Effects of ClC-3 over-expression on structure and function of thyroid in mice

AIM: To study the effect of ClC-3 gene over-expression on thyroid structure and function in mice.METHODS: Three-months-old FVB mice were used to study the difference of thyroid structure and function between wild-type (WT) mouse and ClC-3 transgene mice. The expression and distribution of ClC-3 in the thyroid of mice were determined by the methods of qPCR, Western blot and immunofluorescence. Behavioral monitoring was performed on the daily activities of mice. Serum concentrations of total triiodothyronine (TT3), total thyroxine (TT4) and thyrotropin (TSH) were measured by ELISA.RESULTS: Compared with the WT group, the expression of ClC-3 in the thyroid of ClC-3 transgene group was significantly increased (P<0.05). The thyroid gland showed obvious hyperplasia and the folliculi glandulae thyreoideae was significantly bigger in ClC-3 transgene mice (P<0.05). The weight loss was increased in ClC-3 transgene mice (P<0.05). The expression of TT3 and TT4 were significantly higher than that of WT group (P<0.05), but the change of TSH was not obvious.CONCLUSION: ClC-3 over-expression results in thyroid hyperplasia and thyroid hormone secretion. This study suggests that ClC-3 is likely to be involved in the synthesis of thyroid hormones.     

Histomorphometry investigation and mechanism exploration of postmenopausal osteoporosis

AIM: To explore the pathophysiological mechanism of postmenopausal osteoporosis, we established an animal model for postmenopausal osteoporosis and investigated the loss of bone mass.METHODS: Thirty-one female SD rats (three months old) were randomly divided into two groups: sixteen ovariectomized (OVX) and fifteen sham-operated group as control. At post-operated day 28, seven rats of either OVX group or sham-operated group were sacrificed. At post-operated day 56, the rest of either were sacrificed. The wet weight of the uteruses were measured and the changes of bone micro-architecture were studied by bone mineral density (BMD) and the data of bone histomorphometry. RESULTS: Compared with the sham group, the mean wet weight and the bone density of proximal tibia 1/3 section of the OVX group was significantly decreased (P＜0.05); Data of bone histomorphormetry showed that the trabecular percentage area of metaphysis of both distal femur and proximal tibia was significantly decreased (P＜0.01) both at post-operated day 28 and day 56, so were the number of trabecular bone and the thickness of trabecular bone; but no significance was observed in epiphysis.CONCLUSION: BMD and the data of bone histomorphometry at distal femur decreased steadily while those of at proximal tibia did sharply and unsteadily; the early loss of bone mass after ovariectomy was found in metaphysis of long bones.     

Osteogenic efficiency of antigen-extracted xenogeneic cancellous bone

AIM：To investigate the applicability and degradability of the antigen-extracted xenogeneic bone carrying recombinant human bone morphogenetic protein 2 (rhBMP-2) as a scaffold in repairing the mandibular defect in vivo. METHODS：New Zealand rabbits (n=28) with 28 mandibular defects were divided into 3 groups at random: antigen-extracted xenogeneic cancellous bone/rhBMP-2 group (group A), antigen-extracted xenogeneic cancellous bone group (group B) and blank control group (group C). Twelve bone defects each in group A and group B were classified into 3 time points (4, 8 and 12 weeks). Observation in general, X-ray test and hematoxylin and eosin staining and bone density measurement were conducted on each rabbit in group A and group B. Four bone defects were classified into group C. Observation in general, X-ray test and hematoxylin and eosin staining were also conducted on each rabbit in group C. RESULTS：The X-ray showed that the implanted materials were degraded after a period of time, and were replaced by autogenous bone. At the 12th week, the implanted materials in group A were entirely degraded and replaced by autogenous bone. The bone density measurement showed that the bone density was enhanced after implantation. At the 12th week, there was an obvious difference between group A and group B. The hematoxylin and eosin staining showed there were more neovascularization, new fibrosis and new bone formation in group A than those in group B. The implanted material in group A degraded much faster than that in group B. The significant difference in the new bone area ratio between the 2 groups among all weeks was observed. CONCLUSION：An antigen-extracted xenogeneic cancellous bone has good biocompatibility, which can act as a scaffold in bone repairment. It is the carrier of rhBMP-2 to continue the bone formation. Therefore, antigen-extracted xenogeneic cancellous bone is a kind of good material for bone repairment.     

