Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

SDF-1α/CXCR4 axis promotes migration and invasion of pancreatic cancer cells through inducing epithelial-mesenchymal transition

AIM:To investigate the role of SDF-1α/CXCR4 axis in pancreatic cancer cell migration and invasion.METHODS:The mRNA expression of CXCR4 in 4 pancreatic cancer cell lines was detected by RT-qPCR. The migration and invasion abilities of PANC-1 cells with the axis activated by exogenous SDF-1α or inhibited by CXCR4 inhibitor AMD3100 were detected by Transwell assays. The cell viability was measured by MTS assay. The protein expression of the epithelial-mesenchymal transition (EMT)-related molecules in the cells treated with exogenous SDF-1α or AMD3100 was determined by Western blot.RESULTS:All of the 4 pancreatic cancer cell lines expressed CXCR4 mRNA, while the PANC-1 cell line expressed the most. Exogenous SDF-1α promoted the migration and invasion abilities of PANC-1 cells, which was inhibited by AMD3100. The PANC-1 cells treated with exogenous SDF-1α for 72 h grew faster, while SDF-1α combined with AMD3100 made little significance to the viability of PANC-1 cells. Exogenous SDF-1α induced EMT of PANC-1 cells by up-regulating the expression of SNAIL and TWIST, and AMD3100 reversed this effect.CONCLUSION:SDF-1α/CXCR4 axis enhances the migration and invasion abilities of pancreatic cancer cells through inducing EMT.     

Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Role of purinergic signaling mediated by ATP in Alzheimer's disease-associated colonic motility disorder

AIM: To explore the role of purinergic signaling mediated by ATP in the Alzheimer's disease (AD)-related colon motility disorder and its related molecular mechanisms. METHODS: (1)Clinical trials:AD patients in our hospital were collected and studied. Radioimmunoassay was used for the determination of plasma motilin (MTL), cholecystokinin (CCK), vasoactive intestinal peptide (VIP) and nitric oxide (NO), and high-performance liquid chromatography (HPLC) was applied to test the level of adenosine triphosphate (ATP). The patients were assessed by neuropsychology and scored accordingly. (2)In animal experiments, AD mice received Morris water maze test, and the spatial learning and memory function were evaluated. The plasma levels of MTL, CCK, VIP and NO were examined by radioimmunoassay, and the level of ATP was measured by HPLC. Choline acetyltransferase (ChAT), VIP, nitric oxide synthase (NOS) and ATP synthase were detected by immunohistochemistry. Western blot and immunohistochemistry were used to detect the expression of P2Y receptor. (3)In vitro, organ bath was applied to observe the effect of α,β-methylene ATP (α,β-MeATP), an agonist of P2Y receptor, on both spontaneous and electrically evoked contraction of colonic smooth muscle strip, and the technique of intracellular microelectrode was applied to observe the effect of α,β-MeATP on the membrane potential of colonic smooth muscle cells. RESULTS: Compared with control group, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P < 0.05 or P < 0.01), while the VIP level was not changed. Mini-Mental State Examination (MMSE) score was decreased (P<0.05), Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score, Neuropsychiatric Inventory (NPI) score and Alzheimer's Disease Cooperative Study-Activities of Daily Living Scale (ADCS-ADL) were all increased as compared with control group (P<0.01). The 4~6 d escape latency of APP/PS1 AD mice was significantly prolonged (P<0.05), and the space exploration ability distinctly reduced (P<0.05). In AD mice, the levels of MTL and CCK were decreased (P<0.01), and the levels of NO and ATP were increased (P<0.05 or P<0.01), while the VIP level was not changed. The protein expression of colonic ATP synthase was significantly increased (P<0.05), but the expression of ChAT, VIP and NOS was not changed. The expression of P2Y receptor was increased (P<0.01). The results of in vitro experiment displayed that α,β-MeATP, from 20 μmol/L to 100 μmol/L, inhibited the spontaneous contraction of colonic smooth muscle strip in the normal mice and AD mice (P < 0.05 or P < 0.01), and this inhibition was reversed by Na⁺ channel inhibitor tetrodotoxin (TTX) (P < 0.05 or P < 0.01). In addition, the effect of α,β-MeATP at 100 μmol/L on the AD mice was more obvious than that on the normal mice (P<0.05), and this inhibition was also antagonized by TTX (P < 0.05 or P < 0.01), pro-minent in AD group as compared with control group (P<0.05). In 10 Hz electrically evoked contraction of colonic smooth muscle strip, α,β-MeATP inhibited both the normal and AD mice (P < 0.05 or P < 0.01), while the inhibition was more obvious in the AD mice at the concentration of 40 μmol/L or 100 μmol/L (P < 0.05 or P < 0.01). CONCLUSION: AD patients and AD mice are accompanied by decreased MTL and CCK levels, and enhanced NO level, thus inducing colonic motor dysfunction along with AD. Meanwhile, ATP in plasma, purinergic neurons, and P2Y receptor expression are increased in the AD mice. Purinergic signaling mediated by ATP inhibits colonic smooth muscle strip contraction and further paralyzes the colonic movement function in AD.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

