Effects of bone mesenchymal stem cell transplantation on silicosis fibrosis in rats

AIM: To study the effects of exogenous bone mesenchymal stem cell (BMSC) transplantation on silicosis fibrosis in rats, and to explore the dose-effect relationship. METHODS: BMSCs were isolated and cultured from male 5-week-old SD rats in vitro. Fifty healthy female SD rats were randomly divided into 5 groups: control group, silicosis model group, BMSCs treatment A group (1×10⁹ cells/L), BMSCs treatment B group (3×10⁹ cells/L) and BMSCs treatment C group (5×10⁹ cells/L). The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal intubation, and different doses of BMSCs were given for intervention therapy. All the rats were sacrificed on the 21st day after the model was established. The morphological changes of the lung tissues were observed by HE staining and Masson staining. The localization and distribution of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were determined by the method of immunohistochemistry. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III were detected by Western blotting. The sex-determining region (SRY) protein was searched by an immunofluorescence method to confirm the homing of BMSCs. RESULTS: Compared with control group, the silicosis model group had significant alveolitis changes, silicon nodule formation, collagen deposition and other pathological characteristics. Compared with silicosis model group, the pathological changes in BMSCs treatment A group were improved. The conditions of BMSCs treatment B group were also improved significantly. However,the pathological changes in BMSCs treatment C group were increased obviously. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III in the lung tissues ranked as follows: BMSCs treatment C group > silicosis model group > BMSCs treatment A group > BMSCs treatment B group > control group. The difference between BMSCs treatment C group and silicosis model group was not statistically significant, and the differences between the other groups were statistically significant. The SRY-positive cells were observed in BMSCs treatment B group, but no significant expression in the heart, liver, spleen and kidney tissues was observed. CONCLUSION: The exogenous BMSC transplantation antagonizes the development of silicosis fibrosis in rats, which has dose-effect relationship.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

NK cells from donor liver inhibit NF-κB activity and IL-15 expression after liver transplantation in rats

AIM:To investigate the mechanism that donor liver natural killer (NK) cells alleviate acute rejection after liver transplantation by observing the secretion level of interleukin 15 (IL-15) in peripheral blood, the protein expression of IL-15 in transplanted liver tissues and the activity of NF-κB in spleen tissues in rat acute liver graft rejection model. METHODS:An acute rejection model of liver transplantation in rats was established by the modified two-cuff method, in which Lewis rats were used as donors and BN rats as recipients. The donor leukocytes were depleted by whole body irradiation of ［60Co］ source and the donor liver immunity was reconstituted by transfusion of liver NK cells from the same type of donor (donor type liver NK cells, dtlNKs) via portal vein immediately after grafting the irradiated liver. The rats were divided into the following groups: group A, acute rejection group; group B, BN rats receiving the liver of Lewis rats with ［60Co］ irradiation; group C, BN rats receiving the liver of ［60Co］-irradiated Lewis rats and treated with dtlNKs via the portal vein. The recipients were sacrificed at 1 d, 3 d and 7 d after transplantation. IL-15 level in peripheral blood was detected by ELISA. The expression of IL-15 in the liver grafts was determined by Western blotting. NF-κB activity in the spleen tissues of recipient rats was identified by electrophoretic mobility shift assay. The survival quality and living time in crude survival subgroup were observed. RESULTS:Acute rejection in group B was severer than that in group A and group C. The rats in group B showed significantly shorter average survival time compared with group A and group C. At 3 d and 7 d after transplantation, the IL-15 content in peripheral blood was significantly higher in group B than that in group A and group C. The expression of IL-15 in transplanted liver tissues was significantly higher in group B than that in group A and group C. The activity of NF-κB in the spleen tissues was higher in group B. CONCLUSION: IL-15 might be a significant indicator for monitoring acute rejection after liver transplantation. The donor liver NK cells modulate the immunity of liver transplantation by inhibiting the expression of IL-15 via the suppression of NF-κB activity.     

