concentration of endostatin[(212.80±85.91) μg/L,(293.63±62.53) μg/L, (271.57±32.45) μg/L, respectively] were higher than that of the control group (P<0.05). Along with the development of the tumors, both the levels of endostatin and tumors weight increased. There was a positive correlation between the level of endostatin and tumor weight (r=0.687, P<0.05). CONCLUSION:These results suggested that endostatin links with the development of NPC.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Electrophysiological characters in a noble model of atrial fibrillation induced by left atrium pacing after right atrial infarction

AIM: To explore a noble chronic atrial fibrillation model induced by left atrium pacing after right atrial infarction and to investigate the atrial electrophysiological characters of the model. METHODS: 24 rabbits were randomly divided into 3 groups: control group (C group, n=8), pacing group (P group, n=8), artial infarction+pacing group (I group, n=8). C group: a pacing pole was fixed under adventitia of the left atrium without pacing; P group: a pacing pole was fixed under adventitia of the left atrium with 1 000 beats/min of pacing; I group: the animals were placed under 1 000 beats/min of left atrial pacing after ligating the atrial branch of right coronary artery. The technique of programmed stimulating was used to measure electrophysiological indexes of atrial in the groups. RESULTS: (1) After 3 weeks pacing AF was induced with a higher rates, and reached to 100% in I group. (2) At driving cycle length of 200 ms, ERPA was (115.0±7.6) ms in C group, (81.3±12.5) ms in P group and (87.5±12.8) ms in I group, which were statistically shorter in the later two groups compared to control group (both P<0.01). (3) I group and P group showed a significantly poor performance of frequency adaptability compared to control group after 3 week stimulation (P<0.01, P<0.05, respectively). (4) 3 weeks after pacing, the interval of P-wave in I group was significantly prolonged compared to P group and C group (P<0.05, P<0.01, respectively). (5) ERPA was obviously shortened and RRPA was prolonged significantly in I group compared to control group (P<0.01, respectively). Inter-atrial conduction defect (IACD) was significantly prolonged in I group compared to C group and P group after 1 h to 3 week stimulation (P<0.01, respectively). CONCLUSION: Compared to the traditional AF model induced by pacing only, a noble model of left atrium pacing after right atrial infarction has a higher AF incidence. The apparent electrophysiological changes of the AF model include: shortening of ERPA, the frequency inadaptability, extension of P-wave interval and prolonged RRPA as well as IACD.     

PP2A activation in mesenteric arteries of angiotensin Ⅱ-induced hypertensive rats

AIM: To investigate the mechanism of protein phosphatase 2A (PP2A) activation in mesenteric arteries of angiotensinⅡ(AngⅡ)-induced hypertensive rats. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to AngⅡinfusion (500 ng·kg^-1·min^-1) using osmotic minipump up to 14 d to established the hypertension model. The rats (n=40) were randomly divided into 4 groups:control group (n=10), AngⅡgroup (n=10), candesartan (CAN; AngⅡtype 1 receptor blocker)+AngⅡgroup (n=10) and CAN group (n=10). The rats in CAN+AngⅡgroup and CAN group were administered with candesartan ester at the dose of 10 mg·kg^-1·d^-1 by gavage on the first day after implantation of osmotic minipump. The rats were sacrificed on the 15th day after minipump implantation. Serum and mesenteric arteries were collected. Systolic blood pressure was measured by tail-cuff method. The serum levels of AngⅡ were measured by ELISA. The protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177), PP2A catalytic subunit (PP2Ac), phosphorylated PP2Ac (Tyr307) and PP2A inhibitor 2 (I₂^PP2A) in the mesenteric arteries were determined by Western blot. The activity of PP2A in the arteries was detected using PP2A activity assay kit. RESULTS: Compared with control group, the systolic blood pressure in AngⅡgroup was significantly increased(P<0.05), while those in CAN+AngⅡgroup and CAN group were significantly decreased (P<0.05). The serum levels of AngⅡ in AngⅡ group and CAN+AngⅡ group were significantly higher than that in control group (P<0.05). Compared with control group, the phosphorylation levels of eNOS Ser1177 were decreased in AngⅡgroup (P<0.05), but the activity of PP2A was significantly increased (P<0.05), and Pearson correlation analyses showed a negative correlation between PP2A activity and eNOS S1177 phosphorylation (r=-0.842, P<0.05). Compared with AngⅡgroup, the phosphorylation levels of eNOS Ser1177 in CAN+AngⅡgroup were significantly increased (P<0.05), but the activity of PP2A was reduced (P<0.05). Compared with control group, the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries were decreased in AngⅡgroup (P<0.05), but increased in CAN+AngⅡgroup (P<0.05). No significant difference in all above-mentioned measures between control group and CAN group, nor in the levels of total eNOS and PP2Ac protein expression among all the groups was observed. CONCLUSION: AngⅡmay reduce the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries of AngⅡ-induced hypertensive rats through AngⅡ/AngⅡ type 1 receptor-mediated signaling pathway, resulting in the activation of PP2A, then leading to down-regulation of eNOS S1177 phosphorylation, which ultimately mediates the occurrence of vascular endothelial dysfunction.     