Expression of hepatocyte nuclear factor 4α in experimental colitis in mice

AIM: To investigate the role of hepatocyte nuclear factor 4α (HNF4α) in the pathogenesis of ulcerative colitis (UC) by measuring the expression of HNF4α in the colon tissues in experimental colitis mice. METHODS: BALB/c mice were exposed to 2% or 2.5% (W/V) dextran sulfate sodium (DSS) to induce acute colitis, and the severity of colitis was assessed by observation of disease activity index (DAI), histological injuries and inflammatory cytokines. The correlation between the expression of HNF4α and the severity of disease as well as E-cadherin (E-CAD), junctional adhesion molecule 1 (JAM-1) and desmocollin 2 (DSC-2) was analyzed. RESULTS: Compared with the normal controls, DAI, histological injuries and the mRNA expression of inflammatory cytokines in DSS-treated mice were significantly elevated (P<0.05). The expression of HNF4α at protein and mRNA levels was significantly decreased (P<0.01). The result of Pearson analysis indicated an inverse correlation between the protein expression of HNF4α and the severity of disease (P<0.01). The positive correlation between the mRNA expression of HNF4α and E-CAD/JAM-1/DSC-2 (P<0.01) was also observed. CONCLUSION: There is a close relationship between the expression of HNF4α and the severity of colitis as well as the intercellular linking proteins. The low expression of HNF4α in intestine might aggravate the function of intestinal mucosal barrier, thus promoting the development of UC.     

Effects of erythropoietin on expression of SDF-1 and its receptor in peripheral blood endothelial progenitor cells from rats with chronic renal failure

AIM:To investigate the effects of erythropoietin (EPO) on the expression of homing factors in peripheral blood endothelial progenitor cells (EPCs) from rats with chronic renal failure (CRF). METHODS:The CRF model was established by a two-stage 5/6 nephrectomy procedure in rats. Experimental rats were randomly divided into three groups: sham operation group, CRF model group and EPO treatment group. From the third week after the second stage of 5/6 nephrectomy procedure, rats in EPO treatment group received subcutaneous injection of human recombinant EPO at 50 U/kg every time and three times a week for 6 weeks, and then all the rats were sacrificed. EPCs were isolated from rat peripheral blood and primarily cultured. The mobilization, angiogenesis and functional activity of EPCs in vitro were detected. The mRNA and protein expression of EPO, EPO receptor (EPOR), stromal cell-derived factor 1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in EPCs was also detected by the methods of real-time PCR and Western blotting. RESULTS:Compared with CRF model group, the expression of EPO and EPOR in EPCs in EPO treatment group was significantly up-regulated (P<0.05). Moreover, the expression of SDF-1 and its receptor CXCR4 in EPCs was also up-regulated by administration of EPO (P<0.05). CONCLUSION: EPO can mobilize EPCs from CRF rat peripheral blood, which may be associated with the increased expression of SDF-1 and its receptor CXCR4.     