Tetramethylpyrazine combined with bone marrow mesenchymal stem cell transplantation inhibits neuronal apoptosis in rats with cerebral ischemia

ATM: To investigate the effects of tetramethylpyrazine (TMP) combined with bone marrow mesenchymal stem cells (BMSCs) on neuronal apoptosis, and Bcl-2 and Bax expression in rats with cerebral ischemia. METHODS: The BMSCs were isolated by the whole bone marrow adherent method and cultured, and those in the 3rd passage were used for tail-vein transplantation. The rats were subjected to right middle cerebral artery occlusion (MCAO) using suture method, and the rats except sham group were randomly divided into model group, BMSCs (1×10⁹ cells/L) group, TMP (40 mg/kg) group and combination (TMP+BMSCs) group with 12 rats in each group. Neurological function was evaluated by modified neurological severity scoring (mNSS) on 1 d, 7 d and 14 d after cerebral ischemia. Toluidine blue staining was performed to detect cerebral infarct volume, HE staining was used to observe brain histopathological change, neuronal apoptosis was observed by TUNEL staining, and the mRNA and protein expression of Bcl-2 and Bax was detected by real-time fluorescence quantitative PCR and Western blot at 14 d after cerebral ischemia. RESULTS: Compared with BMSCs group and TMP group, TMP combined with BMSCs significantly reduced the score of mNSS (P<0.01) and the infarct volume (P<0.01), alleviated the pathological damage in the peripheral area of cerebral ischemia, decreased the number of TUNEL positive cells (P<0.01), increased the expression of Bcl-2 and decreased the expression of Bax at mRNA and protein levels (P<0.01).CONCLUSION: Tetramethylpyrazine combined with transplantation of BMSCs improves the functional recovery, reduces the infarct volume, relieves the ischemic injury of the brain tissue, and attenuates neuronal apoptosis in the rats with cerebral ischemia. The mechanism may be related to regulating the expression of Bcl-2 and Bax.     

Difference of immune response to transplantation of BMSCs and non-BMSCs-derived cells in animals

AIM: To analyze the characteristics of immune response in the animals after transplantation of bone marrow stromal cells (BMSCs) or non-BMSCs-derived cells.METHODS: Rat BMSCs and Buffalo rat liver (BRL) cells were transplanted into SD rats and New Zealand rabbits by intravenous and subcutaneous injections. The differences of immune response in the animals received these two types of cells were evaluated by comparative analysis of the lymphocytes and monocytes in peripheral blood as well as the histopathological characteristics of the subcutaneous transplantation sites.RESULTS: The increase intensity of the lymphocyte number in BMSCs group was lower than that in BRL group, regardless of allograft or xenograft. The immune reaction of the animals to BMSCs transplantation was a little slower. It was obviously dissimilar that the increase intensity of monocyte number in BMSCs group was higher than that in BRL group. Pathological cutaneous sections showed that neither obvious necrosis nor inflammatory cells was observed in the vicinity of BMSCs transplantation sites.CONCLUSION: The intensity of immune response induced by BMSCs is lower than that by BRL cells. The anti-rejection ability of BMSCs in cell transplantation is better than that of BRL cells.     

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.     

miR-34a induces senescence of bone marrow-derived mesenchymal stem cells under high glucose condition by SIRT1