Prevention and treatment of Hua Yu Dao Zhi decoction in renal fibrosis induced by gentamicin in rats

AIM: To investigate the effects of Hua Yu Dao Zhi decoction (HYDZD) on renal interstitial fibrosis induced by gentamicin in rats. METHODS: 32 healthy male Wistar rats were randomly divided into 3 groups: control group (n=6), gentamicin group (n=13) and HYDZD+gentamicin group (n=13). The renal tubulointerstitial fibrosis in rats was induced by gentamicin for 9 d. All rats were sacrificed on 30 d after modeling and renal tissues were stained with HE. The renal indexes were calculated and the changes of serum creatinine, blood urea nitrogen, total urinary protein in 24 h, and the level of α-SMA, Smad2, Smad7 were detected. RESULTS: Compared to control group, it was observed that there was mainly necrosis and partly degeneration in the renal tubular epithelial cells, proliferation of fibroblasts and infiltration of the inflammatory cells in the interstitial substance in gentamicin group under the light microscope. The renal indexes, serum creatinine, blood urea nitrogen, total urinary protein in 24 h, and the level of α-SMA, Smad2 were significantly higher than those in control group (P<0.01) and the level of Smad7 was significantly lower than that in control group (P<0.01). Under the light microscope, the results of all mentioned above in HYDZD+gentamicin group were improved significantly (P<0.01), the level of α-SMA, Smad2 were significantly lower than those in gentamicin group (P<0.01). However, no significant difference of Smad7 protein expression between gentamicin group and HYDZD+gentamicin group was observed (P>0.05). CONCLUSION: HYDZD is very effective in preventing and treating renal interstitial fibrosis caused by gentamicin with the decreased level of Smad2.     

Restoration of cardiac function in failing beagle hearts by recombinant adeno-associated viral gene transfer of sarcoplasmic reticulum Ca2+-ATPase

AIM: Abnormal Ca2+ homeostasis is one basic cause of heart failure. Studies have recently shown that overexpression of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) by adenoviral/adeno-associated viral gene transfer restores contractile function ex vivo and in murine or rabbit models. We therefore hypothesized that an increase in SERC A2a protein will improve cardiac function in a pacing-induced big animal model of heart failure.METHODS: 17 beagles were randomized into control group (CG, n=4) and chronic heart failure group (n=11). Four weeks after right ventricular rapid pacing (230 beats/min), 11 beagles all got heart failure (documented by >29.3% decrease in ejection fraction). 4 of 11 were used as heart failure group (HF, n=4). 9 HF beagles were randomized to receive either a recombinant adeno-associated viral carrying the SERCA2a gene (HF+SERC A2a, n=5) or the reporter gene enhanced green fluorescent protein (HF+EGFP, n=4) by thoracotomy. All HF beagles paced by 180 beats/min in order to maintain failing state. Thirty days after infection, parameters of systolic and diastolic function were measured by doppler echocardiography and hemodynamic monitor in all beagles.RESULTS: At 30 days after gene transfer, symptoms of HF+SERCA2a dogs improved. Echocardiogram parameters were superior to those in HF+EGFP group (P<0.05). Cardiac hemodynamic parameters of HF+SERCA2a dogs strikingly improved: LVSP, +dp/dtmax and -dp/dtmax increased, mean value increased respectively 54.12％［(214.72±31.74) mmHg vs (139.32±36.79) mmHg］, 146.81％［(6 779.43±217.58) mmHg/s vs (2 746.85±931.2) mmHg/s］ and 71.52％［(-4 341.42±322.02) mmHg/s vs (-2 531.14±616.15) mmHg/s］; LVEDP lowered 63.43％［(21.86±6.95) mmHg vs (59.78±6.92) mmHg］ compared with the dogs in HF+EGFP group. No significant difference in all parameters compared with those of control group was observed. Under laser confocal microscopy, widespread green fluorescence was observed in the myocardial frozen section of dogs in HF+EGFP group. CONCLUSION: These results support the hypothesis that overexpression of SERCA2a improves cardiac function in big animal model of chronic heart failure. The study demonstrates that gene transfer of SERCA2a into cardiac with recombinant adeno-associated viral vector is a prospective therapy methods.     

