Role of MCU in bupivacaine-induced spinal cord neurotoxic injury in diabetic rats

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in bupivacaine (B)-induced spinal cord injury in diabetic (D) rats.METHODS: Healthy male Sprague-Dawley rats, weighing 180~220 g, were divided into normal rats and diabetic rats. After intraperitoneal injection of streptozotocin for building diabetic rat mo-del, 48 male rats were randomly divided into 6 groups with 8 rats in each group as following:control (C) group (normal rats by intrathecal injection of normal saline), D group (diabetic rats by intrathecal injection of normal saline), C+B group (normal rats by intrathecal injection of bupivacaine), D+B group (diabetic rats by intrathecal injection of bupivacaine), D+R₁+B group (diabetic rats by intrathecal injection of 10 μmol/L Ru360 and bupivacaine) and D+R₂+B group (diabetic rats by intrathecal injection of 50 μmol/L Ru360 and bupivacaine). The changes of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured before modeling, after modeling, and 12 h, 24 h and 48 h after intrathecal injection. Lumbar enlargement was removed from spinal cord after rats were killed. MCU expression was tested by RT-qPCR and Western bolt. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) were determined by ELISA. Spinal neuronal apoptosis was measured using TUNEL assay.RESULTS: Compared with D group, the expression of MCU, the values of PWMT and PWTL, the levels of MDA and 8-OHdG, and the apoptotic rate of spinal cord neurons were significantly increased in D+B group (P<0.05). Compared with D+B group, the expression of MCU, the values of PWMT and PWTL, the levels of MDA and 8-OHdG, and the apoptotic rate of spinal cord neurons were significantly decreased in D+R₂+B group (P<0.05).CONCLUSION: Bupivacaine enhances oxidative stress and aggravates spinal cord injury via up-regulating MCU activity in diabetic rats.     

Relationship between hippocampal structure and high incidence of depression after spinal cord injury in mice

AIM: To explore whether the high risk of depression caused by spinal cord injury is related to structural changes of hippocampus. METHODS: Mouse spinal cord injury model was established. The Basso Mouse Scale (BMS) was used to evaluate the postoperative motor function of mice at different periods after injury. The depression of mice was evaluated by behavior experiment. The morphological changes of cells in hippocampal tissue were observed by HE staining. The structural changes of hippocampal subcellular synapses were observed by electron microscopy. RT-qPCR was used to detect the changes of synaptic protein markers synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at the mRNA level. RESULTS: The mice had significant depressive behaviors in model group, and the depression degree was the most serious on the 7th day (P<0.01). Compared with control group, the arrangement of hippocampal cells was disordered, the cell number was decreased, and the shape was irregular in model group. Compared with control group, the observation results of electron microscopy showed that the number of synapses in the hippocampal ultrastructure was decreased, the length of synaptic activity zone was shortened, the thickness of postsynaptic density was thinned, the width of synaptic gap was increased, and the curvature of synaptic interface was decreased in the model group (P<0.05). The mRNA expression levels of SYP and PSD-95 in model group were lower than those in control group (P<0.05). CONCLUSION: The change of hippocampal structure is one of the important reasons leading to high risk of depression caused by spinal cord injury.     

Protective effect of rapid ischemic preconditioning against spinal cord ischemic injury in rabbits

AIM: To evaluate the protective effect of rapid phase of ischemic preconditioning against spinal cord ischemic injury in rabbits. METHODS: Thirty six male New Zealands white rabbits were randomly assigned to 3 groups (12 in each group): ischemia and reperfusion injury group (IR group), ischemic preconditioning + IR group (IPC+IR group) and sham operation group (sham). In IR group, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min; The rabbits in IPC+IR group underwent a 6 min ischemic preconditioning followed by 30 min of reperfusion before the 20 min clamping; The rabbits in sham group underwent the same procedures as the IR group except for infrarental aortic unclamping. Neurologic status was scored at 8, 12, 24 and 48 h after reperfusion. All animals were sacrificed at 48 h after reperfusion and the spinal cords (L_5-7) were removed for histopathologic study and determination of the activity of Na⁺, K⁺-ATPase. RESULTS: The neurologic function scores in sham group and IPC+IR group at each observation interval were higher than those in IR group (P＜0.01). Compared to IR group, there were more normal neurons in anterior horn of spinal cord in sham group and IPC+IR group (P＜0.01); the activity of Na⁺, K⁺-ATPase in sham group and IPC+IR group were higher than those in IR group (P＜0.01). CONCLUSION: The rapid phase of ischemic preconditioning has a protective effect against spinal cord ischemic injury in rabbits, and this neuroprotection may be related to the maintenance of Na⁺, K⁺-ATPase activity.     