AIM:To detect the effect and potential mechanism of microRNA-34a (miR-34a) on the senescence of bone marrow-derived mesenchymal stem cells (BMSCs) under high glucose condition. METHODS:BMSCs were isolated and cultured from 60~80 g male SD rats. The BMSCs were divided into 5 groups:normal glucose(NG) group, high glucose(HG) group, HG+miR-34a mimic group, HG+miR-34a NC group and HG+miR-34a inhibitor group. In order to confirm whether miR-34a regulated the senescence of BMSCs under high glucose condition by regulating the expression of silent information regulator 1(SIRT1), in addition to the above groups, HG+siRNA-SIRT1 group, HG+siRNA-NT group and HG+miR-34a inhibitor+siRNA-SIRT1 group were added. The expression of miR-34a and SIRT1 mRNA was detected by RT-qPCR. CCK-8 assay and senescence-associated β-galactosidase assay were used to detect cell viability and senescence, respectively. The protein expression of SIRT1, forkhead box O3a (FOXO3a) and P21 in the BMSCs was analyzed by Western blot. RESULTS:The expression of miR-34a in HG group was increased significantly compared with NG group (P<0.01), and long-term exposure of the BMSCs to high glucose lead to decreased cell viability and increased senescence (P<0.05). Compared with HG+miR-34a NC group, the cell viability in HG+miR-34a mimic group was decreased significantly (P<0.01), the senescence of BMSCs was increased significantly (P<0.01), the protein expression of SIRT1 was decreased significantly (P<0.01) and the protein expression of FOXO3a was increased significantly (P<0.01). However, inhibition of miR-34a expression showed the opposite effect to miR-34a mimic. Similar to the HG+miR-34a mimic group, the protein expression of P21 and FOXO3a in HG+siRNA-SIRT1 group were significantly higher than that in HG group (P<0.01). After adding siRNA-SIRT1 into HG+miR-34a inhibitor group, the inhibitory effect of the miR-34a inhibitor on the expression of P21 and FOXO3a in BMSCs were partly weakened (P<0.05). CONCLUSION:miR-34a regulate the senescence of BMSCs under high glucose condition by regulating the expression of SIRT1.     

Effects of meglumine cycle adenylate phosphate combined with transplantation of mesenchymal stem cells on heart functions in rat model of heart failure

AIM: To investigate the therapeutic effects of recombinant meglumine cycle adenylate phosphate (MCA) and bone marrow mesenchymal stem cells (BMSCs) on the enhancement of the cell survival and improvement of the cardiac functions in the rat model of adriamycin-induced cardiomyopathic heart failure. METHODS: BMSCs were isolated and expanded using the pre-plating method. Doxorubicin was used by intraperitoneal injection into the Wistar rats to establish the model of cardiomyopathic heart failure. The model animals randomly received the injection of PBS, MCA, BMSCs or MCA+BMSCs respectively, and normal controls were without any treatment. Four weeks after injection, the cardiac functions were determined by echocardiography and multichannel physiological recorder. The levels of brain natriuretic peptide (BNP) were measured by ELISA. The positive rate of BrdU-labeled BMSCs in the myocardium was analyzed by the method of immunohistochemistry. The expression of myocardium-specific protein, GATA-4, connexin 43(Cx43) and cardiac troponin 1(cTNI), was detected by Western blotting. Myocardial fibrosis was observed with Masson's staining. RESULTS: Compared with other groups, the results of echocardiography and hemodynamic showed that the left ventricular functions in BMSCs+MCA group improved significantly (P<0.05). The BMSCs numbers in the myocardium in BMSCs+MCA group were significantly higher than those in BMSCs group (P<0.05). The level of BNP was significantly lower in BMSCs+MCA group than that in BMSCs group (P<0.05). Compared with other groups, the expression of GATA-4, Cx43 and cTNI was significantly increased in BMSCs+MCA group. CONCLUSION: Combination of MCA with BMSCs transplantation improves the cardiac functions, possibly due to the enhancement of BMSCs survival and the increase in the protein expression of GATA-4.     