Therapeutic effects of modified Bushen Huoxue granules on ovariectomized osteoporosis rats

AIM: To observe the therapeutic effect of modified Bushen Huoxue granules (MBHG) on ovariectomized osteoporosis rats. METHODS: Female SD rats were randomly given sham operation (n=10) and ovariectomy, and then the model rats were further randomly divided into model group, MBHG treatment groups at doses of 200 mg/kg, 100 mg/kg and 50 mg/kg respectively, and positive control (estradiol valerate) group, with 10 rats in each group by intragastric administration for 12 weeks. The morphology, area, thickness, spacing and area percentage of trabecular bone in the rats were observed. The serum levels of calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) were mea-sured by automatic analyzer. Bone mineral density (BMD) was analyzed. Serum estradiol (E₂), osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor κB ligand (RANKL) levels were detected by ELISA. RESULTS: Compared with model group, trabecular bone significantly widened in all treatment groups with large number, and net-like structure restored partially. The thickness, area and area percentages of trabecular bone in treatments groups were higher than those in model group,and trabecular spacing was less than that in model group (P<0.05). The serum Ca, P, E₂ and OPG, and femoral BMD were significantly higher in treatment groups than those in model group, and the levels of ALP, BGP, RANK and RANKL were significantly lower than those in model group (P<0.01).CONCLUSION: MBHG has a significant therapeutic effect on ovariectomized osteoporosis rats. The mechanism may be related to the regulation of OPG and inhibition of RANKL secretion.     

Effects of estrogens on BMP-4 gene expression during osteoporotic fracture healing

AIM: To study the effects of estrogens on expression of BMP-4 in osteoporotic fracture healing, and the pathogenesis of improvement of osteoporotic fracture healing by estrogen. METHODS: 144 female SD rats were divided randomly into ovariectomy group (OVX)， sham-operation group (sham) and estrogen replacement therapy group (OVXE). Closed fractures were produced manually in shaft of both tibias after intra-medullary fixation in all rats one month later. Calluses and soft tissue were collected at the date of 1st, 3rd, 5th, 7th, 14th, 28th, 56th, 112th day after fracture. The histological changes of calluses and the healing of trabecular bone with the structure changes of trabecular bone fiber were observed by microscope and activity of osteoblasts, osteoclasts by electron microscopy. The regularity of BMP-4 expression during osteoporotic fracture and the effect of estrogens on it were studied by in situ hybridization with BMP-4 cDNA probe. RESULTS: (1) Compared to OVXE group, the changes in callus density and arrange of trabecular bone, activity of osteoblasts and osteoclasts in OVX group were significantly different, and more remarkable changes were observed than that in sham group. (2) From 1 to 3 days after fracture, the signals of BMP-4 were positively detected and localized in the cells in parafractures hematoma and in the nearly appeared mesenchymal cells in the muscles adjacent to fracture site, and OVX>OVXE>sham (P<0.01). CONCLUSION: When estrogen deficient, bone turnover was enhanced with the increase in BMP-4 gene expression. Estrogens play an important role in fracture healing, estrogen replacement therapy may be effective.     