Brain injury induced by intrauterine infection affects offspring’s cognitive development and hippocampal neurogenesis

AIM: To explore the effect of brain injury induced by intrauterine infection on offspring’s cognitive development and hippocampal neurogenesis. METHODS: Sprague-Dawley pregnant rats (n=15) were randomly divided into model group (n=8) and control group (n=7). After delivery, the male pups were randomly divided into E. coligroup (n=35) and control group (n=35). Total 20 male pups at postnatal day 28 (P28) were randomly divided into 2 groups for Morris water maze test. Hippocampi of the 2 groups were used for cell apoptosis, neuronal proliferation and survival analysis. RESULTS: (1) The brain tissue was slightly atrophied in E. coligroup. The numbers of TUNEL and caspase-3 positive cells were significantly increased in E. coligroup (P<0.05). (2) In the navigation and memory task, the rats in E. coligroup had longer escape latency than that in control group (P<0.05). (3) There was a significant increase in the BrdU-labeled cells at P3, P7 and P14 in E. coligroup than that in control group (P<0.05). When the cells got mature, no significant difference of BrdU-labeled cells at P28 between the 2 groups was observed (P>0.05). CONCLUSION: Intrauterine infection increases hippocampal neuronal apoptosis, which may be regarded as an etiological factor in the cognitive development impairment. Inflammation-induced neurogenesis may play an important role in neuronal protection and repair in immature brain after intrauterine infection.     

Transfusion of mesenchymal stem cells improve the hematopoiesis in mice with aplastic anemia by regulating immune cells

AIM: To study the possible immunologic mechanism of mesenchymal stem cells (MSCs) by which the hematopoiesis of mice with aplastic anemia (AA) is improved. METHODS: The mice model of immuno-mediated aplastic anemia was established. Thirty BALB/c mice were divided into 3 groups: radiation group, AA model group and MSCs group. The mice in radiation group only had processing of 5 Gy ［60Co］γ radiation. The mice in AA model group were intravenously injected with the cell suspension of thymocyte and lymph-node cell mixture (prepared from DBA/2 mice, 1×106 cells) after 5 Gy ［60Co］γ radiation. The animals in MSCs group received the same treatment as the mice in AA model group did, and afterwards was injected with 1×106 MSCs intravenously at 3 days. All the mice in the 3 groups were observed and analyzed the following parameters: peripheral blood cells, pathological features of bone marrow, the relation between the changes of peripheral CD4+, CD8+, CD4+/CD8+, NK cells and the hematopoiesis. RESULTS: After 5 Gy ［60Co］γ radiation, the peripheral blood cells in all groups decreased at 7 days, while at 21 days those in MSCs group and radiation group restored to normal but those in AA model group was still at lower level. At 7 days, the cells of CD4+, CD8+ and CD4+/CD8+ in each group were similar without statistical difference, but the number of NK cells in MSCs group was quite lower than that in the other 2 groups (P<0.05). At 14 days, the CD4+ cells in all groups were obviously decreased, while the CD8+ cells in MSCs group and AA model group were significantly higher than those in radiation group. At 21 days, the CD8+ and NK cells in MSCs group were quite the same as those in radiation group while those in AA model group were obviously higher compared to the other 2 groups. The number of adipose cells observed in the pathological slice of femoral bone in MSCs group and radiation group was no difference while that in AA model group was much higher. CONCLUSION: MSCs transfusion improves the hematopoiesis in mice with aplastic anemia by regulating the functions of immune cells.     