Migration of enhanced green fluorescent protein labeled bone marrow after transplantation into rat cerebral infarct

AIM:To investigate the role of SDF-1α in migrating of bone marrow stromal cells to the injured areas.METHODS:Ischemic brain lesion model was created in rats by permanent middle cerebral artery occlusion (MCAO).48 SD rats were divided randomly into 2 groups.Group 1:phosphate buffered saline (PBS 1 mL) for control (n=25); Group 2:BMSCs (2×106) were injected intravenously at 24 h after MCAO (n=24).After propagated in BMSCs, Ad5/F35 GFP (green fluorescent protein) was infected to BMSCs.The expression of SDF-1α (stromal cell-derived factor-1α) mRNA in the penrumbral tissue was assayed by real-time quantitative PCR.The expression of CXCR4 on MSCs was detected by flow cytometry.Confocal microscopy was used to detect the GFP-labeled MSCs migration.RESULTS:Ad5/F35 GFP signals was observed in almost infected BMSCs.The expressions of SDF-1α mRNA in the thalamus and hippocampus of the ischemic brains were peaked at 3rd day after stroke, followed by a decrease at 14th day post-ischemia.The expression of SDF-1α mRNA in the cortex of the ischemic brains was peaked at 7th day post-ischemia, still at high level at 14th day post-ischemia.The median percentage of surface CXCR4 expression in BMSCs was 14%.GFP labeled BMSCs were detected in the origination of the middle cerebral artery (olfactory area) at 6 h, after 3 days in the prenumbra tissue such as thalamus, and in the cortex more labeled cells were found after 14 d post-ischemia.CONCLUSION:BMSCs can pass through the blood brain barrier of ischemic rats.Its mechanism might be associated with the expression of SDF-1α in the ischemic brain.     

Molecular mechanism of BMSC intracerebral transplantation in improving learning and memory abilities of AD mice

AIM: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS: C57/BL6 wild-type (WT) and transgenic (Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group, Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracerebroventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9 (MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RESULTS: The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice (P<0.05). Compared with Tg/PBS group, the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05), and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile, the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased (P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95, p85, p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice (P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P<0.05). CONCLUSION: BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic protein expression, thus improving the learning and memory abilities in the APP/PS1 mice, which may be achieved by up-regulating the expression of CX3CL1.     

Salvianolic acid B reduces apoptosis of bone marrow stem cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolic acid B (Sal B) on apoptosis of rat bone mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H₂O₂). METHODS: BMSCs were incubated with Sal B at the concentration of 1, 10 or 100 μmol/L while treated with lethal concentration of H₂O₂ (500 μmol/L). The effect of Sal B at different concentrations on the viability of BMSCs was detected by MTT. Flow cytometry were used to determine the protective role of Sal B in apoptosis of BMSCs. The changes of chromatin distribution in BMSCs were observed by Hoechst 33342 staining. The expression of p-ERK1/2 was detected by Western blotting. RESULTS: Sal B protected the BMSCs against H₂O₂ as the cell viability was increased from (53.60±4.21)% to (85.33±9.08)% or (75.78±6.28)% in a dose-dependent manner. After exposed to H₂O₂, about 50%-65% BMSCs displayed apoptotic morphology. Treatment with Sal B at the concentrations of 10 and 100 μmol/L reduced the cytotoxic effect of H₂O₂ on BMSCs to about 32% and 47%, respectively. The results of flow cytometric analysis confirmed the cytoprotective effect of Sal B. This protective effect was concomitant with significant reduction of ROS generation. Moreover, H₂O₂ time-dependently induced a pronounced increase in ERK1/2 phosphorylation,which was effectively inhibited by Sal B.CONCLUSION: Sal B protects BMSCs against H₂O₂-induced apoptosis. Sal B may exert its protective effect on BMSCs by triggering intracellular anti-apoptosis mechanism as well as reducing the oxidative stress.     

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

Orthotopic transplant fetal rat pancreatic stem cells for treating diabetes mellitus

AIM: To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet-like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16. SRY DNA was examined to discriminate gender by fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult SD rats were divided into three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1×10⁶ male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assessed by Western blotting. RESULTS: 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum (3 weeks after transplantation), culminating (5 weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipients pancreata 8 weeks post-transplantation. The expression levels of insulin mRNA and protein in the orthotopic transplanted recipients pancreata were higher than those in experimental control (P<0.05), and the expressions of PDX-1 mRNA and protein were also higher than those in normal control (P<0.05). CONCLUSION: When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into islet-like cell cluster, gains comparable function with normal islets and reverses experimental diabetes.     