Seipin gene deficiency induces renal injury in mice

AIM:To investigate the effects of seipin gene deficiency on renal injury and the possible mechanisms in seipin-/- mice. METHODS:Six-month-old male seipin knockout (seipin-/-) and wild-type (WT) mice (n=8) were used to study 24 h urinary albumin excretion (UAE), renal functions, pathological changes, and plasma leptin and adiponectin levels. Seipin mRNA expression in different tissues and each part of the kidney was also measured in WT mice. RESULTS:Real-time PCR analysis showed seipin mRNA expression in WT mice was higher in adipose tissue and testicles, and was also found in the kidney, which was mainly in glomeruli. Compared with control group, seipin-/- group showed increased kidney weight/tibia length (P<0.01), 24 h UAE (P<0.01), creatinine clearance (P<0.01), and glomerular and mesangial surface area (P<0.05). Both plasma leptin (P<0.01) and adiponectin (P<0.05) levels were significantly decreased in seipin-/- mice. CONCLUSION: Seipin gene deficiency in mice leads to renal injury probably by decreasing plasma leptin and adiponectin levels due to lack of adipose tissue.     

Expression of MMP3 in subchondral bones in early experimental osteoarthritis

AIM: To demonstrate that matrix metalloproteinase 3(MMP3)plays MMP3 play an important role in the pathogenesis of osteoarthritis by studying the expression of MMP3 in subchondral bones in early experimental osteoarthritis at gene and protein levels. METHODS: The SD rats were randomly divided into 2 groups: experimental group and control group, each containing 30 rats with similar body weight. The right knee joints of the rats in experimental group underwent surgery, which involved in both medial collateral ligament (MCL) transection and medial meniscectomy, while the animals in control group were only carried out a sham operation. The rats were killed at the 1st, 2nd and 4th weeks post-surgery to obtain the right knee joints. Pathological analysis was performed to validate this early osteoarthritic rat model. The expression of MMP3 in subchondral bones at mRNA and protein levels was evaluated by real-time polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: The expression of MMP3 in subchondral bones was significantly increased at both the 1st and 2nd weeks post-surgery in experimental group, with the fold changes of 8.34 (P<0.05) and 4.85 (P<0.05), respectively, as compared with control group. No differentially expression of MMP3 was observed at the 4th week post-surgery between these 2 groups. A lot of MMP3 positive cells, including small mononuclear cells and bigger polynuclear giant cells, were found in subchondral bones in experimental group at the 1st and 2nd weeks post-surgery,but not in control group. At the 4th week post-surgery, no MMP3 positive cells were found in subchondral bones of both groups. CONCLUSION: MMP3 plays an important role in the pathogenesis of early experimental osteoarthritis.     

Relationship between changes of bone mineral density and bone marrow angiogenesis in ovariectomized rats

AIM: To explore the relationship between the changes of bone mineral density (BMD) and bone marrow angiogenesis in ovariectomized rats. METHODS: Thirty female Sprague-Dawley rats (3 month-age) were randomly divided into ovariectomized (OVX) groups and sham operated (sham) groups, and executed after 4 weeks, 8 weeks and 12 weeks respectively. The bone mineral density (BMD) of left femora was measured. The right distal femoral epiphysis was fixed in formalin, decalcificated by EDTA-Na2, dehydrated and embedded in paraffin. Four-μm-thick slices were obtained from the paraffin section and stained with haematoxylin-eosin (HE) for bone marrow pathological examination. The number of bone marrow microvessels was examined by means of immunohistochemical staining for CD34 to stain the endothelial cells of blood vessels to definite the bone marrow microvascular density (MVD). Plasma levels of vascular endothelial growth factor (VEGF) were examined by the method of ELISA. RESULTS: The BMD of femoral in 8-week OVX group were decreased significantly compared to the 8-week sham group (P<0.05), suggesting the establishment of osteoporosis model. Meanwhile, the area of hematopoietic tissue decreased and the area of adipose tissue increased. These changes became obviously in 12-week OVX group, and the area of trabecular bone and the bone marrow MVD significantly decreased compared to the 12-week sham group. A positive correlation between MVD and BMD, area of hematopoietic or trabecular bone as well as a negative correlation between MVD and area of adipose tissue were observed. The plasma levels of VEGF in OVX group were not significantly different from that in sham group, and had no correlation relationship with the indexes of bone marrow pathology. CONCLUSION: There has an increase in MVD companied with the bone mass loss and hematopoietic tissue decreased in ovarietomized rats, which provide experiment proof to treat osteoporosis with the means of promoting of MVD in bone marrow.     