Relationship between the expression of apoptosis regulating proteins and the changes of electrophysiology after acute spinal cord injury

AIM: To observe the expression of apoptosis regulating proteins Bcl-2, Bax and the changes of cortical somatosensory evoked potential (CSEP), motion evoked potential (MEP) after acute spinal cord injury, and to explore the relationship between them. METHODS: Fifty six Wistar rats were randomly divided into 7 groups. Six groups were divided as acute spinal cord injury model, the other group was used as control. CSEP and MEP were monitored after 0 h, 6 h, 12 h, 24 h, 48 h and 72 h. The spinal cord was taken and then HE staining and immunohistochemistry methods were used to study the pathology changes and expressions of Bcl-2 and Bax. RESULTS: The peak of CSEP and MEP showed a immediate drop or disappear, and then partially raised at 6 h, declined again at 12 h and partially raised at 24 h, and then raised obviously at 48 h, the peak still lower than normal at 72 h. Changes of the peak value showed a double posture of decreased-raised- declined again and raised again. The incubation period of peaks extended corresponding to the different degrees. There was a weak positive expression of Bcl-2 and Bax at 6 h after injury. By 24 h after injury, the positive expression of Bcl-2 was maximal and the positive expression of Bax was maximal at 12 h after injury. CONCLUSION: The changes of CSEP and MEP have a very closely correlation to the degree of spinal cord injury. CSEP and MEP may serve as electrophysiological indicators to judge the motor function and the prognosis after acute spinal cord injury.     

Expression of NF-κB,NR2B and iNOS in spinal cord in a rat model of neuropathic pain

AIM: To observe the expression of nuclear factor-kappa B (NF-κB), N-methyl-D-aspartic acid receptor 2B (NR2B) and inducible nitric oxide synthase (iNOS) in the spinal cord in a rat model of chronic constriction injury (CCI) of the sciatic nerve. METHODS: Fifty-six adult male Sprague-Dawley rats weighing 180~220 g were randomly divided into sham group (n=8) and CCI group (n=48). The mechanical withdrawal threshold (MWT) and paw withdrawal latency (PWL) of the hind paws were measured 1 d before CCI and 1 d, 4 d, 7 d, 14 d and 21 d after surgery. The L4~L6 segment of the spinal cord was taken for determining the expression of NF-κB, NR2B and iNOS by RT-PCR and Western blotting. RESULTS: At 1 d, 4 d, 7 d, 14 d and 21 d after surgery, the MWT and PWL in CCI group were obviously lower than those in sham group. The expression of NF-κB, NR2B and iNOS at mRNA and protein levels increased significantly. Positive correlations were found between the mRNA expression of NF-κB and iNOS (r=0.842, P<0.05), and between the mRNA expression of NR2B and iNOS (r=0.833, P<0.05). CONCLUSION: The generation and maintenance of hyperalgesia in sciatic nerve injury rats may attribute to the activation of NF-κB and NR2B and concomitant increase in iNOS.     

Promoting effect of botulinum neurotoxin serotype A heavy chain on neuritogenesis in cultured Neuro-2a cells

AIM: To observe the neuritogenic actions of botulinum neurotoxin serotype A heavy chain (BoNT/A HC) on cultured Neuro-2a cells and to investigate the related signaling mechanisms for the effect of BoNT/A HC. METHODS: Neuro-2a cells were treated with different doses of BoNT/A HC (0.01, 0.1, 1 and 10 nmol/L), and then the cells were harvested at 24 h, 48 h and 72 h of BoNT/A HC exposure for detecting the neurite length and the percen-tage of the cells with neuronal processes by immunofluorescence staining. The most efficient dose of BoNT/A HC was chosen for exposure to Neuro-2a cells as the above. Whole cell protein was harvested at different time points for detecting the protein levels of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated Akt (p-Akt) by Western blot. RESULTS: Low doses of BoNT/A HC stimulated the neurite outgrowth, and increased the percentage of the cells with neurites compared with the negative controls (P<0.05), especially in the group with 1 nmol/L of BoNT/A HC treatment. Meanwhile, the phosphorylation of ERK1/2 and Akt was increased after treated with BoNT/A HC. There was an increasing tendency for the phosphorylation of ERK1/2 after the exposure of the cells to BoNT/A HC. The obvious increase in p-ERK1/2 was seen from 60 min to 5 h with 1 nmol/L of BoNT/A HC treatment (P< 0.05), and the increased protein level of p-Akt was mainly observed at 15 min and 60 min (P<0.05). CONCLUSION: BoNT/A HC stimulates the neuritogenesis. The neuritogenic mechanism of BoNT/A HC on Neuro-2a cells might be realized by activation of the phosphorylation of ERK1/2 and Akt.     