Effects of miR-124 over-expression on right ventricular remodeling in rats with pulmonary hypertension

AIM: To investigate the effect of microRNA-124 (miR-124) over-expression mediated by adeno-associated virus (AAV) on right ventricular remodeling in rats with pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). METHODS: Male SD rats (n=32) were randomly divided into 4 groups:normal control (control) group, MCT+normal saline (NS) group, MCT+AAV-GFP (MCT+GFP) group and MCT+AAV-miR-124 (MCT+miR-124) group. The rats in the latter 3 groups were instilled slowly with 100 μL NS, AAV-GFP and AAV-miR-124 by orotracheal instillation after anesthesia, respectively. Three weeks later, MCT (60 mg/kg) was intraperitoneally injected to establish the PAH model. Right ventricular systolic blood pressure (RVSP) and mean arterial pressure of the rats were measured, and right ventricular hypertrophy index (RVHI) and right ventricular weight index (RVWI) were calculated. The pathological sections of the right heart were stained with Sirius red, and the pathological changes of myocardium were observed under a microscope. The expression of miR-124 in the lung tissues was detected by RT-qPCR. The protein levels of transforming growth factor-β₁(TGF-β₁) and p-Smad2 in right heart tissues were determined by Western blot. RESULTS: Compared with control group, RVSP, RVHI, RVWI and the protein levels of TGF-β₁ and p-Smad2 in MCT+NS group and MCT+GFP group were significantly increased (P<0.05), the right ventricular myocytes were significantly enlarged, and collagen deposition was significantly increased. However, compared with MCT+GFP group, RVSP, RVHI, RVWI and the protein levels of TGF-β₁ and p-Smad2 in MCT+miR-124 group were significantly decreased (P<0.05), the degree of right ventricular myocyte hypertrophy was significantly reduced, and collagen deposition was significantly reduced. CONCLUSION: Over-expression of miR-124 obviously reduces RVSP of rats induced by MCT and relieves myocardial remodeling, which may be related to the down-regulation of TGF-β₁ and p-Smad2.     

Molecular mechanisms of liver regeneration following cold ischemia injury after liver transplantation in rat

AIM: To study the molecular mechanisms of liver regeneration following different cold ischemia (CI) times after liver transplantation in a rat model. METHODS: A model of rat orthotopic liver transplantation was established. The rats were divided into 3 groups: 1 h CI group, 8 h CI group and 16 h CI group. Survival rate in each group was recorded. Specimen were collected at predetermined intervals from 90 min, 1, 4 and 7 d post-reperfusion. The patterns of TNF-α, IL-6 and STAT3 activation were determined in liver grafts with 1 h, 8 h and 16 h CI times. Expression of cyclin D1 and hepatocyte replication with bromodeoxyuridine (BrdU) were confirmed by immunohistochemistry. The results of TNF-α and IL-6 expression in all groups were analyzed after whole liver transplantation. Statistical analysis was used to compare BrdU positively stained hepatocytes at 48 h post-reperfusion. RESULTS: Liver transplantation was successfully performed in all experimental groups. Survival rate in each group was 100% (>14 d). Compared with 1 h CI, TNF-α expressions in whole liver grafts with 8 h and 16 h CI were markedly increased at 90 min after reperfusion(P<0.05). Compared with 1 and 8 h CI, IL-6 expression in liver grafts preserved for 16 h were markedly increased at 90 min after transplantation (P<0.05). With 8 and 16 h CI, STAT3 activity was markedly increased. Cyclin D1 expression in 8 CI group was demonstrated with cytoplasmic and nuclear staining at 24 h in liver grafts. Cyclin D1 expression was mainly nuclear in 16 h CI group. Extensive hepatocyte replication was present. The numbers of hepatocytes with positively stained nuclei in 16 h CI group were more than those in 1 and 8 h CI group at 48 h after transplantation(P<0.05). CONCLUSION: Rat whole liver grafts with 16 h CI injury still initiate and complete liver regeneration and graft recovery after liver transplantation. Liver regeneration following transplantation may be through TNF-α/IL-6/STAT3/cyclin D1/DNA synthesis pathways.     