Resveratrol modulates SDF-1/CXCR4 signaling to inhibit hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats

AIM:To investigate the effect of resveratrol (RSV) on apoptosis and stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway in hypoxia-ischemia-induced neurons from the brain of newborn rats and its mechanism. METHODS:The cortex neurons from the brain of newborn rats were given oxygen-glucose deprivation (OGD) treatment to mimic neonatal hypoxic-ischemic encephalopathy (HIE). The cortex neurons were randomly divided into 4 groups:control group, HIE model group, HIE+RSV-low (10 μmol/L) group, and HIE+RSV-high (50 μmol/L) group. After OGD treatment for 2 h, the neurons were cultured with indicated dose of RSV for 24 h. The apoptosis was analyzed by flow cytometry. Western blot was used to determine the levels of apoptosis-related proteins, SDF-1 and CXCR4. Real-time PCR was used to detect the mRNA expression of SDF-1 and CXCR4. Additionally, to explore the effects of RSV on cell apoptosis and apoptosis-related proteins after the suppression of SDF-1/CXCR4 signaling, a CXCR4 antagonist AMD3100, and RSV were used to co-treat OGD-injured neurons for 24 h. RESULTS:RSV alleviated OGD-induced neuronal apoptosis, down-regulated cleaved caspase-3 and cytochrome C levels, and up-regulated the ratio of Bcl-2/Bax. Compared with the control group, OGD treatment increased the expression of SDF-1 and CXCR4 (P<0.05). Compared with the HIE model group, RSV further up-regulated the expression of SDF-1 and CXCR4 (P<0.05). AMD3100 reversed the effects of RSV on OGD-induced cell apoptosis. CONCLUSION:RSV suppresses hypoxia-ischemia-induced apoptosis of neurons from the brain of newborn rats via up-regulating SDF-1/CXCR4 signaling pathway.     

Effects of procyanidins on oxidative damage of osteocytes caused by TCP wear particles

AIM: To investigate the effects of procyanidins (PC) on oxidative damage of osteocytes caused by tricalcium phosphate (TCP) wear particles, and to explore the underling mechanism. METHODS: Mouse long bone osteocyte MLO-Y4 cells were treated with TCP wear particles (0.1 g/L) for 48 h to establish the model of osteocyte injuries. The MLO-Y4 cells were divided into 4 groups:control group, TCP group, PC (10 μmol/L) group and PC (50 μmol/L) group. Calcein-AM staining and MTT assay were used to observe the viability of MLO-Y4 cells. The levels of dentin matrix protein 1 (DMP-1), sclerostin (SOST) and interleukin-1β (IL-1β) in the culture media were examined by ELISA. The apoptosis of MLO-Y4 cells was analyzed by flow cytometry with Annexin V/PI double staining. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of MLO-Y4 cells, and lactate dehydrogenase (LDH) release in the culture media were measured by chemical colorimetry. The protein levels of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1 and IL-1β in the MLO-Y4 cells were determined by Western blot. RESULTS: Compared with control group, MLO-Y4 cell injuries, apoptosis rate and MDA level were obviously increased in TCP group, while SOD activity was significantly decreased (P<0.05) The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were remarkably up-regulated (P<0.05) in the MLO-Y4 cells, and the level of IL-1β and LDH release were increased in the culture media (P<0.05). Compared with TCP group, the injuries of MLO-Y4 cells, apoptosis rate and MDA level were decreased obviously (P<0.05) in PC groups, whereas SOD activity was increased (P<0.05). The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were down-regulated remarkably in the MLO-Y4 cells (P<0.05), and the level of IL-1β and LDH release were significantly decreased in the culture media (P<0.05). CONCLUSION: PC obviously inhibit oxidative damage of osteocytes caused by TCP wear particles, which may be related to alleviating NLRP3 inflammasome activation and pyroptosis.     