Mechanism of JAK2-STAT3 signaling pathway in ischemic postconditio-ning neuroprotection after cerebral ischemia in tree shrews

AIM: To investigate the regulatory effect of JAK2-STAT3 signaling pathway on the neuroprotection of ischemic postconditioning (IPoC) in tree shrews, and to explore the mechanisms of cerebral injury deterioration after inhibiting the JAK2-STAT3 pathway. METHODS: The model of thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews and the IPoC was established at 4 h after ischemia followed by clipping ipsilateral common carotid artery on the ischemia side for 5 min (3 times). After IPoC and intracerebroventricular injection of AG490 (JAK2 inhibitor), the changes of cerebral infarction area were detected by TTC staining, and the histological and ultrastructural changes of cortical neurons were observed under light and electron microscopes, respectively. The protein levels of t-STAT3 and p-STAT3 in the cortical tissue were determined by Western blot. RESULTS: The neuronal pycnosis, mitochondrial swelling and vanish of the mitochondrial cristae were found in cortical cortex, and the infarction area was (24.78±3.30)% at 24 h after cerebral ischemia. Meanwhile, the phosphorylation level of STAT3 protein in the cortical tissue was significantly increased (P<0.01). The cortical neuronal damage and mitochondrial swelling were decreased after IPoC, the area of cerebral infarction was significantly reduced to (17.67±1.83)% (P<0.01), and the phosphorylation level of STAT3 protein was further increased (P<0.01). However, the neuronal damage was aggravated, the infarction area was expanded to (23.85±2.77)%(P<0.05) after treatment with AG490, and the phosphorylation level of STAT3 protein was also significantly reduced (P<0.05). CONCLUSION: IPoC may reduce cerebral injury by regulating the phosphorylation of STAT3 protein, and inhibition of JAK2-STAT3 signaling pathway may counteract the cerebral protective effect of IPoC and aggravate brain injury.     

Effect of hypothalamic arcuate nucleus damaged by monosodium glutamate on bone histomorphometry parameters in rat

AIM: To investigate the effect of hypothalamic arcuate nucleus(ARC) damaged by monosodium glutamate (MSG) on bone histomorphometry parameters in rats. METHODS: (1) Newborn SD rats of experimental group were hypodermically injected 10% MSG (4 g/kg BW) on the postnatal lst, 3rd, 5th, 7th, 9th day. Meanwhile, the newborn rats of the control group were given equal volume of normal saline and the rats after the postnatal 70th, 72th, 74th, 76th and 78th day were hypodermically injected 10% MSG (4 g/kg BW) as the MSG toxicity control group. After survivial for 160 days, all rats were killed. (2) Morphological methods were used to estimate the ARC neurons and the bone histomorphometry parameters. (3) Radioimmunoassay was used to measure the levels of the serum growth hormoen(GH), estradiol(E2) and testosterone(T). RESULTS: The number of the hypothalamic arcuate nucleus neurons, the levels of serum E2, T, GH and the %Tb·Ar, Tb·Th, Tb·N of proximal tibial metaphysis(PTM) significantly decreased, while Tb·Sp of proximal tibial metaphysis(PTM) significantly increased, and the osteoporotic alterations appeared obviously. All these changes did not appear in the rats of NS group and MSG toxicity control group. CONCLUSION: (1) The changes of the bone in MSG guoup rats are not the effect of the MSG toxicity on the bone directly. (2) The hypothalamic arcuate nucleus participates in the regulation of the bone metabolism. (3) ARC regulates bone metabolism via altering the functions of the hypothalamus-GH-IGF-1 axis and the hypothalamus-pituitary-gonal axis.     