Effect of HO-1 expression on neuronal apoptosis in spinal cord of rats with formaldehyde inflammatory pain

AIM: To observe the change of heme oxygenase(HO-1)expression in the spinal cord during formaldehyde-induced pathological pain and investigate the effect of HO-1 on neuronal apoptosis in the spinal cord induced by formaldehyde inflammatory pain.METHODS: The protein expression of HO-1 in the left and the right spinal dorsal horn was detected by Western blotting. The neuronal apoptosis rate of the spinal cord was determined by flow cytometry.RESULTS: Compared to control group, the protein expression of HO-1 was significantly up-regulated at different time points after the injection of formaldehyde, which was most obviously 24 h after the injection of formaldehyde and was still higher than that in control group at 72 h. Compared to control group, the neuronal apoptosis rate of spinal cord increased in the rats with formaldehyde inflammatory pain. No significant difference of the neuronal apoptosis rate was observed between formaldehyde group and solvent control group . Intrathecal injection of 100 μg ZnppIX, an inhibitor of HO-1, attenuated the degree of spontaneous pain response, but induced an increase in the rate of neuronal apoptosis in spinal cord in the rats with formaldehyde inflammatory pain. CONCLUSION: Formaldehyde inflammatory pain induces the increases in HO-1 expression and neuronal apoptosis in the rat spinal cord. HO-1 promotes the response of spontaneous pain and inhibits the process of neuronal apoptosis in spinal cord induced by formaldehyde inflammatory pain.     

Protective effect of pentoxifylline against spinal cord ischemia/reperfusion injury in rabbits

AIM: To investigate the protection of pentoxifylline against spinal cord ischemia/reperfusion injury.METHODS: Rabbits sustained spinal cord ischemia with 45 min cross-clamping of the infrarenal aorta. Groups were as follows: sham operation (n=8); ischemic control (n=20), receiving only vehicle; PTX A (n=20), receiving PTX before clamping and PTX B (n=20), receiving PTX at the onset of reperfusion. Rabbits were evaluated for hind-limb motor function with the Tarlov scoring system at 48 h. Serum was assayed with ELISA for TNF-α and spinal cords were harvest for MPO activity, histopathologic analysis and TUNEL staining. Immunohistochemistry was used for PECAM-1 and caspase-3 detection, and the numbers of necrosic and apoptotic neuron were counted at 12 h, 24 h, 48 h and 72 h of reperfusion. The necrotic and apoptotic neurons were also observed with transmission electron microscope.RESULTS: Improved Tarlov scores were observed in PTX-treated rabbits as compared with ischemic control rabbits at 48 h. The significant reductions of TNF-α in serum, activity of MPO, immunoreactivity of the PECAM-1 and caspase-3 were found in PTX-treated rabbits. The numbers of necrosic and apoptotic neuron were higher in PTX-treated rabbits than that in the ischemic control rabbits (P<0.05). No necrosic and apoptotic neuron were found in the sham operation group. CONCLUSION: PTX induces protection against ischemia/reperfusion injury in the spinal cord, thereby preventing both necrosis and apoptosis.     

Preliminary study of bone marrow mesenchymal stem cell-derived exosomes in repair of spinal cord injury in rats

AIM:To study the influence of bone marrow mesenchymol stem cell-drived exosomes (BMSC-exosomes) on hindlimb activity, and the numbers of reactive astrocytes and residual neurons in spinal cord injury (SCI) rats. METHODS:BMSCs were cultured using the whole bone marrow adherent culture method and surface markers CD90 and CD34 were verified by flow cytometry. Exosomes were isolated by ultracentrifugation and the morphology of exosomes was observed under transmission electron microscope. The protein markers CD63 and CD9 were verified by Western blot. After exosomes were applied to SCI rats, the Basso, Beattie and Bresnahan locomotor rating scale score, the Nissl staining of the lesion site, and the numbers of reactive astrocytes and residual neurons were assessed at various time points. RESULTS:Transmission electron microscopic observation revealed the presence of saucer-shaped vesicles. BMSC-exosomes were found to express high levels of CD63 and CD9. Compared with injury group, significant improvement of hindlimb activity scores from day 14 after injury in treatment group was observed (P<0.05), and less reactive astrocytes and more residual neurons from day 7 after injury were also observed (P<0.05). CONCLUSION:BMSC-exosomes inhibit reactive astrocytes and death of neurons, and improve hindlimb activity in the rats after SCI.     