Correlation between progression of atherosclerosis accelerated by emotional stimulation and imbalance of SDF-1/CXCR4 in mice

AIM To observe effects of emotional stimulation on expression of stromal cell-derived factor-1（SDF-1） and CXC chemokine receptor 4 （CXCR4） in plasma, platelets, aortas, hippocampus and bone marrow of apolipoprotein E gene knockout （ApoE^-/-） mice, and to reveal the possible mechanism of the aggravated atherosclerotic plaque vulnerability by emotional stimulation. METHODS Thirty 8-week-old male ApoE^-/- mice were randomly divided into normal control group, high fat group, and emotional stimulation group. Ten 8-week-old inbred C57BL/6J mice served as blank control group. After 12 weeks of intervention, the serum levels of SDF-1 and CXCR4 were investigated by ELISA. The protein levels of SDF-1 and CXCR4 in platelets, aortas, hippocampus and bone marrow were determined by Western blot. The pathological damage of aortas was observed by oil red O staining. RESULTS Compared with blank control group, normal control group and high fat group, the mice subjected to emotional stimulation showed more serious atherosclerosis in aortas detected by oil red O staining, and increased levels of SDF-1 and CXCR4 in the plasma and aortas were also observed （P<0.05）. The results of Western blot showed that the protein levels of SDF-1 and CXCR4 in platelets, aortas and hippocampus were increased in the mice subjected emotional stimulation, but the expression of SDF-1 and CXCR4 in the bone marrow was decreased （P<0.05）. CONCLUSION Imbalance of SDF-1/CXCR4 may be the key target by which emotional stimulation accelerates the progression of atherosclerosis.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Effects of Youguiwan on microstructure and Hsp90 of chondrocytes in rats with knee osteoarthritis

AIM: To observe the effect of Youguiwan (YGW) on the expression of heat shock protein 90α (Hsp90α) in articular cartilage tissues of knee osteoarthritis (KOA) rats, and to further reveal its mechanism. METHODS: SD rats (n=60) were randomly divided into 6 groups:sham control (SC) group, model (M) group, glucosamine sulfate (GS) group, and high-, middle-and low-dose YGW (YGW-H, YGW-M and YGW-L) groups. The modified Hulth method was used to make KOA models for 6 weeks. The rats were gavaged with corresponding drugs for 8 weeks. HE staining was used and Makin score was evaluated. The expression of Hsp90α, fibronectin (Fn) and collagen type Ⅱ (COL-II) was determined by immunohistochemistry. The expression of interleukin-1β (IL-1β), matrix metalloproteinase (MMP)-3 and MMP-13 was analyzed by RT-qPCR. The protein expression of Hsp90α and COL-II was determined by Western blot. RESULTS: As compared with SC group, the Makin score was obviously raised in M group, the expression levels of Hsp90α, IL-1β, MMP-3 and MMP-13 were evidently increased, the expression levels of COL-II and Fn were evidently decreased (P < 0.01), articular cartilage was seriously damaged, and the chondrocytes were disarranged. As compared with M group, the Makin score and the expression of Hsp90α were obviously decreased in YGW-H group, the protein expression of Fn was evidently increased in YGW-H group, the protein expression of COL-II was evidently increased in YGW-H and YGW-M groups, the mRNA expressions of IL-1β, MMP-3 and MMP-13 were evidently decreased in YGW-H, YGW-M and YGW-L groups (P < 0.05 or P < 0.01), cartilage structure tended to be normal, the chondrocytes distribution was uneven, and articular cartilage surface was not smooth. CONCLUSION: Youguiwan is effective for treatment of KOA. Youguiwan protects against articular cartilage degeneration by down-regulating the expression of Hsp90α and up-regulating the expression of Fn, and reducing the secretion of inflammatory factors and the degradation of extracellular matrix.     

The SDF-1/CXCR4 axis repairs hypoxia-ischemia brain damage via-regulating oriented differentiation of mesenchymal stem cells