Difference of ClC-3 protein expression and channel function between cisplatin-sensitive and -resistant ovarian cancer cells

AIM: To study the difference of ClC-3 chloride channel protein expression and channel function between cisplatin-sensitive (a2780) and -resistant (a2780cp) ovarian cancer cells. METHODS: The inhibition of a2780 and a2780cp cell proliferation induced by cisplatin were detected by MTT assay. The mRNA and protein expressions of ClC chloride channel families in a2780 cells and a2780cp cells were detected by real-time PCR and Western blot, respectively. The distribution of ClC-3 protein in a2780 cells and a2780cp cells were analyzed by immunofluorescence staining. The whole cell patch-clamp technique was used to record the chloride current in the cells. RESULTS: The sensitivities of a2780 cells and a2780cp cells to cisplatin were different. The IC₅₀ values of a2780 cells and a2780cp cells to cisplatin were 5 μmol/L and 20 μmol/L, respectively (P<0.01). The a2780 cells and a2780cp cells mainly expressed ClC-3 in ClC families. However, the mRNA expression of ClC-3 was much lower in a2780cp cells than that in a2780 cells (P<0.01). Compared with a2780 cells, the protein expression of ClC-3 in a2780cp cells was also significantly decreased (P<0.01). ClC-3 protein was mainly distributed on the membrane in a2780 cells, while was in cytoplasma in a2780cp cells. Cisplatin activated the chloride channel and induced the chloride current in the a2780 cells, but not in the a2780cp cells. Cisplatin did not induced the chloride current in a2780 cells treated with ClC-3 siRNA. CONCLUSION: The differences in protein distribution, expression and function of ClC-3 chloride channel were observed in cisplatin-sensitive and -resistant ovarian cancer cells, which may be one of the underlying mechanisms of cisplatin resistance.     

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.     

Crocin promotes mobilization of EPCs and re-endothelialization of damaged blood vessels in mice with carotid artery injury

AIM: To study the effect of crocin on the mobilization of endothelial progenitor cells (EPCs) in the peripheral blood of the mice with carotid arterial injury and its mechanism.METHODS: The carotid artery injury model of the C57BL/6 mice was established by the method of wire injury. The animals were divided into sham operation group, saline-treated model group, and low dose, medium dose and high dose (10, 50 and 100 μmol·kg^-1·L^-1, respectively) of crocin treatment groups. The mobilization of the EPCs in peripheral blood of the mice with carotid artery injury was detected by flow cytometry at 3 d. The changes of vascular endothelial growth factor (VEGF), stromal-derived factor-1 (SDF-1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9) in the peripheral blood of the mice with carotid artery injury were detected by enzyme-linked immunosorbent assay at 7 d. The vascular re-endothelialization and intimal hyperplasia of the mice with carotid artery injury were detected by Evans blue and hematoxylin-eosin staining. At the same time, real-time PCR was used to detect the mRNA expression of vascular repair factor-related receptors, vascular endothelial growth factor receotor-2 (VEGFR-2), CXC chemokine receptor-4 (CXCR4), basic fibroblast growth factor receptor (bFGFR) and epidermal growth factor receptor (EGFR), in the injured segments of carotid arteries.RESULTS: Compared with sham group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were increased in model group (P<0.05). The area of vascular endothelium was decreased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were increased significantly (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also increased in the injured segments of carotid arteries (P<0.05). Compared with model group, the EPCs mobilization and the content of vascular repair factors VEGF, SDF-1, bFGF, EGF and MMP-9 in peripheral blood were significantly increased in different concentrations of crocin-treated mice with carotid artery injury (P<0.05). The area of vascular endothelium was gradually increased, while the area of intimal hyperplasia and the ratio of intimal to medial membrane area were gradually decreased (P<0.05). The expression levels of VEGFR-2, CXCR4, bFGFR and EGFR were also gradually increased in the injured segments of cartid arteries (P<0.05).CONCLUSION: Crocin promotes the mobilization of EPCs and the re-endothelialization of damaged blood vessels in the mice with carotid artery injury, thus repairing the injured vasculature.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

An activated mutant of SHP-2 tyrosine phosphatase causes murine myeloid abnormal proliferation