Effects of protein kinase C inhibitor,chelerythrine chloride,on nitric oxide synthase expression and nitric oxide content in spinal cord of rats with inflammatory pain induced by formalin

AIM: To study the effects of protein kinase C (PKC) inhibitor， chelerythrine chloride (CH)， on nociceptive response, nitric oxide synthase (NOS) expression and nitric oxide (NO) content in spinal cord of rats with inflammatory pain. METHODS: Inflammatory pain was induced by formalin injection into right hind paw. NADPH-d histochemistry was used to investigate the changes of NOS expression. Nitrate/nitrite (NO2-/NO3-) was assayed to represent NO content. RESULTS: Compared with the control group, the number of NADPH-d positive cells increased significantly in the superficial layer (LaminaeⅠ-Ⅱ) of the spinal cord dorsal horn and the grey matter surrounding the central canal (Laminae Ⅹ) in rats with inflammatory pain, the reactive degree of NADPH-d positive soma and fibers and NO content of the lumbar enlargement of spinal cord also increased significantly. Intrathecal injection of CH inhibited the spontaneous pain response in the second phase induced by formalin injection, and prevented the increases in the number and reactive degree of NADPH-d positive cells, as well as NO content of the lumbar enlargement of spinal cord. CONCLUSION: It is suggested that the activation of PKC promotes NOS expression and NO production in the nociceptive neurons of spinal cord during formalin-induced inflammatory pain.     

Role of interleukin-1β in spinal LTP in rats with neuropathic pain

AIM: To investigate the role of interleukin-1β (IL-1β) in the long-term potentiation (LTP) of C-fiber-evoked field potentials in rats with neuropathic pain. METHODS: The rat model of neuropathic pain was produced by spared nerve injury (SNI) of sciatic nerve or the method of lumbar 5 ventral root transection (L5 VRT). The effect of exogenous IL-1β on C-fiber-evoked field potentials of spinal dorsal horn was tested in both intact rats and the rats with neuropathic pain. The roles of p38 MAPK and NF-κB in the process were also evaluated. RESULTS: IL-1β at concentration of 500 μg/L affected neither basal synaptic transmission mediated by C-fiber nor spinal LTP induced by high frequency stimulation in intact rats. However, low concentration (5 μg/L) of IL-1β induced LTP of C-fiber-evoked field potentials in the rats with neuropathic pain. Pretreatment with either p38 MAPK inhibitor (SB203580) or NF-κB inhibitor (PDTC) completely blocked LTP induced by IL-1β. CONCLUSION: Exogeneous IL-1β might induce spinal LTP of C-fiber-evoked field potentials in the rats with neuropathic pain. p38 MAPK and NF-κB may be involved in the process.     

Expression of GAP-43 mRNA and protein following brachial plexus avulsion injury

AIM:To investigate the expression of GAP-43 mRNA and protein of the motor neurons in spinal cord following the brachial plexus avulsion injury. METHODS:In the present study, three kinds of models of brachial plexus avulsion injury were made: right C₇ anterior root avulsion (group A), C₇ anterior root avulsion with right C₅-T₁ posterior roots breaking (group B), right C₇ anterior root avulsion with hemi-transect between C₅ and C₆ segment of spinal cord (group C). The expression of GAP-43 mRNA in anterior horn of spinal cord was detected at 14 days after operation by SYBR green quantification RT-PCR technique. The amounts of GAP-43 positive neurons in spinal cord were detected at 1, 3, 7 and 14 days after operation by immunohistochemistry technique. RESULTS:In control group, the expression of GAP-43 mRNA was very low in anterior horn. By 14 days after operation, the expression of GAP-43 mRNA was evidently up-regulated compared with control group. GAP-43 positive neuron was observed in control group at 1st day and 3rd day after operation. GAP-43 positive neurons appeared at 7th day and peaked at 14th day after operation. The expression of GAP-43 mRNA and protein were maximum in group C, group B was the lowest. CONCLUSION:The expression of GAP-43 mRNA and GAP-43 protein were up-regulated following brachial plexus injury. The expression of GAP-43 protein results from the recombination of proteins. GAP-43 is closely related to the axon regeneration and functional reconstruction.     