AIM:To explore the effect of stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)axis on the neural differentiation of rat mesenchymal stem cells(rMSCs) and recovery from hypoxia-ischemia brain damage(HIBD). METHODS:The rMSCs were isolated from the rat bone marrow, and expanded in vitro. The mRNA and protein levels of CXCR4 in rMSCs treated with SDF-1α(10 μg/L) and hypoxia for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h were detected by RT-PCR, Western blotting and flow cytometry. The mRNA and protein levels of SDF-1α in the hippocampus of the rats with hypoxia-ischemia for 1 d, 3 d, 5 d, 7 d, 14 d and 21 d were also detected by the same methods. The protein levels of neuron-specific enolase(NSE) and glial fibrillary acidic protein(GFAP), as well as the positive rate of neural-induced rMSCs pretreated with AMD3100(a CXCR4 antagonist) at dose of 5 mg/L were determined by Western blotting and immunocytochemistry. RESULTS:Compared with the normal controls, both mRNA and protein levels of CXCR4 increased in rMSCs exposed to hypoxia for 6 h and 12 h, and the results were also confirmed by flow cytometry. As expected, the mRNA level of SDF-1α in the hippocampus of HIBD rats was higher than that in normal control rats(P<0.01). Moreover, the mRNA expression of CXCR4 was extremely up-regulated in rMSCs treated with SDF-1α at concentration of 10 μg/L, and the results were also confirmed by Western blotting and flow cytometry analysis(P<0.01). The protein levels and positive cell numbers of NSE and GFAP were extremely decreased in rMSCs pretreated with AMD3100 at concentration of 5 mg/L. CONCLUSION:Compared with normal rats, SDF-1α level in the hippocampus of the rats with hypoxia-ischemia is increased. Hypoxia and micro-dose of SDF-1α induce the expression of CXCR4 in rMSCs, while CXCR4 antagonist reduces the neural differentiation of rMSCs, suggesting that SDF-1/CXCR4 axis may be deeply involved in the neural differentiation of rMSCs during the process of repairing HIBD.     

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.     

Protective effect of β-asarone on primary rat hippcampal neurons against hypoxia/hypoglycemia and reperfusion injury

AIM：To investigate the protective effect of β-asarone against hypoxia/hypoglycemia and reperfusion injury in primary rat hippocampal neurons. METHODS：Cell viability, the activity of caspase-3, the protein expression of p-JNK and Bcl-2, and the mRNA expression of Bcl-2 and caspase-3 were determined by MTT assay, spectrophoto-metry, Western blotting and real-time PCR. RESULTS：Compared with normal control group, the cell viability decreased and the activity of caspase-3 increased obviously, the expression of p-JNK protein and caspase-3 mRNA increased obviously, and the expression of Bcl-2 protein decreased obviously in model group (P<0.05). Compared with model group, different doses of β-asarone inhibited the changes of the above indexes (P<0.05). CONCLUSION：β-asarone inhibits JNK-mediated chondrosome signaling pathway, thereby attenuating the process of hippocampal neuron apoptosis after hypoxia/hypoglycemia and reperfusion.     

IL-1β induces human mesangial cell injury via exacerbating lipid-induced endoplasmic reticulum stress

AIM：To investigate the molecular mechanism that interleukin-1 beta (IL-1β) exacerbates lipid-induced endoplasmic reticulum stress (ERS) and the injury of human mesangial cells (HMCs). METHODS：HMCs were cultured and divided into control group, low-density lipoprotein (LDL) group, IL-1β+LDL group and 4-phenyl butyric acid (4-PBA)+IL-1β+LDL group. Oil red O staining was used to evaluate the accumulation of lipid droplet in the cells. The mRNA levels of glucose-regulated protein 78(GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and α-smooth muscle actin (α-SMA) were examined by real-time PCR. Immunocytochemistry was used to observe GRP78 expression. The protein level of NF-κB p65 was measured by Western blotting. The releases of IL-6 and TGF-β1 in the culture supernatants of HMCs were detected by ELISA. RESULTS：Compared with LDL group, the intracellular lipid accumulation, the mRNA levels of GRP78 and PERK, the protein expression of GRP78 and NF-κB p65, and the release of IL-6 were significantly increased in IL-1β+LDL group. Dramatically reduced intracellular lipid accumulation, down-regulated GRP78 and PERK mRNA expression, decreased protein levels of GRP78 and NF-κB p65, and suppressed IL-6 release were observed in 4-PBA+IL-1β+LDL group as compared with IL-1β+LDL group. The mRNA level of α-SMA was higher in IL-1β+LDL group than that in LDL group, and that in 4-PBA+IL-1β+LDL group was significantly depressed. CONCLUSION：IL-1β exacerbates lipid-induced ERS, thus promoting the injury of HMCs.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.