Effect of hydrogen sulfide donor on oxidative stress of myocardium in adriamycin-induced dilated cardiomyopathy rats

AIM: To investigate the effect of hydrogen sulfide (H2S) donor (NAHS) on oxidative stress of adriamycin-induced dilated cardiomyopathy rats. METHODS: Weight-matched adult male Wistar rats were randomly divided into 5 groups as follows: (1) ADR group (n=12), in which 2.5 mg/kg of adriamycin was injected intraperitoneally once a week for 10 weeks (total dose of 25 mg/kg). (2) ADR+small-dose NaHS group (n=12), in which the dosage and the use of adriamycin were as mentioned above, while NaHS solution was injected to rats at a dosage of 2.8 μmol·kg-1·d-1 at the same time. (3) ADR + large-dose NaHS group (n=12), in which the dosage and the use of adriamycin were as mentioned above, while NaHS solution was injected to rats at a dosage of 14 μmol·kg-1·d-1 at the same time. (4) Control group (n=9), in which an equivalent volume of physiological saline was administered weekly for a total of 10 weeks. (5) NaHS group (n=9), in which 14 μmol/kg of NaHS solution was injected to rats intraperitonealy once a week for 10 weeks. Hemodynamic and echocardiographic measurements were obtained 10 weeks after the treatment. Meanwhile, H2S and malondialdehyde (MDA) concentrations, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum and myocardial tissues were evaluated, respectively. RESULTS: The cardiac functions in the group of ADR rats depressed obviously. H2S concentrations, SOD and GSH-Px activities in serum and myocardial tissues of ADR group rats were all significantly decreased as compared with those in the control group (P<0.01). The MDA concentrations in serum and myocardial tissues in ADR group rats were both increased significantly (P<0.01). Exogenous administration of H2S donor NaHS markedly attenuated ADR-induced cardiac dysfunction, and MDA concentration in myocardial tissues was significantly reduced (P<0.01). Serum SOD activity was obviously increased in ADR+large-dose NaHS group compared with control group (P<0.01), and GSH-Px activity in myocardial tissues was markedly increased in ADR+large-dose NaHS group compared with control group (P<0.05). CONCLUSION: H2S might play an important role in the development of adriamycin-induced dilated cardiomyopathy. Administration of exogenous H2S effectively improves myocardial contractile activity, reduces the accumulation of lipid peroxides and increases the capability of antioxidants to inhibit oxidative stress and prevents myocardial damage.     

Protective effect of aliskiren on SH-SY5Y cells injured by oxygen-glucose deprivation and its possible mechanisms

AIM: To investigate the effects of aliskiren on the injury of SH-SY5Y cells induced by oxygen-glucose deprivation (OGD) and its possible mechanisms. METHODS: The SH-SY5Y cells were randomly divided into control group, OGD group and aliskiren (5.0, 10.0 and 20.0 μmol/L) groups. The cell viability was measured by CCK-8 assay. The levels of excitatory amino acid transporter 2 (EAAT2/GLT-1), EAAT3/EAAC1, EAAT4, endothelin-1 (ET-1) and S100 calcium-binding protein β subunit (S-100β) in the SH-SY5Y cells were detected by ELISA. The morphological changes of the cells were observed by Hoechst 33258 staining. Meanwhile, the content of lactic acid (LD) and activity of Na⁺-K⁺-ATPase were also analyzed. RESULTS: The viability of SH-SY5Y cells was not more than 60% after OGD injury for 4 h, so the appropriate time for OGD injury was 4 h. Compared with control group, the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells of OGD group were significantly decreased (P<0.05), but the protein levels of ET-1 and S-100β were significantly increased (P<0.05). Compared with OGD group, treatment with aliskiren dose-dependently increased the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells, but decreases in the levels of ET-1 and S-100β were observed (P<0.05). The results of Hochest 33258 staining showed that aliskiren significantly reduced the apoptosis of SH-SY5Y cells. Compared with control group, a significant increase in the content of LD (P<0.05) and a significant decrease in Na⁺-K⁺-ATPase activity (P<0.05) were found in the SH-SY5Y cells of OGD group. Compared with OGD group, aliskiren dose-dependently decreased the content of LD, but increased the Na⁺-K⁺-ATPase activity in the SH-SY5Y cells (P<0.05). CONCLUSION: Aliskiren has good neuroprotective effects on SH-SY5Y cells after OGD injury. The underlying mechanisms may be associated with the increases in the protein levels of GLT-1, EAAC1 and EAAT4, the enhancement of Na⁺-K⁺-ATPase activity, and the decreases in the levels of ET-1 and S-100β and the content of LD.