Signaling transduction of noxious visceral stimuli in postsynaptic dorsal column neurons in spinal cord

The spinal postsynaptic dorsal column pathway plays a critical role in the visceral pain transmission in spinal cord. The noxious visceral stimuli might induce complex receptor expression and intracellular signaling transductions in postsynaptic dorsal column neurons. It has been demonstrated that neurokinin-1(NK-1) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and intracellular protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (MAPKs) and cAMP response element-binding (CREB) protein are involved in the signaling transduction of visceral stimuli. All these processes contribute to the sensitization of postsynaptic dorsal column neurons and enhance the spinal transmission of visceral pain, indicating a potential and promising way of visceral pain therapy to inhibit the sensitization of postsynaptic dorsal column neurons.     

Fasudil reduces formation of urethral stricture after injury via inhibiting Rho/ROCK pathway activation in rabbit urethra fibroblasts

AIM: To investigate the role of Rho-associated kinase (ROCK) inhibitor fasudil in the formation of rabbit urethral stricture after injury and to observe the cell activity, migration and extracellular matrix synthesis in the rabbit urethra fibroblasts. METHODS: The rabbit model of urethral stricture was established by microsurgical techniques. The rabbits were divided into sham operation group, operation group and fasudil (3 mg/kg, 10 mg/kg, 30 mg/kg) groups. The diameter of the stenosis was measured by retrograde urethrography 3 months after surgery. The fibroblasts were isolated from urethral scar, and then incubated with fasudil (12.5 μmol/L, 25 μmol/L, 50 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L). The untreated cells were used for control. The cell activity was measured by MTT assay. The cell migration ability was tested by the method of Transwell chambers. The protein expression of ROCK, α-smooth muscle actin (α-SMA), collagen I and collagen III was determined by Western blot analysis. RESULTS: Fasudil significantly reduced formation of urethral stricture after injury (P<0.05). Cultured rabbit fibroblasts with different concentrations of fasudil inhibited the cell activity and cell migration ability (P<0.05). The protein expression of ROCK, α-SMA, collagen I and collagen III was also inhibited by treatment with fasudil in a dose-dependent manner (P<0.05). CONCLUSION: Fasudil inhibits the formation of extracellular matrix and reduces the incidence of urethral stricture after injury by down-regulating TGF-β1-induced Rho/ROCK pathway activation in the rabbit urethra fibroblasts.     

Effect of IL-13 on expression of IL-1β in acute renal ischemia/ reperfusion injury in rats

AIM:To investigate the effects of IL-13 on expression of IL-1β in acute renal ischemia/reperfusion injury.METHODS:Fifty-seven male Wistar rats were randomly divided into 8 group: normal group, sham operation group, ischemia group, ischemia/reperfusion injury group(I/R), normal saline(NS)-treated group 1(C-1), NS-treated group 2(C-2), IL-13-treated group1(T-1)and IL-13-treated group 2(T-2).Rats were subjected to 45 min bilateral renal ischemia followed by reperfusion. rmIL-13 (1.5 μg/50 g body weight )was injected into the renal arteries through the abdominal aorta before ischemia(T-1) or immediately afterischemia(T-2).The serum level of IL-1β and the renal expression of IL-1β were determined in each group at 24 h post-ischemia. In addition, BUN, Cr and renal histology were also measured.RESULTS:(1)The serum level of IL-1β, gene expression and protein production of IL-1β in kidney decreased markedly in IL-13-treated groups.(2)Renal function and histology were significantly improved in IL-13-treated groups, renal injury scores decreased significantly.(3)A positive correlation were found between the serum level of IL-1β and BUN, SCr(r=0.708, P＜0.01;r=0.770, P＜0.01).CONCLUSION:These data suggest that IL-13 inhibit the expression of IL-1βand improve func-tion and histology of kidney in acute renal ischemia/reperfusion injury.     