Effect of zacopride on angiotensin Ⅱ-induced myocardial fibrosis

AIM:To investigate the effect of inwardly rectifying potassium channel (I_K1) agonist zacopride (Zac) on angiotensin Ⅱ (Ang Ⅱ)-induced viability and apoptosis of cardiac fibroblasts (CFb) and to explore the underlying anti-fibrosis mechanism.METHODS:The ventricular fibroblasts from neonatal SD rats were isolated and cultured by tissue digestion and differential adherence methods. The model of rat cardiac fibroblast activation induced by angiotensin Ⅱ was established. The CFb were randomly divided into control group, Ang Ⅱ model group, Ang Ⅱ+Zac group, Ang Ⅱ+Zac+BaCl₂ group, AngⅡ+Zac+chloroquine group and Ang Ⅱ+captopril group. CCK-8 assay was used to detect the effect of Zac on the viability of CFb. The amount of collagen I and collagen Ⅲ secreted by CFb was determined by ELISA. The apoptosis of the CFb was analyzed by flow cytometry. The protein expression of Kir2.1 was determined by Western blot. RESULTS:Compared with the control group, the viability and collagen synthesis of the CFb were significantly increased, along with decreased Kir2.1 expression (P<0.05). Compared with the Ang Ⅱ model group, Zac treatment inhibited the viability and collagen synthesis of the CFb, induced apoptosis and up-regulated Kir2.1 expression (P<0.05). I_K1 blockers BaCl₂ and chloroquine reversed the effect of Zac. CONCLUSION:By enhancing I_K1 (Kir2.1) expression, Zac attenuates Ang Ⅱ-induced ventricular fibrosis, in response to the inhibition of cell viability and induction of apoptosis.     

Proliferation and differentiation of bone marrow mesenchymal stem cells in process of bone loss in ovariectomized rats

AIM: To study the function of proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) for bone loss in the pathogenesis of osteoporosis (OP) in ovariectomized rats. METHODS: Animal model of OP was established by ovariectomy (OVX,bilateral ovarian resection) in 10-week-old healthy female Sprague-Dawley (SD) rats.BMSCs were isolated, cultured and purified by the combination of density gradient centrifugation, adhesion separation and limited dilution method, and cultured in vitro to the 3rd~4th passage in all experiments. The BMSCs phenotype appraisal was studied by flow cytometry. Colony-forming assay was applied to detect the BMSCs proliferation ability. The MTT method was used to analyze the growth curves of BMSCs. After adipogenic induction (ADI), lipid drops were observed by oil red O staining to compare the adipogenic potential between the 2 kinds of BMSCs. After osteogenic induction (OSI), calcium nodules were observed by alizarin red staining (ARS). The mRNA expression levels of BMSCs osteogenesis-related proteins, for instance, Runx2, osteocalcin (OCN) and osteopontin (OPN) were measured by RT-PCR. RESULTS: Compared with sham group, the colony-forming ability of BMSCs in OVX group became decreased, the proliferation capacity was declined, the osteogenic potential was decreased, and the adipogenic potential was increased(P<0.05). CONCLUSION: In ovariectomized OP rats, the proliferation and osteogenesis of BMSCs decrease, and the adipogenesis of BMSCs increases, which may cause rapid bone loss and play an important role in the pathogenesis of OP.