AIM: To investigate whether an activated mutant of SHP-2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODS: Wild-type (WT) and SHP-2^D61G/+mutant mice aged 8 weeks and 16 weeks were used. The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively. The surface markers (Mac-1 and Gr-1 for myeloid, Ter119 for erythroid, CD3 for T-lymphocyte and B220 for B-lymphocyte) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry. The rate of Mac-1 or Gr-1 positive cells in the peripheral blood and the rate of Mac-1, Gr-1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed. The ability of colony formation unit (CFU) of the bone marrow was also observed by CFU assay. Finally, the expression of p-Akt and p-ERK in the peripheral blood leukocytes induced by interleukin-3 (IL-3, 5 μg/L) was detected by Western blotting.RESULTS: The number of leukocytes in peripheral blood of 16-week-old mice was more (P<0.01) and the spleens were bigger in mutant SHP-2^D61G/+ mice than those in WT mice. The rate of Mac-1 and Gr-1 positive cells in peripheral blood leukocytes of 16-week-old SHP-2^D61G/+ mice were dramatically increased (P<0.05). Mac-1 and Gr-1 positive cell rates in bone marrow of SHP-2^{D61G / +} mice were much higher (P<0.05) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells. The number of CFU-GM (represents myeloid) was increased in mutant mice. The expression of p-Akt and p-ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL-3.CONCLUSION: These results suggest that activated mutant SHP-2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Effect of ligustrazine on expression of LFA-1 and ICAM-1 in bone marrow of radiation injured mice

AIM: To investigate the effect of ligustrazine on the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in bone marrow and on the mechanism of hematopoietic reconstitution in radiation injured mice.METHODS: The 24 mice (clean class) were randomly divided into 3 groups: normal group, radiation injured group and ligustrazine group. After irradiation by 6.0Gy ［⁶⁰Co］ γ-ray, the radiation injured animals were given normal saline (0.2 mL, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine through gastric tube (0.2 mL, twice a day). The mice in normal group received no treatment. At the 7th, 14th, 21st day after irradiation, the femur were taken and the bone marrow mononuclear cells (BMNCs) suspension were made to culture bone marrow stromal cells (BMSCs). The mRNA and protein expressions of ICAM in BMSCs were assayed by RT-PCR and Western blotting. The expression levels of LFA-1 in BMNCs were evaluated by flow cytometry analysis.RESULTS: In ligustrazine group the expression levels of LFA-1 at the 7th, 14th and 21st days after irradiation were higher than those in radiation injured group (P<0.01 or P<0.05). However, the expression level of ICAM-1 was lower than that in the compared group (P<0.01 or P<0.05).CONCLUSION: Ligustrazine can increase the LFA-1 expression level of BMNCs, decrease the ICAM-1 expression level in BMSCs, indicating that ligustrizine promotes the recovery of hematopoietic cells in bone marrow, then improves the bone marrow microenvironment and enhances hematopoietic reconstitution.     

Microvessel density and expression of vascular endothelial growth factor in glomeruli of diabetic mice

AIM: To investigate the relationship between microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in glomeruli of diabetic mice. METHODS: Streptozotocin-induced diabetic mice as well as the control mice were involved in this study for 6 weeks. The body weight and blood glucose level of the mice in each group were weekly measured at certain time point. The morphological changes of the kidney were observed under light microscope, and the diameter, perimeter and area of the glomeruli were detected by an image analysis system. The expression of CD34 and VEGF in glomeruli was examined by immunohistochemistry method, and MVD and VEGF index were also calculated. RESULTS: In comparison with the control mice, the blood glucose level was significantly increased,and the body weight was decreased in diabetic mice(P<0.01). The diameter, perimeter and area of glomeruli in diabetic mice were significant greater than those in control mice (P<0.05). Increased expression of CD34 and VEGF in the glomeruli of diabetic mice was observed. Glomerular MVD of diabetic mice was significantly higher than that of the controls (P<0.01), and was positively correlated with the VEGF index (r=0.9979, P<0.05). CONCLUSION: VEGF may promote the angiogenesis in glomeruli of diabetic mice. The increase in VEGF expression may play a role in the pathogenesis of diabetic nephropathy.