Knockdown of Atg5 aggravates cerebral ischemia and reperfusion injury in mice

AIM: To study the role of autophagy-related gene 5 (Atg5) in cerebral ischemia and reperfusion injury in mice. METHODS: BALB/c male mice (weighing 18~22 g) were randomly divided into sham group, ischemia/reperfusion (I/R) group, Atg5 siRNA group and control siRNA group. Focal cerebral ischemia was performed using the method of middle cerebral artery occlusion (MCAO) for 60 min and reperfusion for 24 h. In siRNA group and control group, 5 μL Atg5 siRNA or scrambled siRNA was administered by intracerebroventricular injection 24 h before MCAO. The expression of Atg5 at mRNA and protein levels in ischemic cortex at 24 h after reperfusion was determined by real-time PCR and Western blot. The infarct volume and edema were evaluated by TTC staining, and motor deficits were evaluated by neurological scoring. RESULTS: The expression of Atg5 at mRNA and protein levels was significantly increased 24 h after reperfusion in I/R group compared with sham group. Atg5 siRNA obviously decreased the expression of Atg5 at mRNA and protein levels induced by I/R. Inhibition of Atg5 exacerbated the infarct volume and ameliorated the neurological symptoms. CONCLUSION: Atg5 has neuroprotective effect on focal cerebral ischemia and reperfusion injury.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Inhibitory effect of IGF-1 overexpression on oxidative damage in umbilical cord mesenchymal stem cells

AIM: To analyze the inhibitory effect of insulin-like growth factor-1 (IGF-1) overexpression in umbilical cord mesenchymal stem cells (UC-MSCs) on oxidative damage and to develop new application model for UC-MSCs. METHODS: UC-MSCs were isolated from human umbilical cord with enzymatic digestion, and further characte-rized with flow cytometry. IGF-1-overexpressing UC-MSCs (UC-MSCs-IGF-1) were established by retrovirus infection. IGF-1 expression of UC-MSCs-IGF-1 was evaluated by real-time quantitative PCR and flow cytometry, and its surface markers, as well as osteogenic and adipogenic differentiation ability, were further analyzed. The proliferation, anti-oxidative damage and anti-apoptosis abilities of UC-MSCs-IGF-1 were evaluated when treated with H₂O₂ at different concentrations (0 μmol/L, 10 μmol/L, 50 μmol/L and 100 μmol/L). RESULTS: UC-MSCs showed positive expression of CD29, CD90 and CD105, but negative expression of CD34, which coincided with the normal phenotype of mesenchymal stem cells. UC-MSCs-IGF-1 established with retrovirus infection showed much higher expression of IGF-1 compared with normal UC-MSCs, and expressed the same surface markers as UC-MSCs.The osteogenic and adipogenic differentiation abilities were also observed. With the oxidative damage by H₂O₂ treatment, UC-MSCs-IGF-1 showed more strong proliferation, anti-oxidative damage and anti-apoptosis abilities as compared with normal UC-MSCs. In addition, the activity of SOD in UC-MSCs-IGF-1 was a little higher than that in control group. CONCLUSION: IGF-1 overexpression in UC-MSCs inhibits oxidative damage and cell apoptosis. UC-MSCs-IGF-1 may have more advantagies in clinical application.     

Roles of Akt pathway and cell apoptosis in leptin-mediated chronic morphine antinociceptive tolerance

AIM: To explore the roles of Akt (also called protein kinase B) and active caspase-3 in the leptin-mediated chronic morphine antinociceptive tolerance in rats. METHODS: A model of chronic morphine antinociceptive tolerance was established in the SD rats. The protein levels of spinal Akt and cleaved caspase-3 were tested by Western blotting. The technique of immunohistochemical staining was used to detect the immunoreactivity positive cells of phosphorylated (p)-Akt and cleaved caspase-3 in the spinal cord. Double staining of immunohistochemistry was used to examine the cellular location of the p-Akt and cleaved caspase-3 positive cells. RESULTS: The chronic intrathecal injection of morphine (15 μg) for 7 d markedly upregulated the spinal protein levels of p-Akt and cleaved caspase-3 in the rats. Thirty min before injection of morphine, intrathecal injection of leptin antagonist (3 μg) for 7 d significantly attenuated the upregulation of the protein levels of p-Akt and cleaved caspase-3 induced by chronic morphine treatment. The p-Akt was exclusively observed in the spinal neurons. The cleaved caspase-3 was only localized with the spinal astrocytes. Intrathecal injecting the inhibitors of leptin, Akt and caspase-3 ameliorated the chronic antinociceptive tolerance. CONCLUSION: The spinal Akt pathway and active caspase-3 are involved in the leptin-mediated chronic morphine antinociceptive tolerance in rats.